25-Hydroxvitamin D3 Promotes the
Long-Term Effect of Specific
Immunotherapy in a Murine Allergy Model
This information is current as
of June 16, 2017.
Guido Heine, Christoph Tabeling, Bjoern Hartmann, Carla
R. González Calera, Anja A. Kühl, Juliane Lindner, Andreas
Radbruch, Martin Witzenrath and Margitta Worm
J Immunol published online 20 June 2014
http://www.jimmunol.org/content/early/2014/06/19/jimmun
ol.1301656
http://www.jimmunol.org/content/suppl/2014/06/19/jimmunol.130165
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Supplementary
Material
Published June 20, 2014, doi:10.4049/jimmunol.1301656
The Journal of Immunology
25-Hydroxvitamin D3 Promotes the Long-Term Effect of
Specific Immunotherapy in a Murine Allergy Model
Guido Heine,*,1 Christoph Tabeling,†,1 Bjoern Hartmann,‡ Carla R. González Calera,†
Anja A. K€
uhl,x Juliane Lindner,* Andreas Radbruch,{ Martin Witzenrath,† and
Margitta Worm*
T
he active metabolite of vitamin D termed calcitriol
(1a,25-dihydroxyvitamin D3) mediates anti-inflammatory
functions after binding to the vitamin D receptor (VDR),
which then acts as a transcription factor regulating specific target
gene expression (1). We have previously shown that vitamin D receptor activation inhibits IgE expression in B cells (2) via recruitment of a VDR-DNA complex to the ε-switch transcript promoter
(3). Furthermore, VDR activation enhances IL-10 expression in
B cells after binding to its promoter (4). Accordingly, we determined
*Department of Dermatology, Venerology and Allergy, Allergy-Center-Charité,
Charité Campus Mitte, Charité - Universitätsmedizin, D-10117 Berlin, Germany;
†
Department of Infectious Diseases and Pulmonary Medicine, Charité - Universitätsmedizin, D-10117 Berlin, Germany; ‡Division of Immunology and Rheumatology,
Stanford University School of Medicine, Stanford, CA 94305; xDepartment of Internal
Medicine, Rheumatology and Clinical Immunology, Research Center Immuno Sciences, Charité Campus Benjamin Franklin, Charité - Universitätsmedizin, D-10117 Berlin, Germany; and {Deutsches Rheuma-Forschungszentrum Berlin, D-10117 Berlin,
Germany
1
G.H. and C.T. contributed equally to this work.
Received for publication June 24, 2013. Accepted for publication May 23, 2014.
This work was supported by Deutsche Forschungsgemeinschaft Grants SFB650 TP5
(to M. Worm, A.R., and G.H.), SFB-TRR130 (to M. Worm and G.H.), and SFB-TR84
C3, C6 (to M. Witzenrath).
Address correspondence and reprint requests to Prof. Dr. Margitta Worm, Charité Universitätsmedizin Berlin, Klinik f€ur Dermatologie, Venerologie und Allergologie,
Allergie-Centrum-Charité, Charité Campus Mitte, Charitéplatz 1, D-10117 Berlin,
Germany. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: AHR, airway hyperresponsiveness; calcitriol,
1a,25-dihydroxyvitamin D3; BALF, bronchoalveolar lavage fluid; CYP27B1, cytochrome P450 subfamily 27 B polypeptide 1, also referred to as 25-hydroxyvitamin
D3-1a-hydroxylase; D-SIT, 25(OH)D in combination with specific immunotherapy;
HPF, high-power field; MCh, methacholine; O/O, OVA-sensitized and challenged
mice; PBS/O, nonsensitized and OVA-challenged mice; 25(OH)D, 25-hydroxyvitamin D3 (inactive precursor); SIT, specific immunotherapy; VDR, vitamin D receptor.
Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301656
that allergen-specific IgE was diminished upon VDR activation in
mice (5).
Data from humans indicate that calcitriol pulse therapy induced
IL-10–expressing T cells in patients with steroid-resistant allergic
asthma, which was associated with an improved lung function (6).
However, the long-term therapeutic use of calcitriol is limited by
hypercalcemic side effects. We and others demonstrated that activated B lymphocytes (4), T cells (7), and myeloid APCs (8, 9) can
synthesize biologically active calcitriol from 25-hydroxyvitamin
D3 (inactive precursor) [25(OH)D]; Supplemental Fig. 1). Thus,
25(OH)D may modulate immune reactions. Epidemiologic data
from a representative cohort suggested increased serum IgE concentrations in vitamin D–deficient individuals (,50 nmol/L 25(OH)
D) in contrast to vitamin D–sufficient individuals (105–115 nmol/L
25(OH)D) (10). Cord blood 25(OH)D levels were correlated with
IL-10 levels and inversely with total IgE titers (11). In allergic
asthma patients, vitamin D deficiency has been associated with
increased requirement of inhaled corticosteroids (12) severe exacerbations, hospitalizations, and elevated IgE serum concentrations
(13, 14).
To date, the only available treatment, which targets the cause
of type I allergy, modifies the disease and exceeds the treatment period, is specific immunotherapy (15). By repetitive allergen exposition immune cells are targeted and modulated in their
function (15). The exact cellular and molecular mechanisms of
specific immunotherapy (SIT) are not fully understood, but accepted parameters include changes on the humoral level like the
induction of specific IgG4 (16) in conjunction with increased frequencies of allergen-specific IL-10–secreting (17) and/or Foxp3+ Th
cells (18). Recent data from a murine model of SIT suggested that
calcitriol administered in conjunction with the allergen, further reduced allergic airway inflammation (19).
In the current study, we investigated whether and to what extent
25(OH)D might be relevant during the process of type I sensiti-
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Calcitriol (1a,25-dihydroxyvitamin D3) is the active vitamin D metabolite and mediates immunological functions, which are
relevant in allergy. Its therapeutic use is limited by hypercalcaemic toxicity. We have previously shown that the activation of
the vitamin D receptor inhibits IgE production and that B cells can synthesize calcitriol from its precursor 25-hydroxyvitamin D3
(inactive precursor) [25(OH)D] upon antigenic stimulation. In this study, we address the impact of 25(OH)D on the development of
type I sensitization and determine its role in allergen-specific immunotherapy. BALB/c mice were sensitized to OVA, under 25(OH)Ddeficient or sufficient conditions. The humoral immune response over time was measured by ELISA. OVA-specific immunotherapy
was established and studied in a murine model of allergic airway inflammation using lung histology, pulmonary cytokine expression analysis, and functional parameters in isolated and perfused mouse lungs. In 25(OH)D-deficient mice, OVA-specific IgE and
IgG1 serum concentrations were increased compared with control mice. OVA-specific immunotherapy reduced the humoral
immune reaction after OVA recall dose-dependently. Coadministration of 25(OH)D in the context of OVA-specific immunotherapy
reduced the allergic airway inflammation and responsiveness upon OVA challenge. These findings were paralleled by reduced Th2
cytokine expression in the lungs. In conclusion, 25(OH)D deficiency promotes the development of type I sensitization and correction of its serum concentrations enhances the benefit of specific immunotherapy. The Journal of Immunology, 2014, 193: 000–000.
2
zation and desensitization. Our data show that both the sensitization
and the desensitization by SIT are modulated by 25(OH)D leading
to reduced humoral specific IgE and IgG1 induction and reduction
of allergic airway inflammation following allergen airway exposure
in vitamin D–deficient mice.
Materials and Methods
Animals and treatments
Collection of lung tissue and histology
Lungs were inflated through the trachea with 4% paraformaldehyde at
a pressure of 20 cmH2O for 15 min and embedded in paraffin. The tissue
sections (1–2 mm) were deparaffinized and blocked after a heat-induced
retrieval step with either avidin ⁄ biotin blocking kit (Vector Laboratories,
Burlingame, CA) or peroxidase block (DakoCytomation, Hamburg,
Germany). After 1 h of incubation at room temperature, Abs against CD3
(polyclonal rabbit Ab; DakoCytomation), B220 (RA3-6B2; eBioscience,
San Diego, CA), or cytochrome P450 subfamily 27 B polypeptide 1
(CP27B1; PC290; Binding Site, Schwetzingen, Germany) sections were
blocked with 10% donkey and rabbit serum, respectively, followed by
incubation with secondary Abs (all Dianova, Hamburg, Germany) for
30 min at room temperature. For detection, Streptavidin-AP kit and EnVision kit (DakoCytomation) were used. Nuclei were counterstained with
hematoxylin and slides mounted with gelatin (Merck, Darmstadt, Germany). Negative controls were performed omitting primary Abs. Images
were acquired using an AxioImager Z1 microscope (Carl Zeiss MicroImaging, Jena, Germany). Positive cells were quantified per high-power
field (HPF = 0.237 mm2), and 5 HPF were averaged in each case. In situ
detection of IgG1/IgA/B220 was performed using immunofluorescence.
One to 2-mm paraffin sections were deparaffinized and subjected to a heatinduced retrieval step. Slides were incubated overnight at 4˚C with Abs
directed against IgG1 (A85-1; BD Biosciences) and IgA (polyclonal goat;
Southern Biotechnology Associates) in optimal dilution followed by incubation with secondary Abs (Alexa488-labeled donkey anti-rat and
Alexa555 labels donkey anti-goat; both Life Technologies, Darmstadt,
Germany) for 30 min at room temperature. After rinsing, Cy5-labeled
primary Ab against B220 (RA3.6B2) was applied for 30 min at room
temperature. Nuclei were stained with DAPI (Sigma-Aldrich, Munich,
Germany) and slides mounted with Fluoromount G (Sigma-Aldrich). Negative controls were performed omitting primary Abs. Images were acquired
using an AxioImager Z1 microscope (Carl Zeiss MicroImaging, Jena,
Germany). Positive cells were quantified per HPF (0.237 mm2), and 5 HPF
were averaged in each case. All immunohistochemical evaluations were
performed in a blinded manner.
Statistical analysis
Ig measurement
Serum concentrations of murine total and OVA-specific IgE, IgG subclasses,
IgM and IgA were determined by ELISA as described previously (21, 23).
Briefly, 96-well Maxisorb plates (Nunc, Darmstadt, Germany) were coated
with anti-Ig mAbs or OVA (all 5 mg/ml from Southern Biotechnology Associates, Eching, Germany, or Sigma-Aldrich, respectively). Serum samples
and standards were diluted serially in 1% skimmed milk/PBS and incubated
for 2 h. The amounts of subclass-specific Ig and OVA-specific IgG1, IgG2a,
IgM, and IgA were detected by a biotinylated isotype-specific Ab (Southern
Biotechnology Associates, Birmingham, AL), respectively. OVA-specific IgE
serum levels were determined from serially diluted sera using anti-IgE (2 mg/ml,
clone R35–72; BD Biosciences, Heidelberg, Germany)–coated 96-well
Maxisorb plates and biotinylated-OVA (1.25 mg/ml). Diluted pooled sera of
OVA-sensitized mice or purified Igs (all from BD Biosciences) were used to
calculate the Ig concentrations.
Normal distribution was tested by Kolmogorov–Smirnov test. Parametric
values were tested by Student t test and non-parametric values by Mann–
Whitney U test. Two-way ANOVA was used for comparison of doseresponse curves (GraphPad Prism 5.0, GraphPad Software, La Jolla, CA;
SPSS19, IBM). The p values depicted as *p # 0.05, **p # 0.01, and ***p #
0.001 were considered statistically significant.
Results
25(OH)D deficiency promotes the IgE response after
sensitization
Murine lungs were isolated as previously described (24, 25) perfused with
37˚C sterile Krebs–Henseleit hydroxyethyl amylopectin buffer in a nonrecirculating fashion (1 ml/min) and ventilated with constant negative
pressure (24.5/29 cm H2O, 90 breaths/min) in a closed chamber. Left
atrial pressure was adjusted at +2.2 cm H2O. Airway resistance (resaw),
dynamic lung compliance (Cdyn), tidal volume (TV), and mean pulmonary
arterial pressure (Ppa mean) were monitored continuously (26). After
a steady-state period of 20 min, baseline parameters were assessed and
methacholine (MCh) or serotonin (Sigma-Aldrich) was administered to the
perfusate for 0.5 min at 8-min intervals. Differences in resaw (Dresaw) and
Ppa mean (DPpa mean) were expressed in cmH2O/ml/s and cmH2O, respectively. The area under curve was calculated.
To investigate the impact of 25(OH)D on the humoral type I allergic immune reaction, we first established 25(OH)D deficiency
[serum concentration , 50 nmol/l (20)] in BALB/c mice by nutritional restriction. 25(OH)D deficiency (mean 15.2 6 0.9 nmol/l),
which was achieved within 2 wk, was stable over the course of the
experiment and clearly differing from control mice kept on regular
chow (p # 0.001) (Fig. 1A, 1B). The following OVA sensitization
induced OVA-specific IgE and IgG1 serum concentrations in all
mice in comparable amounts (Fig. 1C, 1D). After OVA recall on
day 64 and 95, the OVA-specific IgE and IgG1 serum concentrations increased significantly in 25(OH)D-deficient mice compared with control mice leading to maximum values on day 101
(+174 and +433%; p # 0.01–0.001). The sensitization-induced
OVA-specific and total IgE, IgM, IgA, and IgG2a serum concentrations were comparable between all groups after OVA recall (see
Supplemental Fig. 2A–E; data not shown).
Leukocyte analysis in the bronchoalveolar lavage fluids
Efficient specific immunotherapy in 25(OH)D-deficient mice
Bronchoalveolar lavage was performed twice using 800 ml PBS as described previously (24, 25). Leukocytes were differentiated by means of
microscopic analysis in a blinded manner (400 cells counted/individual).
The following cytokines from bronchoalveolar lavage fluid (BALF) supernatant were quantified in duplicate using a cytokine multiplex assay
(Bioplex; Bio-Rad, Hercules, CA): IL-4, IL-5, IL-10, IL-12p40, and IL-13.
To investigate the role of 25(OH)D on the type I allergic immune
reaction when combined with allergen-specific immunotherapy
(SIT), we analyzed the specific humoral response of 25(OH)Ddeficient and sufficient control mice that received 3 weekly OVASIT injections in increasing dosages (Fig. 2A). In SIT dosages
Airway and pulmonary vascular responsiveness in isolated
perfused and ventilated mouse lungs
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All procedures were in accordance with the local State Office of Health and
Social Affairs. Female BALB/c mice (strain cByJRj; Elevage Janvier, Le
Genest St. Isle, France) were held in a specific pathogen–free environment.
Mice were fed with a vitamin D–deficient diet (Altromin, Lage or Sniff,
Soest) or regular chow (control: 1000 IU/kg vitamin D). Murine 25(OH)D
serum concentrations were determined by EIA (IDS Hamburg) and deficiency was considered at concentrations below 50 nmol/l (1 ng/ml = 2.5
nmol/l) (20). OVA-specific type I sensitization was performed as described
previously (21). Because the sensitization-induced plasmablast response is
known to decline 2–3 wk after sensitization (22), OVA-SIT was initiated
3 wk after type I sensitization to target generated memory lymphocytes and
avoid anergy. OVA-SIT was performed on days 42, 49, and 56 by injecting
10–1000 mg OVA/PBS (grade V; Sigma-Aldrich) s.c. (nuchal), alone, or in
combination with 0.05–50 mg/kg body weight 25(OH)D (Biomol, Hamburg, Germany; dissolved in 10% DMSO, 5% Tween 80 in 0.9% NaCl).
Control mice received PBS/solvents accordingly. To target resting specific
memory B cells, OVA recalls were performed after allergen-free intervals
of 3–4 wk on day 64 s.c. (ear) and on day 95 or 88 i.p. as indicated in
Figs. 1B and 2A. The OVA recall on day 88 in the experiments considering
the airway inflammation was performed i.p. to reactivate specific memory
cells systemically without their homing primarily to the lung and to avoid
interference of newly formed infiltrating cells attracted by the airway
challenge. In these experiments, mice were exposed to aerosolized OVA
(10 mg/ml dissolved in PBS) for 20 min/d 2 wk after OVA recall on days
102–104 and lung function was determined in isolated perfused and ventilated mouse lungs on day 106 (Fig, 3A).
D-SIT IN MURINE AIRWAY INFLAMMATION
The Journal of Immunology
3
thus, this group was excluded from further analysis. These data
suggest that strong Ag signaling during SIT may override 25(OH)Dmediated immunomodulation, which itself is depending on simultaneous Ag exposure. Thus, we chose a suboptimal SIT protocol using 100 mg OVA in all further experiments in 25(OH)Ddeficient mice and extended the analysis to its impact on murine
airway inflammation.
FIGURE 1. Enhanced humoral allergic immune response in 25(OH)Ddeficient mice. (A) BALB/c mice were fed with a 25(OH)D restriction
(black dotted line) or regular diet (black line). Plasma 25(OH)D concentration (deficiency , 50 nmol/l, gray dotted line) were monitored over
time (n = 32). (B) Experimental setup. Arrows indicate OVA sensitization
(i.p., 10 mg) and OVA challenges (s.c., 10 mg) on day 64 and OVA recall
(i.p., 10 mg) on day 95. Serum concentrations of OVA-specific IgE (C)
and IgG1 (D) were measured by ELISA from the 25(OH)D restriction or
regular diet group. Data show mean 6 SEM, n = 8. **p # 0.01, ***p #
0.001.
of $100 mg, serum concentrations of allergen-specific IgE were
significantly reduced in 25(OH)D-deficient mice, whereas specific
IgG1 was enhanced (p # 0.05; Fig. 2B, 2C). Serum OVA-IgE was
comparable between 25(OH)D-deficient mice after 1 mg SIT and
sufficient controls without SIT (p . 0.05; Supplemental Fig. 2). In
25(OH)D-sufficient control mice, the humoral immune response
was only marginally altered by SIT (Supplemental Fig. 2). The
OVA-specific IgG2a, IgM, and IgA (Supplemental Fig. 2) and the
polyclonal Ig-profile including IgE, IgG1, IgG2a, IgM, and IgA
(data not shown) did not differ significantly between both groups.
The humoral-specific IgE response is not altered significantly,
especially not rescued completely, by treatment of sensitized mice
with 25(OH)D without Ag (p = 0.11; Supplemental Fig. 3), and
The humoral immune reaction is reduced following SIT and
adjuvantive 25(OH)D
Subsequently, we investigated the impact of 25(OH)D on the
humoral immune reaction when applied in conjunction with SIT—
25(OH)D in combination with SIT [D-SIT]—in 25(OH)D-deficient
mice and its functional relevance in a model of allergic airway
inflammation (Fig. 3A). Initial dose-response analysis indicated that
three weekly administrations of .25 mg/kg bodyweight 25(OH)D
were required to achieve sufficiency (.80 nmol/l (20); data not
shown). Treatment with D-SIT containing 50 mg/kg body weight
25(OH)D resulted in serum concentrations of 238.0 6 12.6 nmol/l
(Fig. 3B). In all other groups, 25(OH)D deficiency remained stable
over time (mean, 38.6 6 0.9 nmol/l) (Fig. 3B). In all sensitized
groups, the allergen-specific Ig profile was comparable until day
88 (data not shown). Because of OVA recall on day 88, specific
IgE and IgG1 concentrations were strongly induced in OVAsensitized and challenged mice (O/O) but not in mice that received immunotherapy without (O/O+SIT) or with 25(OH)D
(O/O+D-SIT) (Fig. 3C, 3D). In addition, serum OVA-IgG1 levels
in the D-SIT group were lower compared with SIT alone (Fig. 3D).
In mice with pronounced vitamin D deficiency (serum 25(OH)D
concentrations , 15 nmol/l), the OVA recall induced humoralspecific IgE, and IgG1 response was blocked by both SIT alone or
in combination with 25(OH)D, with little stronger effects by D-SIT
(Supplemental Fig. 4).
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FIGURE 2. Dose-dependent modulation of humoral allergen-specific
Igs by immunotherapy. (A) Experimental setup as described in Fig. 1A.
25(OH)D-deficient BALB/c mice were OVA sensitized. On day 42, a SIT
was applied by weekly injections of increasing doses OVA (s.c., 10–
1000 mg). Arrows indicate OVA injections. Monitoring of OVA-specific
IgE (B) and IgG1 (C) serum concentrations. Data show mean 6 SEM,
n = 8. *p # 0.05, **p # 0.01, ***p # 0.001 compared with control
(without SIT).
4
25(OH)D enhances the effects of SIT
To prove clinical efficacy of D-SIT, we investigated its impact on
murine allergic lung inflammation in OVA-sensitized 25(OH)Ddeficient mice following immunotherapy, OVA recall to the immune cells (i.p.) and airway exposure with aerosolized OVA (Fig. 4).
Functional lung parameters at baseline levels showed decreased
dynamic lung compliance (226.9 6 4.0%) and tidal volume per
body weight (225.6% 6 4.0) in lungs of O/O compared with lungs
of nonsensitized and OVA-challenged control mice (PBS/O, p #
0.01; Fig. 4A, 4B). Although only marginally influenced by SIT
(O/O+SIT), D-SIT (O/O+D-SIT) significantly improved dynamic
lung compliance and tidal volume per body weight compared with
OVA-sensitized and challenged mice without SIT (O/O, p # 0.01–
0.05) to an extent comparable with nonsensitized control mice
(PBS/O, both p = 0.4; Fig. 4A, 4B). No difference in baseline
airway resistance was observed between the groups (data not
shown).
Airway hyperresponsiveness (AHR) upon MCh provocation was detected in all OVA-sensitized groups (O/O, O/O+SIT, and O/O+DSIT) compared with nonsensitized mice (PBS/O, all p # 0.01 and
p # 0.05) (Fig. 4C). Reflecting our findings on the baseline parameters, D-SIT, in contrast to SIT alone, decreased AHR compared with
OVA-sensitized mice without SIT (O/O). In OVA-sensitized mice
FIGURE 4. 25(OH)D given in conjunction with allergen-specific immunotherapy further improves lung function. Experimental setup as shown
in Fig. 3A. Lung function parameters were determined in isolated perfused
and ventilated mouse lungs. After a steady-state period of 20 min, dynamic
lung compliance (Cdyn) (A) and tidal volume (TV) (B) were determined
under baseline conditions. (C) Airway hyperresponsiveness (AHR) upon
MCh provocation. (D) Pulmonary vascular hyperresponsiveness upon serotonin (5-HT) administration. Data are shown as mean fold induction 6
SEM of the area under the curve (AUC), n = 7. *p # 0.05, **p # 0.01.
with pronounced 25(OH)D deficiency (,15 nmol/l), distinct AHR
was observed and the impact of D-SIT was pronounced compared
with SIT alone as reflected by significantly diminished AHR (Supplemental Fig. 4D).
The baseline pulmonary arterial pressure in isolated perfused
lungs was comparable between all groups (data not shown). Pulmonary vascular hyperresponsiveness to serotonin (5-hydroxytryptamine), reflecting Th2-induced pulmonary vascular disease, was
detected in all OVA-sensitized and challenged mice compared with
nonsensitized mice (Fig. 4D), as shown previously (25, 27). Again,
D-SIT reduced pulmonary vascular hyperresponsiveness following
serotonin administration compared with OVA-sensitized mice
without SIT (O/O, p # 0.05) but not SIT alone (O/O+SIT).
Immune cell infiltration and cytokine expression is reduced in
the lungs following D-SIT but not SIT alone
We investigated the impact of D-SIT on the pulmonary immune
reaction by analyzing cellular subsets and cytokine expression in
the BALF from mice treated as described in Fig. 3. The immune
cell counts were 8-fold higher in OVA-sensitized and challenged
mice (O/O) compared with nonsensitized mice (PBS/O, p #
0.001) (Fig. 5A). SIT and D-SIT strongly reduced the immune cell
influx (p # 0.001 and p # 0.05), mainly of eosinophils. BALF
concentrations of IL-4, IL-12p40, and IL-13 were strongly in-
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FIGURE 3. D-SIT modulates the SIT-induced humoral allergen-specific
immune response following recall. (A) Experimental setup. 25(OH)D-deficient BALB/c mice were OVA-sensitized and monitored without immunotherapy (O/O) or with OVA-specific immunotherapy (O/O+SIT, 100 mg
on days 42, 49, 56) or with additional 25(OH)D (O/O+D-SIT, 50 mg/kg in
conjunction with SIT) or not sensitized mice. (B) 25(OH)D serum concentrations over time. OVA-specific serum concentrations of IgE (C) or
IgG1 (D) were determined on day 88 before OVA recall (i.p.) and on day
106. Data are shown as mean fold induction 6 SEM, n = 7. *p # 0.05,
**p # 0.01 (compared with O/O group unless marked otherwise).
D-SIT IN MURINE AIRWAY INFLAMMATION
The Journal of Immunology
5
lungs of sensitized mice (Fig. 6C). The number of CYP27B1+ cells
was decreased by D-SIT compared with SIT alone or without immunotherapy (D-SIT: mean 25.7; SIT: 61.5; O/O: 58 cells per 5
HPFs). These data show that allergen-driven inflammation facilitates the local synthesis of calcitriol from 25(OH)D by immune
cells.
Discussion
creased in sensitized mice (O/O) compared with nonsensitized
mice (PBS/O). Paralleling the lung function parameters, these Th2
cytokines were reduced by D-SIT, but not by SIT alone, compared
with sensitized mice without SIT (O/O) (all p # 0.05, IL-12p40
p = 0.054; Fig. 5B–D), BALF concentrations of IL-5 and IL-10
were comparable between all sensitized mice (O/O, O/O+SIT, and
O/O+D-SIT; data not shown).
Histological analysis showed a strong pulmonary CD3+ T cell infiltration in OVA-sensitized mice (O/O, O/O+SIT, and O/O+D+SIT)
compared with nonsensitized mice (PBS/O) (Fig. 6A). Mean CD3+
cell infiltration was 15% lower after D-SIT compared with SIT
alone (p . 0.05). Interestingly, B cell influx in OVA-sensitized
mice (O/O) was increased after D-SIT (p # 0.05), but not after
SIT alone (O/O+SIT), reflecting reduced lung inflammation
(Fig. 6B). The ratio of IgG1+/IgA+ infiltrating B220+ B cells in
sensitized mice (O/O) was increased by SIT and D-SIT but failed
statistical significance (from 0.4 to 1.0 and 1.5. p = 0.31 and 0.28;
data not shown).
Remarkably, cytochrome P450 subfamily 27 B polypeptide 1, also
referred to as 25-hydroxyvitamin D3-1a-hydroxylase (CYP27B1),
the essential enzyme to convert 25(OH)D to active calcitriol, is
expressed in myeloid and lymphoid immune cells infiltrating the
FIGURE 6. D-SIT reduced the influx of inflammatory cells into the lung. Experimental setup and groups
as described in the legend of Fig. 3A. Lung histology
was determined 2 d after OVA exposure on day 106
regarding expression of CD3 (A), B220 (B cells) (B),
and CYP27B1 (endogenous calcitriol-synthesis) (C).
Representative stainings from one mouse of the O/O
group are shown in 3100 original magnification (inset,
3400). Data are shown as mean cells per 5 HPFs 6
SEM, n = 7. *p # 0.05, **p # 0.01.
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FIGURE 5. D-SIT reduced lung infiltration and inflammatory cytokine
expression. Experimental setup as shown in Fig. 3A. BALFs were obtained
on day 106. Cellular differentiation (A). IL-13 (B), IL-4 (C), and IL-12p40
(D) concentrations. Dotted line indicates sensitivity threshold. Data are
shown as mean fold induction 6 SEM, n = 7. *p # 0.05, ***p # 0.001.
n.d., below detection limit.
We show that 25(OH)D deficiency results in enhanced humoral IgE
and IgG1 responses following type I sensitization. Furthermore, we
demonstrate that coadministration of 25(OH)D enhances the effect
of specific immunotherapy in a murine model of allergic airway
inflammation. Importantly, these data suggest that 25(OH)D, the
precursor of calcitriol, exerts beneficial immunomodulatory functions without toxic side effects and is therefore superior to calcitriol
in a therapeutic context.
In this study, 25(OH)D deficiency was achieved by nutritional
restriction within 2 wk. This is attributed to its large volume
of distribution [e.g., in the serum, muscles, and fat tissue (28)].
D-SIT–induced 25(OH)D serum concentrations are in a physiological range also observed after UV exposure in humans (20, 29).
Direct VDR activation by 25(OH)D requires concentrations . 400
nM in vivo and in vitro (30–32), which is 1.5-fold more than
observed using D-SIT in this study. Thus, direct functions of
25(OH)D alone on immune cells are unlikely, as confirmed by
the observed lack of specific IgE regulation by 25(OH)D alone
without the Ag. To exert functions on immune cells, 25(OH)D
requires enzymatic conversion to calcitriol, which is expressed in
activated B cells (4), T cells (7), and myeloid APCs (8, 9). Our
data support the concept that allergen-specific immune cells are
activated by the SIT allergen, express CYP27B1, and synthesize
calcitriol from 25(OH)D endogenously. Indeed, we were able to
detect CYP27B1 expression in the lung after allergen challenge.
On the humoral level, OVA-specific IgE and IgG1 serum concentrations are reduced in the presence 25(OH)D following Ag
challenge in sensitized mice compared with the deficient control.
These data are in line with previous data from mice treated with a
synthetic low-calcaemic calcitriol-derivative (5) or vdr knockout
mice (33). As 25(OH)D alone the absence of Ag is inactive in the
concentrations determined, the reduced Ig response in the presence
of 25(OH)D following Ag challenge supports the hypothesis that
endogenous calcitriol signaling is dependent on CYP27B1 expression in activated immune cells, which leads to reduced IgE
expression, as we have shown in human B cells (3, 4). In 25(OH)Ddeficient mice, the OVA-specific serum IgE and IgG1 concentrations were comparable before the final OVA recall between
6
D-SIT may be therapeutically superior to vitamin D3 alone by
induction of a long-lasting immune modulation of memory cells and
by reduction the risk of potential vitamin D–associated side effects
by long-term use (e.g., kidney stones).
However, our findings are consistent with previous reports in
mice showing the effects of SIT in the presence of calcitriol including the reduction of the Th2 cytokine IL-13 in the BALF (19, 36).
Because IL-13 expression is critical for the induction of AHR (38),
the reduced IL-13 levels might at least partly explain the ameliorated AHR in D-SIT–treated mice. There is no consensus on the
impact of VDR on purified T cells in allergy [e.g., naive T cells
differentiated into GATA3+ Th2 cells (39) or into IL-10–secreting
Tr1 cells (40)]. In this study, we determined reduced OVA-specific
Ig serum concentrations of the Th2-induced isotypes IgE and
IgG1 after D-SIT compared with SIT alone. In addition, the Th2
cell cytokine expression was reduced after OVA airway challenge.
In line, VDR activation reduced the infiltration of Th2 cells into
inflamed tissue in a murine model of inflammatory bowel disease
(41). Taken together, our data suggest that the specific Th2 response is decreased by 25(OH)D coadministered with SIT.
Pulmonary vascular hyperresponsiveness has been described
earlier in OVA-sensitized and challenged mice (25) and correlates
with pulmonary arterial remodeling (27) while dissociated from
AHR (27, 42). To date, its pathogenic role in allergic lung inflammation is not fully understood, but it might be associated with
pulmonary arterial remodeling observed in severe asthmatic patients
(43). By diminishing pulmonary vascular hyperresponsiveness,
D-SIT could additionally provide protective effects on pulmonary vasculature. Inconsistent with our findings, less IL-5 and
eosinophils were previously observed upon calcitriol treatment by
Taher et al. (19). However, in VDR knockout mice, VDR signaling
was positively associated with allergen-induced airway inflammation,
splenic Th2 cytokine expression, and influx of eosinophils into the
lung (33). Consistently, we observed a marginally higher eosinophilic
infiltration of the lungs after D-SIT compared with SIT alone
(p = 0.20), which was significantly decreased compared with
OVA-sensitized mice without SIT (p , 0.05).
Taken together, our study provides evidence that 25(OH)D deficiency enhances type I sensitization and that applying 25(OH)D
promotes the effects of allergen-specific immunotherapy in deficient
mice suggesting enhanced immune tolerance induction. This function of 25(OH)D in additional sensitization routes and Ag models
(e.g., intranasal dust mite allergen) is not known yet, but it provides
an interesting topic for future investigations. Because 25(OH)D
deficiency (,50 nmol/l as investigated in this study) is frequent in
latitudes above 37˚ from November till February, ranging from 50
to 75% in healthy individuals and type I allergic patients (29, 44,
45) and during that period, pollen-specific SIT is regularly initiated
as seasonal pollen are absent, the data of this study suggest that
correction of 25(OH)D deficiency may enhance the efficacy of SIT.
Acknowledgments
We thank Dennis Ernst, Katja Grollich, and Simone Spieckermann for outstanding technical assistance. We also thank Tim Hollstein, Kiran Kumar,
René Riedel, and Sandra Zehentmeier for support during the studies on
murine allergic airway inflammation.
Disclosures
The authors have no financial conflicts of interest.
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