Oral Administration of an Active Form of Vitamin D3

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Oral Administration of an Active Form of Vitamin D3
(Calcitriol) Decreases Atherosclerosis in Mice by Inducing
Regulatory T Cells and Immature Dendritic Cells With
Tolerogenic Functions
Masafumi Takeda, Tomoya Yamashita, Naoto Sasaki, Kenji Nakajima, Tomoyuki Kita,
Masakazu Shinohara, Tatsuro Ishida, Ken-ichi Hirata
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Objective—To determine whether the administration of an active form of vitamin D3 (calcitriol) could prevent
atherosclerosis through anti-inflammatory actions.
Methods and Results—Recent clinical studies have shown that lack of vitamin D3 is a risk factor for cardiovascular events.
Oral calcitriol administration decreased atherosclerotic lesions, macrophage accumulation, and CD4⫹ T-cell infiltration
at the aortic sinus, when compared with the corresponding observations in control mice. We observed a significant
increase in Foxp3⫹ regulatory T cells and a decrease in CD80⫹CD86⫹ dendritic cells (DCs) in the mesenteric lymph
nodes, spleen, and atherosclerotic lesions in oral calcitriol–treated mice in association with increased interleukin 10 and
decreased interleukin 12 mRNA expression. CD11c⫹ DCs from the calcitriol group showed reduced proliferative
activity of T lymphocytes, suggesting the suppression of DC maturation. Neutralization of CD25 in vivo revealed that
calcitriol inhibited atherosclerosis mainly in a regulatory T cell– dependent manner but also partly because of a decrease
in DC maturation.
Conclusion—Oral calcitriol treatment could prevent the development of atherosclerosis by changing the function or
differentiation of DCs and regulatory T cells. These findings suggest that intestinal and systemic immune modulation
by calcitriol may be a potentially valuable therapeutic approach against atherosclerosis. (Arterioscler Thromb Vasc
Biol. 2010;30:2495-2503.)
Key Words: atherosclerosis 䡲 regulatory T cells 䡲 dendritic cells 䡲 immune system 䡲 calcitriol
T
T-lymphocyte–mediated pathogenic immune response plays
a critical role. Clinical strategies developed to modulate the
immune response have been insufficient for preventing atherosclerosis. Cumulative data based on experimental animal
models suggest that CD4⫹ T cells are present within plaques
from the initial stages of the disease in mice, and adaptive
transfer of these cells is potentially proatherogenic.8 Accumulating evidence has revealed novel functions of several
subsets of regulatory T cells (Tregs), which maintain immunologic tolerance to self-antigens and inhibit atherosclerosis
development by suppressing the inflammatory response of
effector T cells.9 –12 These studies have provided new insights
into the immunopathogenesis of atherosclerosis and imply
that promotion of regulatory immune responses may have
therapeutic potential for suppression of atherosclerotic
diseases.
In addition to Tregs, dendritic cells (DCs) are also reportedly involved in maintaining immune tolerance to self-
he active form of vitamin D3, 1,25(OH)2-dihydroxyvitamin
D3, is a secosteroid hormone that not only plays a central
role in bone and calcium metabolism but also modulates the
immune response. Recent epidemiological studies have
shown a relationship between low plasma levels of vitamin
D3 and a predisposition to cardiovascular events.1–3 This
finding is supported by a meta-analysis showing that oral
vitamin D3 treatment contributes to the improvement of
mortality from all causes, in part by decreasing cardiovascular deaths.4 Transgenic rats constitutively expressing vitamin
D-24-hydroxylase, a model of vitamin D3 deficiency, showed
aggravated atherosclerosis under a high-fat and high-cholesterol diet, when compared with control rats.5 However, there
are no reports about the direct effects of an orally administered active form of vitamin D3 on atherosclerosis.
See accompanying article on page 2317
It is widely recognized that atherosclerosis is a complex
inflammatory disease of the arterial wall,6,7 in which the
Received on: June 2, 2010; final version accepted on: September 23, 2010.
From the Division of Cardiovascular Medicine, Department of Internal Medicine (M.T., T.Y., K.N., T.K., M.S., T.I., and K.-i.H.), Kobe University
Graduate School of Medicine, Kobe, Japan; and the Department of Experimental Pathology (N.S.), Institute for Frontier Medical Science, Kyoto
University, Kyoto, Japan.
Correspondence to Tomoya Yamashita, MD, PhD, Division of Cardiovascular Medicine, Department of Internal Medicine, Kobe University Graduate
School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. E-mail [email protected].ac.jp
© 2010 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
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DOI: 10.1161/ATVBAHA.110.215459
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Arterioscler Thromb Vasc Biol
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antigens. DCs are the most potent antigen-presenting cells.
They efficiently stimulate the differentiation of effector T
cells from naı̈ve T-cell precursors. DCs are also thought to
perform the important function of presenting antigens to T
cells, which leads to peripheral tolerance by inducing Tregs
or inhibiting effector T cells.13 DCs can be tolerogenic and
immunogenic.14 In particular, immature DCs, which downregulate major histocompatibility complex class II molecules
and costimulatory molecules (eg, CD40, CD80, and CD86)
have had tolerogenic properties associated with decreased
interleukin (IL) 12 and enhanced IL-10 production. Recently,
the contribution of DCs to atherogenesis has been extensively
examined.15–17 Furthermore, DCs and Tregs may be helpful in
inducing tolerance against self-antigens within plaques.
Therefore, an agent able to modulate the function of DCs and
Tregs may be beneficial in the treatment of atherosclerotic
disease.
Recently, several articles have reported that 1,25(OH)2dihydroxyvitamin D3 induced reciprocal differentiation
and/or expansion of Tregs and induced tolerogenic DCs
characterized by downregulation of costimulatory molecules.18 Calcitriol and its analogues have inhibited autoimmune disease, allergy, and the rejection of transplanted
organs in animal models via induction of tolerogenic DCs and
Tregs.19 –21 However, the interaction between DCs and Tregs
remains to be fully elucidated. Given this background, we
examined whether orally administered calcitriol would induce Tregs and tolerogenic DCs in apolipoprotein E– knockout (ApoE⫺/⫺) mice and how these immune cells might
contribute to the inhibition of atherosclerosis. We focused not
only on systemic immune responses but also on regulation of
the intestinal immune system as therapeutic targets for
suppressing the formation of atherosclerotic lesions. Our
findings provide the first evidence that oral calcitriol administration induces Tregs and immature DCs via the intestinal
immune system and that these 2 immune cells are induced
both systemically and locally in atherosclerotic lesions, resulting in mutually suppressing pathogenic immune processes
that play a pivotal role in the progression of atherosclerosis.
Methods
Detailed Methods are provided in the supplemental data (available
online at http://atvb.ahajournals.org).
Experimental Design
We fed 6-week-old female ApoE⫺/⫺ mice 20 or 200 ng of calcitriol
dissolved in carboxymethylcellulose or vehicle alone by gastric
intubation with a plastic tube twice a week for 12 weeks. Mice under the
same protocol as previously described were injected with either 100 ␮g
of neutralizing CD25 monoclonal antibody (clone PC61) to deplete
CD4⫹CD25⫹ Tregs or 100 ␮g of isotype-matched control rat IgG once
every 4 weeks at the age of 6, 10, and 14 weeks. Mice were euthanized
at the age of 18 weeks, and atherosclerotic lesions were examined as
previously described.12 All animal experiments were conducted according to the Guidelines for Animal Experiments at Kobe University
School of Medicine, Kobe, Japan.
Cell Isolation Using In Vitro Cell Functional
Experiments and Flow Cytometry Analyses
Purified CD4⫹ T cells and CD11c⫹ DCs were isolated from
mesenteric lymph nodes (MLNs) and spleens. A cell proliferation
assay was performed by assessing [3H]thymidine incorporation.
Table.
Body Weight, Blood Pressure, and Plasma Analyses*
D3 Group
Characteristics
Control Group
20 ng
200 ng
Body weight, g
21.2⫾1.1
20.8⫾1.0
21.5⫾1.0
109.3⫾1.4
106.8⫾6.8
103.1⫾1.4
Blood pressure, mm Hg
Systolic
Diastolic
65.4⫾11.0
57.1⫾9.7
1,25(OH)2-dihydroxyvitamin D3,
pg/mL
85.1⫾40.5
78.8⫾56.4 180.6⫾75.5†
57.3⫾12.7
25(OH)-hydroxyvitamin D3,
ng/mL
29.7⫾5.4
30.2⫾9.7
25.6⫾3.2
Intact PTH, pg/mL
49.5⫾8.1
51.9⫾9.1
50.0⫾8.8
Calcium, mg/dL
7.2⫾0.5
7.4⫾0.9
7.7⫾1.2
Phosphorus, mg/dL
7.7⫾1.0
7.3⫾2.7
7.7⫾2.3
Cholesterol, mg/dL
Total
422.7⫾45.4
400.4⫾61.5 534.1⫾53.5†
Low-density lipoprotein
74.6⫾16.9
67.2⫾13.5
80.2⫾15.2
High-density lipoprotein
10.0⫾3.7
10.2⫾3.3
11.7⫾3.6
Triglyceride, mg/dL
38.2⫾11.6
47.5⫾24.7
39.3⫾22.6
Renin mRNA levels
1.00⫾0.48
NA
0.88⫾0.36
NA indicates not applicable; PTH, parathyroid hormone.
*Data are given as mean⫾SEM (6 to 9 mice per group).
†P⬍0.05 vs control mice.
Fluorescence-activated cell sorter (FACS) analysis was performed.
Total RNA was extracted from these cells for real-time RT-PCR.
Statistical Analysis
Data were expressed as mean⫾SEM. To detect significant differences between 2 groups or among 3 groups, an unpaired Student t
test, a Mann–Whitney U test, or a 1-way ANOVA with a post hoc
test was used when appropriate. P⬍0.05 was considered statistically
significant.
Results
Effects of Oral Calcitriol Treatment on General
Conditions and Plasma Examination Values
Calcitriol at various doses inhibits autoimmune disease (0.03
␮g/kg PO per day), allergy (0.5 ␮g/kg SC per day), and
rejection of transplanted organs (5 ␮g/kg PO 3 times per
week) in animal models.19 –21 Compared with previous reports, the dose used in our study (10 ␮g/kg [200 ng] PO 2
times per week) was somewhat higher. However, severe
adverse effects, such as loss in body weight and hypercalcemia, were not observed in the present study. Notably, 200 ng
of calcitriol (2 times per week) significantly increased its
plasma level without affecting plasma levels of 25(OH)hydroxyvitamin D3, intact parathyroid hormone, calcium, or
phosphorus. Although plasma 25(OH)-hydroxyvitamin D3
levels were inversely associated with renin activity in hypertensive patients,2 and calcitriol administration led to suppression of renin in an animal model,22 in the present study,
calcitriol administration affected neither blood pressure nor
renin mRNA expression in the kidney (Table). The effect of
vitamin D3 supplementation on blood pressure may be limited
only to subjects under hypertensive conditions or vitamin D3
insufficiency. Contrary to our expectations, the administra-
Takeda et al
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Figure 1. Oral calcitriol treatment inhibits atherosclerotic plaque
formation. Images are representative photomicrographs of oil
red O staining and measurement analysis of atherosclerotic
lesion size in the aortic sinus of female ApoE⫺/⫺ mice treated
with vehicle (control), 20 ng of calcitriol (D3 20 ng), or 200 ng of
calcitriol (D3 200 ng) for 12 weeks. The black bar on the left represents 200 ␮m; and horizontal bars on the right, means.
*P⬍0.05 vs controls.
tion of 200 ng of calcitriol significantly increased plasma
total cholesterol level (Table).
Oral Calcitriol Treatment Inhibits Atherosclerotic
Plaque Formation in ApoEⴚ/ⴚ Mice
To determine the effect of calcitriol administration on the
development of atherosclerosis, 6-week-old female ApoE⫺/⫺
mice receiving a standard diet were orally treated with 20 or
200 ng of calcitriol dissolved in carboxymethylcellulose or
with vehicle alone twice a week for 12 weeks. At the age of
18 weeks, the mice were euthanized; and cryosections of the
aortic root from the 3 groups were stained with oil red O and
analyzed. Surprisingly, 200 ng of calcitriol induced a marked
39.1% reduction in atherosclerotic lesion formation (plaque
area, 1.84⫾0.53⫻105 ␮m2), when compared with the values
in controls (2.88⫾0.90⫻105 ␮m2 [calcitriol, 20 ng] and
2.66⫾0.67⫻105 ␮m2 [vehicle]; P⬍0.05; Figure 1).
Effects of Calcitriol on Tregs in the Small
Intestine, MLNs, and Spleen
To investigate the effects of 200 ng of calcitriol treatment on
Tregs in ApoE⫺/⫺ mice, we performed immunohistochemical
studies in the small intestine and flow cytometry analyses in
MLNs and spleens. FACS analyses revealed that all of the
Treg subsets, such as CD4⫹CD25⫹, CD4⫹CD25⫹Foxp3⫹,
and CD4⫹Foxp3⫹, were significantly increased in the calcitriol group in MLNs and spleen (compared with the control
group: P⬍0.05; Figure 2A–2D). A further detailed investigation just after starting calcitriol treatment revealed that the
number of Tregs first increased in MLNs (but not thymus),
suggesting that the active form of vitamin D3 mainly induces
Tregs peripherally (supplemental Figure I). We also demonstrated a significant increase in intestinal Foxp3⫹ Tregs by
Vitamin D3 Inhibits Atherosclerosis
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immunohistochemistry in the calcitriol group (P⬍0.05, Figure 2E). Next, to confirm the induction of Tregs, we applied
quantitative RT-PCR to assess mRNA levels of Tregassociated markers in CD4⫹ T cells from MLNs after
positively separating them with anti–CD4⫹ microbeads (Figure 2F). We found a significant increase in mRNA levels of
Treg-associated markers (ie, CD25, Foxp3, and cytotoxic
lymphocyte antigen 4 [CTLA4]) in the calcitriol group
(P⬍0.05). In addition, mRNA expression of IL-10 and
transforming growth factor (TGF)-␤ tended to increase in
CD4⫹ T cells from the MLNs of calcitriol-treated mice
(P⫽0.0816 and P⫽0.510, respectively), suggesting the possibility of Treg induction. To determine whether a calcitriolinduced increase in Tregs contributes to the suppression of
T-cell function, we performed in vitro proliferation assays of
CD4⫹ T cells from MLNs and spleen of the calcitriol or
control group. As shown in Figure 2G, CD4⫹ T-cell proliferation was suppressed in the calcitriol group, suggesting an
increased number of Tregs by calcitriol might contribute to
the suppressive potential of T lymphocytes.
Effects of Calcitriol on DCs in the Small Intestine,
MLNs, and Spleen
CD80 and CD86 are recognized as important costimulatory
molecules related to the maturation of DCs. We performed
FACS analyses using MLN cells (Figure 3A) and splenocytes
and found a significant decrease in CD80⫹CD86⫹ mature
DCs within the CD11c⫹ DC population from calcitrioltreated mice (P⬍0.05, Figure 3B). To confirm the effects of
calcitriol on inhibition of DC maturation, we assessed mRNA
levels of DC-associated markers in CD11c⫹ DCs from
spleens after positive selection with anti–CD11c⫹ microbeads (Figure 3C). RT-PCR analyses revealed that CD80
and CD86 mRNA levels tended to decrease in the calcitriol
group (P⫽0.098 and P⫽0.1023, respectively). Furthermore,
we found a significant decrease in IL-12p40 mRNA levels
and an increase in those of IL-10 in the calcitriol group
(P⬍0.05). Notably, we also found a significant increase in
CC-chemokine ligand (CCL)17 and CCL22 mRNA levels,
which were recognized as Treg-attracting chemokines, in the
calcitriol group (P⬍0.05). Consistent with previous reports,23,24 these results indicate that calcitriol induced immature DCs, which interact with Tregs to suppress activation of
immune reactions via their tolerogenic properties. To determine whether a calcitriol-induced increase in the number of
tolerogenic DCs contributes to the suppression of T lymphocytes, we performed in vitro proliferation assays of CD4⫹ T
cells from Balb/c mouse spleens stimulated by CD11c⫹ DCs
from the spleens of mice from the calcitriol or control group
(Figure 3D). CD4⫹ T-cell proliferation was suppressed to a
greater extent when cocultured with DCs from the calcitriol
group than when cultured with DCs from the control group.
These results indicated that DCs from calcitriol-treated mice
resulted in reduced proliferation activity of T lymphocytes.
Taken together, tolerogenic DCs induced by oral calcitriol
administration suppressed T-cell immune responses and
worked as antiatherogenic factors mainly by changing their
cytokine and chemokine productions. We further evaluated
the number of CD11c⫹CD86⫹ mature DCs in the small
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Figure 2. Effects of calcitriol on Tregs in the
small intestine, MLNs, and spleen. A, Representative results of CD4, CD25, and Foxp3⫹
expression in MLNs from the control or calcitriol group, assessed by flow cytometry. B
through D, The graphs represent the percentage of CD25⫹ cells within the CD4⫹ population
(B), CD25⫹Foxp3⫹ cells within the CD4⫹ population (C), and Foxp3⫹ within the CD4⫹ population (D) in MLNs and spleen. For all FACS analyses, data represent mean⫾SEM of at least 9
mice in each group. The horizontal bars represent means. *P⬍0.05 vs control. E, Representative photomicrographs and measurement
analyses of Foxp3⫹ Treg immunostaining in
the small intestine from control and calcitrioltreated mice. The white bar represents
100 ␮m. Data represent mean⫾SEM of 8 mice
in each group. *P⬍0.05 vs control mice. F,
Total RNA extracted from CD4⫹ T cells from
MLNs of control or calcitriol-treated mice.
Expressions of CD25, Foxp3, CTLA4, IL-10,
and TGF-␤ were quantified by quantitative
RT-PCR and normalized to GAPDH. Fold
changes relative to the control group are
shown; n⫽5 to 9 per group. *P⬍0.05 vs control. G, CD4⫹ T cells were positively selected
with anti–CD4⫹ antibody microbeads. Proliferation of CD4⫹ T cells from MLNs and spleen of
control or calcitriol-treated mice was assessed
by [3H]thymidine incorporation, stimulated with
anti–CD3 and anti–CD28 antibodies; n⫽3 per
group. *P⬍0.05 vs control mice. APC indicates
allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin.
intestine by immunohistochemistry and revealed that there
were no significant differences in the number of CD11c⫹
DCs and CD11c⫹CD86⫹ mature DCs between the 2 groups
(Figure 3E). However, the percentage of CD86⫹ cells in
CD11c⫹ DCs was lower in the calcitriol group.
Effects of Calcitriol on Tregs, DCs, and
Inflammatory Cells in Atherosclerotic Plaques
To evaluate the effects of calcitriol on Tregs and DCs in
atherosclerotic plaques, we conducted immunohistochemical
studies and RT-PCR in atherosclerotic lesions. The immunohistochemical analyses of Foxp3⫹ Tregs in atherosclerotic
lesions showed a significantly increased number of Foxp3⫹
Tregs in the calcitriol group (P⬍0.05, Figure 4A and 4B). We
further examined the expression of Treg-associated markers,
such as CD25, Foxp3, and CTLA4, in atherosclerotic lesions
and demonstrated that mRNA levels of all Treg-associated
markers significantly increased in the calcitriol group when
compared with the control group (P⬍0.05, Figure 4C).
Recent studies have suggested that antigen presentation may
occur within atherosclerotic plaques and in lymphoid organs
and that Tregs migrate to atherosclerotic lesions to suppress
local immune responses.25 Our finding supports this notion
and implies that Tregs could be a promising target in treating
atherosclerotic plaques. Next, we investigated the effect of
calcitriol on DCs in atherosclerotic lesions and found that the
numbers of CD11c⫹ DCs and CD11c⫹CD86⫹ mature DCs
and the percentage of CD86⫹ cells among CD11c⫹ DCs were
significantly decreased in the calcitriol group (P⬍0.05, Figure 4D and 4E). These results indicated that calcitriol decreased the number of DCs recruited into the plaque and that
they were maintained in an immature state in atherosclerotic
lesions. To reveal the effects of oral calcitriol treatment on
inflammatory cells, such as macrophage and CD4⫹ cells, we
conducted immunohistochemical studies of the atherosclerotic lesions (Figure 4F and 4G). Interestingly, the 200-ng
calcitriol group showed a 29.0% reduction in the accumulation of macrophages (P⬍0.01) and also a 26.4% decrease in
CD4⫹ T-cell infiltration (P⫽0.02), compared with the values
recorded for the control group.
Next, to reveal the mechanisms of reduced atherosclerotic
plaque development and inflammatory cell recruitment, we
Takeda et al
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Figure 3. Effects of calcitriol on DCs in the
small intestine, MLNs, and spleen. A, Representative results of CD80 and CD86 expression
in CD11c⫹ DCs of MLNs from the control or
calcitriol group, assessed by flow cytometry.
B, Measurement analyses of CD80⫹CD86⫹
cells in the CD11c⫹ population in MLNs and
spleens. For all FACS analyses, data represent
mean⫾SEM of at least 6 mice in each group.
The horizontal bar represent means. *P⬍0.05
vs control. C, Total RNA was extracted from
CD11c⫹ DCs from spleens of control or
calcitriol-treated mice. Expressions of CD80,
CD86, IL-10, TGF-␤, IL-12p35, IL-12p40,
CCL17, and CCL22 were quantified by quantitative RT-PCR and normalized to GAPDH. Fold
changes relative to the control group are
shown; n⫽5 to 7 per group. *P⬍0.05 vs control. D, Proliferation of CD4⫹ T cells from
spleens of Balb/c mice with CD11c⫹ DCs from
spleens of control or calcitriol-treated mice, as
assessed by [3H]thymidine incorporation; n⫽3
per group. *P⬍0.05 vs control mice. E,
Measurement analyses of CD11c⫹ DCs,
CD11c⫹CD86⫹ DCs, and percentage of CD86⫹
within the CD11c⫹ DC population in the small
intestine from control and calcitriol-treated
mice. Data represent mean⫾SEM of 4 mice in
each group. APC indicates allophycocyanin; PE,
phycoerythrin.
examined mRNA expression of the proinflammatory molecules in atherosclerotic plaques by quantitative RT-PCR
(Figure 4H). We found that adhesion molecule and proinflammatory chemokine and cytokine, such as vascular cell
adhesion molecule-1, monocyte chemoattractant protein-1,
and interferon-␥, were markedly reduced (P⬍0.05) and that
anti-inflammatory cytokines, such as TGF-␤ and IL-10, were
significantly increased (P⬍0.05) in the calcitriol group when
compared with the control group. The expression of DCderived chemokine CCL22 mRNA was significantly increased (P⬍0.05); and the expression of its receptor, CCchemokine receptor (CCR) 4, tended to increase in
atherosclerotic lesions of the calcitriol group (P⫽0.0983).
Taken together, these results indicated that calcitriol might
inhibit the migration of effector T cells and macrophages into
the plaques by increasing the proportion of immature DCs
and Tregs systemically and locally in atherosclerotic plaques.
Inhibition of Tregs by Injection of Neutralizing
Anti–CD25 Antibodies
To clarify the interaction between Tregs and DCs in preventing atherosclerosis after calcitriol treatment, we conducted an
in vivo CD25 neutralization study with the injection of
anti–CD25 antibody. According to a previous report,9 the
effect of a single intraperitoneal injection of 100 ␮g of
CD25-depleting PC61 antibody was maintained for 4 weeks.
In our experiment, we injected CD25-depleting PC61 antibody or isotype-matched control antibody into mice once
every 4 weeks at the ages of 6, 10, and 14 weeks. Both the
calcitriol and control groups receiving anti–CD25 antibody
showed significantly increased atherosclerotic lesion formation when compared with the calcitriol group receiving
isotype-matched control antibody (P⬍0.05, Figure 5A).
These results suggested that Tregs had pivotal and major
roles in inhibiting atherosclerosis after calcitriol treatment.
Notably, when mice were injected with neutralizing antibody,
there remained significant differences in atherosclerotic lesion formation between calcitriol-treated and control mice,
indicating that calcitriol partially inhibited atherosclerotic
lesion formation independent of Tregs (P⬍0.05). We also
measured the percentage of Tregs by FACS in the MLNs and
spleens after the injection of anti–CD25 antibody (Figure
5B–5E). In contrast with a previous report,9 we observed a
60% to 80% depletion in CD4⫹CD25⫹ Tregs and CD4⫹
CD25⫹Foxp3⫹ Tregs in MLNs and spleens at 1 week after a
single intraperitoneal injection of 100 ␮g of CD25-depleting
PC61 antibody. However, all changes had reversed by 2
weeks after injection (supplemental Figure II).
We further analyzed DC maturation after partial depletion of
CD4⫹CD25⫹ Tregs and observed that the rates of maturation
were increased by anti–CD25 antibody administration and that
the calcitriol group receiving anti–CD25 antibody still showed a
decrease in the number of CD80⫹CD86⫹ mature DCs when
compared with the control group receiving anti–CD25 antibody
injection (Figure 5F). These data suggest that DC maturation
was highly associated with the induction of Tregs, although
calcitriol may partially decrease DC maturation independent of
Tregs. It is likely that calcitriol mainly inhibits the progression of
atherosclerosis via a Treg-dependent pathway but also a partly
DC-mediated and Treg-independent manner.
Discussion
Recent clinical studies and experimental investigations have
indicated that vitamin D3 insufficiency increases the inci-
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Figure 4. Effects of calcitriol on Tregs, DCs, and
inflammatory cells in atherosclerotic plaques. A,
Representative photomicrographs of Foxp3⫹ Tregs
in the atherosclerotic plaques of the aortic sinus
from control and calcitriol-treated mice. The white
bar represents 100 ␮m. B, Measurement analyses
of Foxp3⫹ Tregs in the plaques of the aortic sinus
from control and calcitriol-treated mice. Data represent mean⫾SEM of 5 mice in each group. The horizontal bars represent means; and white bar,
100 ␮m. *P⬍0.05 vs control. C, Total RNA was
extracted from the aortic roots of control or
calcitriol-treated mice. The expressions of CD25,
Foxp3, and CTLA4 were quantified by quantitative
RT-PCR and normalized to GAPDH. Fold changes
relative to the control group are shown; n⫽6 to 10
per group. *P⬍0.05 vs control. D, Representative
photomicrographs of CD11c⫹ (green), CD86⫹ (red),
and CD11c⫹CD86⫹ DCs (yellow) in the plaques of
the aortic sinus from control and calcitriol-treated
mice. E, Measurement analyses of CD11c⫹ DCs
and CD11c⫹CD86⫹ DCs and the percentage of
CD86⫹ in CD11c⫹ DCs in the plaques of the aortic
sinus from control and calcitriol-treated mice. Data
represent mean⫾SEM of 5 mice in each group.
*P⬍0.05 vs control mice. F and G, Representative
photomicrographs of atherosclerotic lesions stained
with antibodies specific for MOMA-2 for macrophages (F) and CD4⫹ T cells (G) treated with control or calcitriol. Data represent mean⫾SEM of 12
mice in each group. The black bar represents
200 ␮m. Right panels indicate the measurement
analyses of MOMA-2 and CD4⫹ T cells of the left
histological assays, respectively. The horizontal bars
represent means. *P⬍0.05 vs control. H, Total RNA
was extracted from the aortic roots of control or
calcitriol-treated mice. Expression of typical adhesion molecules, cytokines, chemokines, and their
receptors in atherosclerotic lesions was quantified
by quantitative RT-PCR and normalized to GAPDH.
Fold changes relative to the control group are
shown; n⫽5 to 10 per group. *P⬍0.05 vs control.
ICAM indicates intercellular adhesion molecule; IFN,
interferon; MCP, monocyte chemoattractant protein; VCAM, vascular adhesion molecule.
dence of cardiovascular events.1–3 Supplementation with an
active form of vitamin D3 (calcitriol) should have beneficial
effects on cardiovascular disease (CVD) through antiinflammatory and vasculoprotective actions.4 However, there
have been no clinical or animal studies showing the beneficial
actions of vitamin D3 in the treatment of CVD, including
atherosclerosis. In the present study, we showed, for the first
time to our knowledge, that oral calcitriol administration
inhibited atherosclerosis in an animal model, suggesting a
beneficial effect of vitamin D3 on clinical CVD. At least 2
different cells (ie, Tregs and immature DCs) were involved in
the antiatherogenic mechanisms of vitamin D3 treatment.
Although the antiatherogenic mechanism of Tregs’ effect and
the functional role of DCs in atherogenesis still remain to be
determined, we have revealed, for the first time to our
knowledge, that calcitriol-induced tolerogenic DCs and Tregs
might have antiatherogenic properties.
Accumulating evidence has revealed that several subsets of
Tregs have beneficial effects on atherogenesis.9 –12 Both
naturally occurring CD4⫹CD25⫹ Tregs and IL-10 –producing Tregs (Tr1) have inhibited atherosclerosis in mouse
models.10,11 Recently, it was demonstrated that an orally
administrated immunomodulatory agent, anti–CD3 antibody,
inhibits atherosclerosis by inducing Tregs, especially latencyassociated peptide Tregs.12 Vitamin D3 receptor (VDR)
ligands induce the differentiation of Foxp3⫹ Tregs18 and Tr1
in the presence of dexamethasone,26 and both types of Tregs
contribute to T-cell immune response inhibition. In the
present study, FACS analyses in MLNs and spleens documented that Foxp3⫹ Tregs were significantly increased in the
calcitriol group. However, it is unlikely that Tr1 and latencyassociated peptide Tregs play central roles in Treg induction
by calcitriol (supplemental Figure III). Foxp3⫹ Tregs were
significantly increased in systemic lymphoid organs and
atherosclerotic lesions in the calcitriol group. Although the
actual roles of intraplaque Tregs in atherogenesis have not yet
been clarified, a previous study suggested that increasing
numbers of Tregs might suppress pathogenic T-cell immune
responses or macrophage activation in atherosclerotic
lesions.12
Several articles have previously reported that VDR ligands
inhibit the differentiation and maturation of DCs.18 –21 These
Takeda et al
Vitamin D3 Inhibits Atherosclerosis
2501
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Figure 5. Inhibition of Tregs by injection of
neutralizing anti–CD25 antibodies. A, Measurement analysis of atherosclerotic lesion size
in the aortic sinus of female ApoE⫺/⫺ mice
treated with calcitriol and anti–CD25 antibodies
(D3 200 ng plus anti–CD25 antibody [Ab]), control with anti–CD25 antibodies (control plus
anti–CD25 Ab), and calcitriol with isotypematched control antibodies (D3 200 ng plus rat
IgG). The horizontal bar represents means. B,
At 1 week after a single intraperitoneal injection
of 100 ␮g of anti–CD25 or isotype-matched
antibodies, lymphoid cells from spleens were
prepared and stained with fluorescein isothiocyanate (FITC)–anti-CD4, phycoerythrin (PE)–
anti-CD25, and allophycocyanin (APC)–antiFoxp3. Representative results of CD4, CD25,
and Foxp3⫹ expression in MLNs from the control or calcitriol group with anti–CD25 antibodies, as assessed by flow cytometry. C through
E, The graphs represent the percentage of
CD25⫹ cells within the CD4⫹ population (C),
CD25⫹Foxp3⫹ cells within the CD4⫹ population (D), and Foxp3⫹ within the CD4⫹ population (E) in MLNs and spleen. Data represent
mean⫾SEM of 5 mice in each group. F, The
graph represents the percentage of CD80⫹ and
CD86⫹ cells within the CD11c⫹ population in
MLNs and spleen. Data represent mean⫾SEM
of 5 mice in each group. *P⬍0.05 vs control
plus anti–CD25 Ab. †P⬍0.05 vs D3 200 ng plus
anti-CD25. FSC, forward scatter.
results included the observation that treatment of DCs with
calcitriol leads to low surface expression of costimulatory
molecules, such as CD80 and CD86; decreased IL-12 production; and enhanced secretion of IL-10 and CCL22, resulting in T-cell hyporesponsiveness. The presence of such
costimulatory molecules on DCs is required for T-cell activation and for differentiation from naı̈ve T lymphocytes into
effector T cells. In the absence of costimulation, T cells
interacting with DCs undergo anergy or apoptosis. IL-12p70,
a heterodimeric cytokine consisting of p35 and p40, is
released mainly by activated macrophages and DCs. IL12p70 is a key mediator in inducing T-helper type 1 response
and stimulates the production of interferon-␥ from T-helper
type 1. Inhibition of DC-derived IL-12p70 production is
followed by a downregulated response of T-helper type 1.
Furthermore, the anti-inflammatory cytokine IL-10 also inhibits T-cell immune response by acting on antigen-presenting cells, such as macrophages and DCs. Our FACS
analysis revealed that CD80⫹CD86⫹ DCs are decreased in
MLNs and spleens of calcitriol-treated mice, indicating augmentation of the immature DC phenotype. Expression of
IL-12p40 mRNA in splenic DCs was decreased, and expression of IL-10 was increased. We clearly demonstrated that
DCs from the calcitriol treatment group had less T-cell
proliferation activity, which might indicate that DCs changed
their phenotypes to tolerogenic. Recently, the functional
importance of DCs in atherosclerosis has been highlighted in
several animal and human studies.15–17 Accumulating evi-
dence suggests that DCs in atherosclerosis are involved in
antigen presentation to T cells within plaques.15 DCs present
in normal arteries are immature and become activated during
atherogenesis, and DCs in vessels have been involved in the
initiation and progression of atherosclerosis.16,17
Regarding the interactions between Tregs and tolerogenic
DCs, immature DCs lacking sufficient expression of costimulatory molecules have induced Tregs13,14 and affected suppressive immunoresponses through the induction of antiinflammatory cytokines, such as IL-1027 and TGF-␤.28 In our
study, we demonstrated that expression of IL-10 mRNA in
DCs was significantly increased, whereas expression of
TGF-␤ mRNA was not increased. In accordance with previously published findings that naı̈ve T cells in the periphery
can differentiate into Foxp3⫹ Tregs in the presence of
IL-10,29 the increase of IL-10 in tolerogenic DCs might
contribute to the induction of Tregs, including Tr1. Tregs
play critical roles in the differentiation of immature DCs
through the interaction of CTLA4 and CD80/CD86. Tregs
constitutively express high levels of CTLA4 that bind to
CD80 and CD86 with high affinity. CTLA4 activity is
important for Treg-induced tolerance in several animal models.30 The interaction of CTLA4 on Tregs with CD80/CD86
on CD11c⫹ DCs conveyed a negative signal to DCs and
reduced the expression of these costimulatory molecules on
DCs in vitro.31 In our study, CTLA4 mRNA expression in
CD4⫹ T cells was significantly increased in the calcitriol
group. Taken together, we conclude that Tregs and tolero-
2502
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December 2010
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genic DCs might interact via CTLA4 and CD80/CD86 to
induce each other, resulting in effective inhibitory immunoresponses in both cell types. We also found another important
interaction between Tregs and tolerogenic DCs: expression of
CCL17 and CCL22 mRNA in the splenic DCs was enhanced
in the calcitriol group in this study. These chemokines are
ligands for CCR4 and CCR8 expressed on Tregs.32 DCs are
the major source of CCL22 in vitro and in vivo,33 and
transcriptional changes and production of CCL22 in human
myeloid DCs have been induced by calcitriol.23,24 In the
present study, calcitriol increased CCL22 expression in DCs,
suggesting that DCs released Treg-attracting chemokines and
attracted Tregs via CCR4. Tregs were also promoted in vivo
by calcitriol administration. In addition, the percentage of
immature DCs was increased in atherosclerotic lesions in the
calcitriol group. Interestingly, the expression of CCL22
mRNA was significantly increased and its chemokine, CCR4,
tended to increase in atherosclerotic lesions of the calcitriol
group. It is likely that tolerogenic DCs release CCL22 to
interact with CCR4-expressing Tregs in spleen and within
atherosclerotic plaques; thus, DCs may function as antiatherogenic agents.
To clarify the roles of Tregs in the antiatherogenic effects
of calcitriol and DC maturation, we examined the effect of
CD25-neutralizing monoclonal antibody. Tregs characteristically express Foxp3 and, in most cases, CD25. Functional
studies in which CD4⫹CD25⫹ Tregs were depleted with
anti–CD25 antibodies in ApoE⫺/⫺ mice resulted in an increase of atherosclerotic lesions.9 In contrast to the previous
study, in our model, Tregs were not completely deleted by
anti–CD25 antibodies after 4 weeks; however, partial inhibition of Tregs by injection of anti–CD25 antibodies partially
reversed the beneficial effects of calcitriol on atherogenesis
and slightly increased DC maturation at 1 week after anti–
CD25 antibody injection. These data suggest that Tregs
induced by oral calcitriol administration may have an important role in inhibiting atherogenesis and that calcitriol directly
decreased DC maturation and inhibited the progression of
atherosclerosis, independent of Tregs; however, partly immature DCs were induced via a Treg-dependent pathway.
In the present study, oral administration of calcitriol
definitely regulated and affected the function and proportion
of DCs and Tregs in MLNs and reduced atherosclerotic lesion
formation. It is likely that modulation of both intestinal and
systemic immune systems is critical to calcitriol-induced
antiatherogenic properties. In a previous article, it was reported that oral administration of anti–CD3 antibody induced
latency-associated peptide T cells in MLNs and spleens.12 We
also confirmed that the maturation of DCs was inhibited and
that the proportion of CD11c⫹ CD80⫺CD86⫺ DCs in MLNs
was increased at the same time, suggesting that an orally
administered small amount of antibody reached the intestine
(but not in the blood), possibly regulated intestinal immunity,
and resulted in the mutual differentiation of tolerogenic DCs
and Tregs in MLNs (N.S., unpublished data). In the present
study, we demonstrated that oral calcitriol administration
increased the number of Tregs in MLNs but not in the thymus
(supplemental Figure I). Taken together, although we should
be careful about the interpretation of our results, the intestinal
immune system might be a novel therapeutic target for
treatment of CVDs and atherosclerosis. This possibility must
be further investigated.
In summary, we have demonstrated that oral administration
of the active form of vitamin D3 inhibits atherosclerosis
development by inducing tolerogenic DCs and Tregs. We
report herein, for the first time to our knowledge, that Tregs
and tolerogenic DCs work as antiatherogenic agents and that
both cells may play key roles in the beneficial effects of
calcitriol on atherogenesis. It is likely that tolerogenic DCs
recruit Tregs through chemokine CCL22 and its receptor,
CCR4; both cells interact through the cell-to-cell contact of
CTLA4 and CD80/CD86 at lymphoid organs and atherosclerotic lesions. These data indicate that calcitriol could be used
clinically as a promising therapy for preventing atherosclerotic CVD. Clinical studies in humans are required to identify
the efficacy of oral calcitriol in the prevention of atherosclerotic diseases and to evaluate the relationship between plasma
levels of calcitriol and CVDs.
Sources of Funding
This study was supported by a Grant-in-Aid for Scientific Research
in Japan; research grants from the Mitsubishi Pharma Research
Foundation; the Medical Research Fund of the Hyogo Medical
Association; the Cardiovascular Research Fund; and the Takeda
Science Foundation.
Disclosures
None.
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Oral Administration of an Active Form of Vitamin D3 (Calcitriol) Decreases
Atherosclerosis in Mice by Inducing Regulatory T Cells and Immature Dendritic Cells
With Tolerogenic Functions
Masafumi Takeda, Tomoya Yamashita, Naoto Sasaki, Kenji Nakajima, Tomoyuki Kita,
Masakazu Shinohara, Tatsuro Ishida and Ken-ichi Hirata
Arterioscler Thromb Vasc Biol. 2010;30:2495-2503; originally published online October 7,
2010;
doi: 10.1161/ATVBAHA.110.215459
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
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Copyright © 2010 American Heart Association, Inc. All rights reserved.
Print ISSN: 1079-5642. Online ISSN: 1524-4636
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Supplement Methods
Animals
ApoE-/- mice (offspring of homozygous ApoE-/- mice, backcrossed onto the C57BL/6
background)1 were kept in a specific pathogen-free animal facility at Kobe University
institute, were fed a normal chow (Oriental Yeast, Tokyo, Japan) and water ad libitum,
and were maintained on a 12:12-h light-dark cycle in the animal facility.
All animal
experiments were conducted according to the Guidelines for Animal Experiments at
Kobe University School of Medicine.
Experimental Design
We fed six-week-old female ApoE-/- mice 20ng or 200ng calcitriol dissolved in
carboxymetylcellulose or vehicle alone by gastric intubation with a plastic tube twice a
week for 12 weeks.
Mice under the same protocol as described above were injected
with either 100μg of neutralizing CD25 monoclonal antibody (clone PC61; Bio
Express Inc., West Lebanon, NH) to deplete CD4+CD25+ Tregs or 100 μ g of
isotype-matched control rat IgG (Bio Express Inc.) once every 4 weeks at 6, 10 and 14
week-old.
Mice were killed at 18 weeks of age, and atherosclerotic lesions were
examined.
Assessment of Biochemical and Physiological Parameters
Blood was obtained on the day after the last feeding with calcitriol or vehicle under
overnight fasting condition and was collected by the cardiac puncture under anesthetic
conditions using pentobarbital sodium (80mg/kg intraperitoneal injection).
was obtained through centrifugation and stored at -80°C.
Plasma
Concentrations of plasma
total cholesterol, high density lipoprotein cholesterol, low density lipoprotein
cholesterol, triglyceride, calcium and phosphorus were determined enzymatically by
using
an
automated
chemistry
analyzer
(SRL,
Tokyo,
Japan).
1,25(OH)2-dihydroxyvitamin D3 and 25(OH)-hydroxyvitamin D3, the principal
circulating form of vitamin D3, were analyzed by radioimmunoassay (SRL).
Plasma
intact parathyroid hormone (PTH) was determined using a commercial ELISA kit
(Alpco Diagnostics, Salem, NH).
Systolic and diastolic blood pressure was measured
with the tail cuff method (Softron blood pressure-98A, Tokyo, Japan).
Atherosclerotic Lesions Assessments
At 18 weeks of age, mice were anesthetized and the aorta was perfused with saline.
For aortic root lesion analysis, samples were cut from the ascending aorta, and the
proximal samples containing the aortic sinus were embedded in OCT compound
(Tissue-Tek; Sakura Finetek, Tokyo, Japan).
Five consecutive sections (thickness, 10
μm), spanning 550μm of the aortic sinus, were collected from each mouse and stained
with Oil Red O (Sigma, St Louis, MO).
For quantitative analysis of atherosclerosis,
the total lesion area of 5 separate sections from each mouse was obtained and
quantitatively analyzed with the use of the Image J software (National Institutes of
Health) as previously described.1,2
comparison among the groups.
We assessed the aortic sinus mean plaque area in
Lesion analysis was conducted by a single observer
blinded to the group of the mice.
Immunohistochemical Analyses of Atherosclerotic Lesions
Immunohistochemistry was performed on acetone-fixed cryosections (10μm) of mouse
aortic roots using antibodies to identify macrophages (MOMA-2 at a dilution of 1:400;
BMA Biomedicals, Augst, Switzerland), and T cells (CD4, 1:100; BD Biosciences, San
Jose, CA), followed by detection with biotinylated secondary antibodies and
streptavidin-horseradish peroxidase.
For natural Tregs, acetone-fixed cryosections of
mouse aortic roots were incubated with rat anti-Foxp3 antibody (clone FJK-16s, 1:100;
eBioscience, San Diego, CA), followed by Alexa Fluor 588 anti-rat secondary antibody
(1:500; Molecular Probes, Eugene, OR) as described.2
For mature DCs, slides were
stained by using TSATM- kit (PerkinElmer LAS, Inc., Boston, MA) according to the
manufacturer’s instructions. In brief, endogenous peroxidase activity was quenched
0.5% H2O2 for 30 minutes.
Sections were blocked with TNB buffer (TSATM- kit) and
hamster anti-CD11c antibody (clone N418, 1:200; BioLegend, San Diego, CA) and rat
anti-CD86 (clone PO3.1, 1:200; eBioscience, San Diego, CA) of primary antibodies
were applied for 1 hour at room temperature.
Slides were washed and incubated with
HRP-labeled goat-anti-rat IgG (American Qualex, San Clemente, CA) for 30 minutes.
CD86 were detected with Cy3-Tyramide.
Next, primary HRP was deactivated by
treatment with 0.5% H2O2 for 30 minutes, and the sections were then incubated with
biotinylated anti-hamster IgG (Vector Laboratories, Inc., Burlingame, CA).
were washed and incubated with streptavidine-HRP (TSATM- kit).
Slides
Staining by
hamster anti-CD11c was visualized by amplification of the signal with FITC-Tyramide.
Nuclei were counterstained with DAPI (Molecular Probes). The appropriate fixation
reagent depending on the primary antibodies was used.
Negative controls were
prepared with substitution with an isotype control antibody.
Staining with Masson’s
trichrome was used to delineate the fibrous area as previously described.2
Sections
were observed under an All-in-one Type Fluorescence Microscope (BZ-8000; Keyence,
Osaka, Japan) using BZ Analyzer Software (Keyence).
Stained sections were digitally
captured, and the percentage of the stained area (the stained area per total
Quantification of CD4+ T cells, Foxp3+
atherosclerotic lesion area) was calculated.
Tregs, CD11c+ DCs and CD11c+CD86+ DCs in atherosclerotic lesions was done by
counting positively stained cells, which was divided by total plaque area.
Cell Isolation and Flow Cytometry Analyses
Purified CD4+ T cells and CD11c+ DCs were isolated from mesenteric lymph nodes
(MLNs) and spleens.
CD4+ T cells and CD11c+ DCs were positively selected with
anti-CD4+ antibody microbeads and with anti-CD11c+ microbeads (Miltenyi Biotec,
Inc., Auburn, CA) using an AutoMACS separator (Miltenyi Biotec) according to the
manufacturer’s instructions.
For fluorescence-activated cell sorter (FACS) analyses of
lymphoid organs, MLN cells and splenocytes were isolated at 18 weeks of age.
were stained in PBS containing 2% FCS.
Cells
FACS analysis was performed with a
FACSCalibur using CellQuest Pro software (BD Bioscience). The antibodies used
were as follows; anti-CD16/CD32 (clone 2.4G2; BD Bioscience), FITC-conjugated
anti-CD4, Peridinin Chlorophyll Protein cyanin 5.5 (PerCP Cy5.5)-conjugated anti-CD4
(clone H129.19; BD Bioscience), PE-conjugated anti-CD8 (clone 53-6.7; BD
Bioscience),
phycoerythrin
(PE)-conjugated
anti-CD25,
allophycocyanin
(APC)-conjugated anti-CD25 (clone PC61; BD Bioscience), (APC)-conjugated Foxp3
(clone FJK-16s; eBioscience), PE-conjugated anti-IL-4 (clone BVD4-1D11; BD
Bioscience),
PE-conjugated
PE-conjugated
anti-IFN- γ
anti-IL-10
(clone
(cloneXMG1.2;
JES5-16E3;
BD
BD
Bioscience),
Bioscience),PE-conjugated
streptavidin (BD Bioscience), biotinylated anti-human LAP (BAF 246; R&D Systems),
FITC-conjugated anti-CD3e (clone 17A2; BD Bioscience), PE-conjugated anti-NK1.1
(clone PK136; eBioscience), APC-conjugated CD80 (clone 16-10A1; eBioscience),
PE-conjugated CD86 (clone PO3.1; eBioscience), and isotype matched control
antibodies.
For CD80 and CD86 staining, CD11c+ DCs were positively selected with
anti-CD11c+ microbeads as described above and were stained. Intracellular staining of
Foxp3 or cytokines such as IL-4, IL-10 and IFN-γ was performed using the Foxp3
staining buffer set (eBioscience) or intracellular cytokine staining kit (BD Bioscience)
and APC-conjugated Foxp3, PE-conjugated anti-IL-4, PE-conjugated anti-IL-10,
PE-conjugated anti-IFN- γ
or antibody as described above according to the
manufacturer’s instructions. All staining procedures were performed after blocking Fc
receptor with anti-CD16/CD32 antibody.
Surface staining was performed according to
standard procedures at a density of 1x106 cells per 100μl, and volumes were scaled up
accordingly.
Cell Proliferation Assays and Cytokine assays
In all cell culture experiments, we used RPMI 1640 medium (Sigma) supplemented
with 10% FCS, 10mM Hepes, 50μmol/L 2β-mercaptoethanol and antibiotics.
For
analysis of in vitro suppressive function of CD11c+ DCs, CD11c+ cells from spleens of
calcitriol-treated or control mice were cultured with 1x105 of CD4+ T cells from spleens
of Balb/c mice at various ratios (total volume, 200μL/well).
These cells were
cultured at various ratios in flat-bottomed 96-well plates at 37°C with 5% CO2 for 72
hours.
In these experiments, CD11c+ DC were irradiation with 18.5 Gy before
co-culture.
The cells were pulsed with 1μCi of [3H]-thymidine (GE Healthcare,
Buckinghamshire, UK) for the last 16 hours, and thymidine incorporation was assessed
with a LS 6500 liquid scintillation counter (Beckman Coulter, Inc, Brea, CA). For
measurement of cytokine such as IL-17, IL-10, IFN-γ, and IL-6, splenocytes were
cultured at a concentration of 1×106 cells/ml for 72 hours with 2μg/ml concanavalin A
(Con A; Sigma).
Culture supernatants were collected and analyzed by enzyme-linked
immunosorbent assay (ELISA) using paired antibodies specific for corresponding
cytokines according to the manufacturer’s instructions (R&D Systems).
RT-PCR Analysis
At 18 weeks old, mice were anesthetized and the aortic roots were excised as described
above.
Total RNA was extracted from CD4+ T cells, CD11c+ DCs, and mouse aortic
roots after perfusion with RNA-later (Ambion, Austin, TX) using TRIzol reagent
(Invitrogen, Carlsbad, CA).
Quantitative PCR was performed using One Step SYBR
PrimeScript RT-PCR Kit (Takara, Shiga, Japan) and an ABI PRISM 7500 Sequence
Detection system (Applied Biosystems, Foster City, CA) according to the
manufacturer’s protocol as described previously.2
The following primers were used to
amplify renin, CD25, Foxp3, Cytotoxic lymphocyte antigen 4 (CTLA4), CD11c, CD80,
CD86, interleukin (IL)-6, IL-10, IL-12p35, IL12p40, interferon (IFN)-γ, transforming
growth factor (TGF)-β, intracellular adhesion molecule (ICAM)-1, vascular cell
adhesion molecule (VCAM)-1, monocyte chemoattractant protein (MCP)-1, thymus and
CC-chemokine ligand (CCL)17, CCL22, CC-chemokine receptor (CCR)4, CCR8 and
GAPDH: Renin, 5’-CTC CTG GCA GAT CAC GAT GAA G-3’ and 5’-GGA GCT
CGT AGG AGC CGA GAT A-3’; CD25, 5’-CTG ATC CCA TGT GCC AGG AA-3’
and 5’-AGG GCT TTG AAT GTG GCA TTG-3’; Foxp3, 5’-CTC ATG ATA GTG
CCT GTG TCC TCA A-3’ and 5’-AGG GCC AGC ATA GGT GCA AG-3’; CTLA4,
5’- CCT CTG CAA GGT GGA ACT CAT GTA-3’and 5’-AGC TAA CTG CGA CAA
GGA TCC AA-3’; CD11c, 5’-AGA CGT GCC AGT CAG CAT CAA C-3’ and
5’-CTA TTC CGA TAG CAT TGG GTG AGT G-3’; CD80, 5’-AGT TTC CAT GTC
CAA GGC TCA TTC-3’ and 5’-TTG TAA CGG CAA GGC AGC AAT A-3’; CD86,
5’-TGG CAT ATG ACC GTT GTGTGT G-3’; IL-6, 5’-CCA CTT CAC AAG TCG
GAG GCT TA-3’ and 5’-GCA AGT GCA TCA TCG TTG TTC ATA C-3’; IL-10,
5’-GAC CAG CTG GAC AAC ATA CTG CTA A-3’ and 5’-GAT AAG GCT TGG
CAA CCC AAG TAA-3’; IL-12p35, 5’-TGT CTT AGC CAG TCC CGA AAC C-3’
and 5’-TCT TCA TGA TCG ATG TCT TCA GCA G-3’; IL-12p40, 5’-GCT CGC
AGC AAA GCA AGG TAA-3’ and 5’-CCA TGA GTG GAG ACA CCA GCA-3’;
IFN-γ, 5’-CGG CAC AGT CAT TGA AAG CCT A-3’ and 5’-GTT GCT GAT GGC
CTG ATT GTC-3’; TGF-β, 5’-GTG TGG AGC AAC ATG TGG AAC TCT A-3’ and
5’-TTG GTT CAG CCA CTG CCG TA-3’; ICAM-1, 5’-CAA TTC ACA CTG AAT
GCC AGC TC-3’ and 5’-CAA GCA AGT CCG TCT CGT CCA-3’; VCAM-1, 5’-TGC
CGG CAT ATA CGA GTG TGA-3’ and 5’-CCC GAT GGC AGG TAT TAC CAA
G-3’; MCP-1, 5’-GCA TCC ACG TGT TGG CTC A-3’ and 5’-CTC CAG CCT ACT
CAT TGG GAT CA-3’; CCL17, 5’-CCG AGA GTG CTG CCT GGA TTA-3’ and
5’-AGC TTG CCC TGG ACA GTC AGA-3’; CCL22, 5’-GGC ACC TAT CCA GTG
CCA CA-3’ and 5’-TGG TGG ACC AGC CTG AAA CTC-3’; CCR4, 5’-TGC TCG
CCT TGT TTC AGT CAG-3’ and 5’-AGC CAT CTT GCC ATG GTC TTG-3’; CCR8,
5’-CAG ACC CAC AAC CTG CTG GA-3’ and 5’-GAC AGC GTG GAC AAT AGC
CAG A-3’;GAPDH, 5’-TGT GTC CGT CGT GGA TCT GA-3’ and 5’-TTG CTG TTG
AAG TCG CAG GAG-3’. Amplification reactions were performed in duplicate and
fluorescence curves were analyzed with included software.
GAPDH was used as an
endogenous control reference.
Statistical Analysis
Data were expressed as means±SEM.
To detect significant differences between 2
groups or among 3 groups, unpaired Student t test, Mann-Whitney U test, or one way
ANOVA with post hoc test was used when appropriate. Statistical values of p<0.05
were considered statistically significant. For statistical analysis, GraphPad Prism 4.0
was used.
Supplemental References
1.
Ozaki M, Kawashima S, Yamashita T, Hirase T, Namiki M, Inoue N, Hirata K,
Yasui H, Sakurai H, Yoshida Y, Masada M, Yokoyama M. Overexpression of
endothelial nitric oxide synthase accelerates atherosclerotic lesion formation in
apoE-deficient mice. J Clin Invest. 2002;110:331-340.
2.
Sasaki N, Yamashita T, Takeda M, Shinohara M, Nakajima K, Tawa H, Usui T,
Hirata K. Oral anti-CD3 antibody treatment induces regulatory T cells and
inhibits
the
development
2009;120:1996-2005.
of
atherosclerosis
in
mice.
Circulation.
CD25+ cells / CD4+ cells (%)
MLNs
*
Base 2W 4W
Base 2W 4W
CD25+ Foxp3+ cells /
CD4+ cells (%)
Foxp3+/ CD4+ CD8- cells (%)
Base 2W 4W
CD25+ Foxp3+ cells /
CD4+ cells (%)
CD25+ cells / CD4+ cells (%) CD25+ cells / CD4+ cells (%)
Thymus
Base (4 weeks of age)
Control
D3 200ng
Base 2W 4W
*
Base 2W 4W
Spleen
Base 2W 4W
Supplemental Figure I
CD25+ cells / CD4+ cells (%)
CD25+ cells / CD4+ cells (%)
MLNs
*†
*†
*†
Spleen
*†
Supplemental Figure II
IL-4 / CD4+cells (%)
C
(pg/ml) IL-10
MLN Spleen
(pg/ml) IL-17
CD25+LAP+ / CD4+cells (%)
MLN Spleen
MLN Spleen
LAP+ / CD4+cells (%)
B
IL-10 / CD4+cells (%)
MLN Spleen
Control
D3 200ng
MLN Spleen
(pg/ml) IFN-γ
*
CD25-LAP+ / CD4+cells (%)
IFN‐γ / CD4+cells (%)
A
MLN Spleen
(pg/ml) IL-6
*
CD3- NK1.1+ (%)
CD3+ NK1.1+ (%)
D
MLN Spleen
MLN Spleen
Supplemental Figure III
Supplemental Figure Legends
Supplemental Figure I. Effects of Calcitriol on stimulating the expansion of
thymus and peripherally induced Tregs.
We
fed
4
week-old
female
ApoE-/-
mice
200ng
calcitriol
dissolved
in
carboxymetylcellulose or vehicle alone by gastric intubation with a plastic tube twice a
week for 4 weeks. For FACS analyses of lymphoid organs, thymus cells, MLN cells
and splenocytes were isolated at 4, 6 and 8 weeks of age. Thymus cells were stained
with FITC-conjugated anti-CD4, PE-conjugated anti-CD8 and APC-conjugated antiFoxp3. MLN cells and splenocytes cells were stained with FITC-conjugated anti-CD4,
PE-conjugated anti-CD25 and APC-conjugated anti-Foxp3. For all FACS analyses,
data represent means ± SEM of 3 mice in each group. Horizontal bars represent means.
*p<0.05 vs. control.
Supplemental Figure II. The dynamics of the alteration of Tregs after a single
intraperitoneal injection of 100 μg of CD25-depleting PC61 antibody.
MLN cells and splenocytes from calcitriol with anti-CD25 antibodies (D3 200ng+antiCD25 Ab; closed circle), control with anti-CD25 antibodies (Control+anti-CD25 Ab;
open circle), and calcitriol with isotype-matched control antibodies (D3 200ng+rat-IgG;
gray circle) were isolated 1, 2, and 4 weeks after a single intraperitoneal injection of 100
µg of CD25-depleting PC61 antibody. Data represent means ± SEM of 3 mice in each
group.
*p<0.05 vs. D3 200ng+anti-CD25. † p<0.05 vs. control+anti-CD25 Ab.
Supplemental Figure III. Effects of calcitriol on the phenotypes of T lymphocytes
and other cells in MLNs and spleen.
For FACS analyses of lymphoid organs, MLN cells and splenocytes were isolated at 18
weeks of age. CD4+ T cells were stained with PE-conjugated anti-IFN-γ for Th1
lymphocytes, PE-conjugated anti-IL-4 for Th2 lymphocytes, PE-conjugated anti-IL-10
for Tr1 cells, APC-conjugated anti-CD25 and PE-conjugated anti-LAP for Th3, or
FITC-conjugated anti-CD3e and PE-conjugated anti-NK1.1 for Natural Killer (NK)
cells or NKT cells (A, B, and D).
There was no significant differences in IFN-γ
producing CD4+ T cells (Th1) and IL-4 producing CD4+ T cells (Th2) in the calcitrioltreated group and control group (A). Furthermore, there was no obvious induction of
IL-10 producing CD4+ T cells (Tr1) and LAP+ Tregs (mainly Th3) in calcitriol-treated
mice (B). Furthermore, there were no significant difference in NK cells (CD3-NK1.1+)
and NKT cells (CD3+ NK1.1+) between the two groups (D). Data represent means ±
SEM of 3 mice in each group.
Horizontal bars represent means. *p<0.05 vs. control.
Splenocytes were stimulated with Con A in vitro for 72 hours. IL-17, IL-10, IFN-γ, and
IL-6 productions in supernatants were measured by ELISA (C). The productions of IL10 and IL-17 from splenocytes were similar between the two groups, suggesting it is
unlikely calcitriol affected Tr1 and Th17 in the spleen. Productions of inflammatory
cytokine such as IFN-γ and IL-6 from splenocytes were significantly decreased in
calcitriol-treated mice. Data represent means±SEM of at least 6 mice in each group.
Horizontal bars represent means. *p<0.05 vs. control.

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