Chlamydial infection in vitamin D receptor knockout mice is more intense and prolonged than in wild-type mice

Abstract

Vitamin D hormone (1,25-dihydroxyvitamin D) is involved in innate immunity and induces host defense peptides in epithelial cells, suggesting its involvement in mucosal defense against infections. Chlamydia trachomatis is a major cause of bacterial sexually transmitted disease worldwide. We tested the hypothesis that the vitamin D endocrine system would attenuate chlamydial infection. Vitamin D receptor knock-out mice (VDR(-/-)) and wild-type mice (VDR(+/+)) were infected with 10(3) inclusion forming units of Chlamydia muridarum and cervical epithelial cells (HeLa cells) were infected with C. muridarum at multiplicity of infection 5:1 in the presence and absence of 1,25-dihydroxyvitamin D(3). VDR(-/-) mice exhibited significantly higher bacterial loading than wild-type VDR(+/+) mice (P < 0.01) and cleared the chlamydial infection in 39 days, compared with 18 days for VDR(+/+) mice. Monocytes and neutrophils were more numerous in the uterus and oviduct of VDR(-/-) mice than in VDR(+/+) mice (P < 0.05) at d 45 after infection. Pre-treatment of HeLa cells with 10 or 100nM 1,25-dihydroxyvitamin D(3) decreased the infectivity of C. muridarum (P < 0.001). Several differentially expressed protein spots were detected by proteomic analysis of chlamydial-infected HeLa cells pre-treated with 1,25-dihydroxyvitamin D(3). Leukocyte elastase inhibitor (LEI), an anti-inflammatory protein, was up-regulated. Expression of LEI in the ovary and oviduct of infected VDR(+/+) mice was greater than that of infected VDR(-/-) mice. We conclude that the vitamin D endocrine system reduces the risk for prolonged chlamydial infections through regulation of several proteins and that LEI is involved in its anti-inflammatory activity.

Full text

Chlamydial infection in vitamin D receptor knockout mice is
more intense and prolonged than in wild-type mice
Qing Hea,d,*, Godwin A. Ananabab, John Patricksonc, Sidney Pittsc, Yeming Yid, Fengxia
Yane, Francis O. Ekoa, Deborah Lyna, Carolyn M. Blackd, Joseph U. Igietsemea,d, and
Myrtle Thierry-Palmera
aDepartment of Microbiology, Biochemistry, and Immunology, Morehouse School of Medicine,
Atlanta, GA 30310, United States
bDepartment of Biology, Clark Atlanta University, Atlanta, GA30314, United States
cDepartment of Anatomy and Neuroscience, Morehouse School of Medicine, Atlanta, GA 30310,
United States
dCenters for Disease Control and Prevention, Atlanta, GA 30333, United States
eClinical Research Center, Morehouse School of Medicine, Atlanta, GA 30310, United States
Abstract
Vitamin D hormone (1,25-dihydroxyvitamin D) is involved in innate immunity and induces host
defense peptides in epithelial cells, suggesting its involvement in mucosal defense against
infections. Chlamydia trachomatis is a major cause of bacterial sexually transmitted disease
worldwide. We tested the hypothesis that the vitamin D endocrine system would attenuate
chlamydial infection. Vitamin D receptor knock-out mice (VDR−/−) and wild-type mice (VDR+/+)
were infected with 103 inclusion forming units of Chlamydia muridarum and cervical epithelial
cells (HeLa cells) were infected with C. muridarum at multiplicity of infection 5:1 in the presence
and absence of 1,25-dihydroxyvitamin D3.VDR−/− mice exhibited significantly higher bacterial
loading than wild-type VDR+/+ mice (P<0.01) and cleared the chlamydial infection in 39 days,
compared with 18 days for VDR+/+ mice. Monocytes and neutrophils were more numerous in the
uterus and oviduct of VDR−/− mice than in VDR+/+ mice (P< 0.05) at d 45 after infection. Pre-
treatment of HeLa cells with 10nM or 100nM 1,25-dihydroxyvitamin D3 decreased the infectivity
of C. muridarum (P< 0.001). Several differentially expressed protein spots were detected by
proteomic analysis of chlamydial-infected HeLa cells pre-treated with 1,25-dihydroxyvitamin D3.
Leukocyte elastase inhibitor (LEI), an anti-inflammatory protein, was up-regulated. Expression of
LEI in the ovary and oviduct of infected VDR+/+ mice was greater than that of infected VDR−/−
mice. We conclude that the vitamin D endocrine system reduces the risk for prolonged chlamydial
infections through regulation of several proteins and that LEI is involved in its anti-inflammatory
activity.
© 2012 Elsevier Ltd. All rights reserved.
*Corresponding author at: Department of Microbiology, Biochemistry & Immunology, Morehouse School of Medicine, 720 Westview
Drive, S.W., Atlanta, GA 30310, United States. Tel.: +1 404 756 8959; fax: +1 404 752 1149. qhe@msm.edu (Q. He).
NIH Public Access
Author Manuscript
J Steroid Biochem Mol Biol. Author manuscript; available in PMC 2014 June 20.
Published in final edited form as:
J Steroid Biochem Mol Biol. 2013 May ; 135: 7–14. doi:10.1016/j.jsbmb.2012.11.002.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Keywords
Chlamydial infection; 1,25-Dihydroxyvitamin D3; Vitamin D receptor knock out mouse; HeLa
cells; Leukocyte elastase inhibitor
1. Introduction
The vitamin D hormone (1,25-dihydroxyvitamin D, 1,25-(OH)2D)1 is a fat-soluble
secosteroid and a major regulator of mineral homeostasis through its actions in the kidney,
intestines, bone, and parathyroid glands. The biological effects of 1,25-(OH)2D3 are
mediated by the vitamin D receptor (VDR), a member of the super family of nuclear
hormone receptors that function as agonist-activated transcription factors. Recent studies
indicate that vitamin D hormone regulates the immune system through induction of natural
host defense peptides, activation of pattern recognition toll-like receptors, and anti-
inflammatory activity [1, 2]. Liu et al. [3] reported that toll-like receptor activation of human
macrophages up-regulated expression of VDR and the 25-hydroxyvitamin D-1-hydroxylase
(required for synthesis of 1,25-(OH)2D), leading to induction of cathelicidin, an
antimicrobial peptide, and killing of intracellular Mycobacterium tuberculosis. Subsequent
animal and clinical studies have provided further evidence of the role of the vitamin D
endocrine system in innate immunity [4–9]. It has also been reported that 1,25-
(OH)2D3/VDR modulates the capacity of antigen presenting cells to induce T cell activation,
cytokine secretion, and localized antigen-specific immune responses [10–13].
Chlamydia trachomatis is a major infectious bacterial agent of sexually transmitted disease
(STD) in industrialized and developing nations. An estimated three million new cases of
chlamydial infection occur annually in the United States [14]. Chlamydial infection, the
most common STD in the United States, can cause severe health consequences for women,
including pelvic inflammatory disease, ectopic pregnancy, chronic pelvic pain, and
infertility [15]. The local mucosal epithelial cells of the genital tract are important in
chlamydial infectivity, acting as sentinels to recognize pathogens and send signals to
underlying immune cells [16, 17]. We report here on the use of the vitamin D receptor
knock-out (VDR−/−) mouse and HeLa cells (human cervical epithelial cells) to test the
hypothesis that the vitamin D endocrine system attenuates chlamydial infection.
2. Materials and methods
2.1. Chemicals
1,25-Dihydroxyvitamin D3 was obtained from MP Biomedicals (LLC, Solon, OH). Anti-
mouse antibody to leukocyte elastase inhibitor (LEI) was purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). An antibody to Chlamydia conjugated with FITC was
obtained from Bio-Rad (Hercules, CA).
2.2. Animals
Female VDR−/−[18, 19] and VDR+/+ mice on a C57BL/6J background (6 week old) were
purchased from Jackson Laboratory (Bar Harbor, MA), fed food and water ad libitum, and
maintained in laminar flow racks under pathogen-free conditions with a 12 h light and 12 h
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dark cycle. All VDR−/− and VDR+/+ mice were fed a rescue diet [20] high in calcium,
phosphate, and lactose (Harlan Laboratories, Madison, WI) to achieve normal plasma
calcium levels in the VDR−/− mice. The mice (n = 6/group) were infected at 8 week old and
killed on d 45 after infection by cervical dislocation. The protocols involving mice were
approved by the Institutional Animal Care and Use Committee (IACUC) of Morehouse
School of Medicine.
2.3. Infectivity assay in VDR−/− and VDR+/+ mice
Female VDR−/− and VDR+/+ mice (n = 6/group, 8 week old) were subcutaneously
administered 2 mg of medroxyprogesterone acetate (Sigma-Aldrich Co, St Louis, MO) 7 d
prior to infection and infected intra-vaginally with 103 inclusion forming units (IFU) of
Chlamydia muridarum in 20 μl of PBS. Bacterial shedding was assessed by performing
vaginal swabs on d 3, 6, 9, 12, 15, 18, 25, 32, and 39 after infection. Tissue culture isolation
of C. muridarum was by standard procedures and the number of inclusions per group per
time point was determined [21].
2.4. Histology and immunohistochemistry
The entire genital tract was collected and fixed in 4% formaldehyde. The samples were
embedded in paraffin, cut longitudinally into 4 μm sections, and stained with hematoxylin
and eosin. The right and left uterine horns and oviducts were individually evaluated in a
blinded method for the presence of acute inflammation (neutrophils), chronic inflammation
(monocytes), and plasma cells. A four-tiered semiquantitative scoring system was used to
quantitate the inflammation [22]: 0, normal; 1+, rare foci (minimal presence) of
inflammatory cells; 2+, scattered (1–4) aggregates or mild diffuse increase in parameter; 3+,
numerous aggregates (>4) or moderate diffuse or confluent areas of parameter; 4+, severe
diffuse infiltration or confluence of parameter.
Tissue slides from the oviducts were incubated with a mouse anti-LEI (serpinB1a) primary
antibody, followed by incubation for 30 min with a horseradish peroxidase-conjugated goat
anti-mouse secondary antibody in accordance with the manufacturer’s directions. Images of
representative fields were obtained using an Olympus Provis AX70 microscope equipped
with a Leica DFC 320 Digital Camera system (Leica Camera AG, Solms, Germany).
2.5. Infectivity in HeLa cells
HeLa cells were pre-treated with 1,25-(OH)2D3 at different concentrations for 24 h. The
treated and un-treated cells were washed and infected with C. muridarum at multiplicity of
infection (MOI) 5:1 for 48 h. The intensity of infection was measured by staining with a
chlamydial antibody conjugated with fluorescein isothiocyanate. The number of inclusions
was calculated as previously described [23].
2.6. Proteomic analysis
HeLa cells that had been pre-treated with 1,25-(OH)2D3 (100nM) for 24 h or un-treated
were infected with C. muridarum for 2 h. Proteins were extracted with a Bio-Rad protein
extraction kit and cleaned with 2-D Clean Up Kit, according to the manufacturer’s protocol.
Protein concentration was determined by 2D Quant Kit from GE Healthcare (GE Healthcare,
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Piscataway, NJ). Samples (80 μg) were labeled with Cy3 or Cy5 fluorescence dyes, mixed in
a rehydration buffer, and subjected to two-dimensional fluorescence differential gel
electrophoresis analysis (2D-DIGE) [24, 25]. The spots corresponding to differentially
expressed proteins were digested and analyzed by nanocapillary LC-MS/MS (Xevo G2 Tof,
Waters, Milford, MA). Protein candidates were identified with automated database
searching against a NCBI database using MASCOT Daemon software (Matrix Sciences,
Boston, MA).
2.7. Western blot analysis
HeLa cells that had been pre-treated with 1,25-(OH)2D3 (100 nM) or un-treated for 24 h
were pulsed with Chlamydia muridarum agent (MOI 5:1) for up to 2h. Protein expression of
LEI at 0.5, 1, and 2 h after infection with Chlamydia was determined by Western blot
analysis. The cell lysates were subjected to electrophoresis for protein separation on 4–20%
Mini-PROTEAN TGX Precast Gels (Bio-Rad, Hercules, CA) in running buffer (Tris/
glycine/SDS). Proteins were then transferred for 1 h onto nitrocellulose membranes in
transfer buffer (Tris/glycine/methanol). Non-specific binding was blocked by incubating the
membranes in 5% non-fat dried milk, 0.05% (v/v) Tween 20 in 1X TBS for 1 h at room
temperature. After several washes with buffer (TBS Tween 0.05%), membranes were
incubated with the primary antibodies overnight at 4°C. The polyclonal antibodies used
were: serpinB1 (1:200) (Santa Cruz Biotech. Inc., Santa Cruz, CA); glyceraldehide-3-
phosphate dehydrogenase (GAPDH) (1:200) (R&D Systems, Minneapolis, MN).
Horseradish peroxidase-conjugated secondary antibodies were utilized in accordance with
the manufacturer’s directions (Santa Cruz Biotechnology, Santa Cruz, CA).
2.8. Statistical analysis
A mean ± SD was calculated for each group and statistical significance (P< 0.05) evaluated
(SAS 9.2, SAS Institute Inc., Cary, NC). Repeated measures ANOVA was used to compare
intensity and duration of infection in VDR−/− and VDR+/+ mice and one way ANOVA to
assess the effect of 1,25-(OH)2D3 on intensity of infection in HeLa cells. A t-test was used
to compare the pathology scores of VDR−/− and VDR+/+ uterus and oviducts.
3. Results
3.1. Chlamydial infection and inflammatory response in VDR−/−and VDR+/+ mice
VDR−/− mice that were genitally infected with C. muridarum had significantly higher
bacteria loads at d 6, 9, 12, 15 post-infection than VDR+/+ mice, indicating a greater
intensity of infection (Fig. 1). VDR−/− mice also exhibited a slower rate of clearance of the
genital chlamydial infection than VDR+/+ mice. Whereas VDR+/+ mice had cleared the
infection by d 18, VDR−/− mice cleared the infection by d 39. The uterus of uninfected
VDR+/+ and VDR−/− mice revealed normally observed branched folia with intact epithelial
lining composed of both secretory and ciliated cells (Fig. 2). Significantly higher numbers of
monocytes (pathology scores: 2.5 ± 0.3 vs. 0.7 ±0.7, P<0.01) and neutrophils (pathology
scores: 1.2 ±0.6 vs. 0.2 ±0.2, P<0.05) were observed in the infected uterus of VDR−/−mice,
compared with that of infected VDR+/+ mice. No inflammation was observed in the oviduct
of un-infected VDR−/− and VDR+/+ mice (Fig. 3). Significantly higher numbers of
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monocytes (pathology scores: 3.2 ±0.6 vs. 1.7 ±0.7, P<0.01) and neutrophils (pathology
scores: 1.5 ± 0.3 vs. 0.3 ± 0.3, P< 0.01) were observed in the oviduct of infected VDR−/−
mice, compared with that of infected VDR+/+ mice.
3.2. Effect of 1,25-(OH)2D3 on chlamydial infection in HeLa cells
Chlamydial infectivity of HeLa cells pre-treated with 10nM or 100nM 1,25-(OH)2D3 for
24h was significantly less than chlamydial infectivity of un-treated HeLa cells or HeLa cells
pre-treated with 1.0nM 1,25-(OH)2D3(Fig. 4). Vitamin D hormone treatment of chlamydial-
infected HeLa cells resulted in numerous differentially expressed protein spots, of which 25
have been identified, using LC-MS/MS ion search software and associated databases (Table
1). Fewer differentially expressed protein spots were observed for pre-treated un-infected
HeLa cells (Supplemental Fig. 1).
Supplementary material related to this article found, in the online version, at http://
dx.doi.org/10.1016/jjsbmb.2012.11.002.
3.3. Leukocyte elastase inhibitor expression
Western blots indicated significantly more LEI in infected HeLa cells pre-treated with 1,25-
(OH)2D3 than in un-treated infected HeLa cells (Fig. 5). Pre-treatment with 1,25-(OH)2D3
did not, however, significantly increase LEI in un-infected HeLa cells. LEI was detected in
the oviduct and ovary of infected VDR+/+ mice by immunohistochemistry, but not in the
oviduct and ovary of infected VDR−/− mice (Fig. 6).
4. Discussion
In this study, vitamin D receptor knock-out mice and control mice (VDR+/+) were used to
evaluate the 1,25-(OH)2D3/VDR effect on genital chlamydial infection. Homozygous
VDR−/− mice are phenotypically normal at birth and survive for at least six months [18].
These mice exhibit hypocalcemia, hyperparathyroidism, rickets and osteomalacia. One
feature seen in VDR−/− mice, but not seen in mice made vitamin D deficient by dietary
means, is alopecia, which develops progressively from the age of four weeks [18]. Feeding
VDR−/− mice a rescue diet high in calcium, phosphorus, and lactose [20] normalized all of
the symptoms above except alopecia [19]. In the present study, VDR−/− mice fed the rescue
diet suffered a more intense and prolonged genital chlamydial infection compared with
VDR+/+ mice, suggesting that attenuation of genital chlamydial infectivity in the VDR+/+
mice is not an indirect effect of maintaining normal calcium, but is a process which requires
a functional VDR.
Intensive chlamydial infection and a prolonged infection course trigger inflammatory
responses that are thought to be largely res,ponsible for the chlamydial induced pathology of
the genital tract. Inflammation was not observed in the uterus and oviduct of un-infected
VDR−/− and VDR+/+ mice, but higher numbers of monocytes and neutrophils were observed
in the infected uterus and oviduct of VDR−/− mice, compared with that of VDR+/+ mice. The
more intense and prolonged chlamydial infection in the VDR−/−mice was thus accompanied
by a more prolonged inflammatory response.
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Genital epithelial cells are the first line of defense against microbial invasion, so we
conducted chlamydial infection assays with HeLa cells, a genital epithelial cell line. Pre-
treatment of HeLa cells with 1,25-(OH)2D3 dramatically inhibited intracellular replication of
C. muridarum and inclusion development. It is possible that 1,25-(OH)2D3 mediates the
protective response of epithelial cells to bacterial replication [26]. HeLa cells pre-treated
with 1,25-(OH)2D3 and infected with C. muridarum exhibited differential expression of 65
protein spots, whereas fewer differentially expressed protein spots were detected in un-
infected HeLa cells pre-treated with 1,25-(OH)2D3. Several categories of proteins were
differentially expressed: cytoskeleton proteins that affect microfilament network and cell-
cell junctions (tubulin, vimentin, tropomyosin chain, vinculin); stress response proteins
related to protein folding and chaperones (heat shock proteins, protein disulfide isomerase,
T-complex protein-1); proteins involved in oxidation/reduction (peroxiredoxin-2 and -6,
thioredoxin-dependent peroxide reductase); proteins involved in growth and regulation of
cell proliferation (tumor protein D54, 60S acidic ribosomal protein P0, proliferation-
associated protein 2G4); proteins involved with ATP (ATP synthase subunit beta,
transitional endoplasmic reticulum ATPase); calcium binding protein (protein S100-A7);
anti-inflammatory protein (LEI); serum albumin precursor. A caveat to be considered in
conducting complementary experiments with HeLa cells is that human monocytes and
macrophages show vitamin D hormone induced expression of antimicrobial proteins (e.g.
cathelicidin, beta-defensin), but these genes are not regulated by 1,25-(OH)2D3 in mice.
We selected for further study LEI, an anti-inflammatory protein that was up-regulated in
chlamydial infected HeLa cells pre-treated with 1,25-(OH)2D3, compared with un-treated
cells. LEI is a 42 kDa member of the serine protease inhibitor (serpin) superfamily [27] and
is also known as monocyte/neutrophil elastase inhibitor (MNEI) and serpinB1. It inhibits
neutrophil proteases (elastase, cathepsin G, proteinase-3) that are involved in killing
phagocytosed microbes by forming irreversible covalent complexes with them. LEI is
abundant in the cytoplasm of monocytes, neutrophils, and macrophages and attenuates the
inflammatory response to microbial infection by limiting the activity of the proteases, thus
limiting degradation of host defense and matrix proteins [28]. Recombinant MNEI
administered daily by aerosolization to rats previously inoculated with Pseudomonas
aeruginosa decreased the inflammatory injury and enhanced the clearance of bacteria from
the infected rat lungs [29]. SerpinB1−/− mice exhibited higher mortality relative to wild type
mice, associated with late-onset failed clearance of P. aeruginosa. Co-administration of
recombinant serpinB1 with the P. aeruginosa inoculum normalized bacterial clearance in
serpinB1−/− mice [30]. Gong et al. [31] reported that serpinB1−/− mice died earlier and in
greater numbers than wild-type mice when infected with high-dose surfactant protein-D-
sensitive influenza A/Philadelphia/82(H3N2). They concluded that serpinB1 plays a critical
role in mitigating inflammation and restricting pro-inflammatory cytokine production in
influenza infection. L-DNase II, an endonuclease involved in the degradation of genomic
DNA during apoptosis, is derived from LEI by an acidic-dependent post-translational
modification or by digestion with elastase [32–36]. Appearance of the endonuclease activity
results in loss of the anti-protease activity [32]. Benarafa et al. [37] have indicated that
human MNEI is encoded by a single SERPINB1 gene, whereas four murine genes have been
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identified and fully sequenced, with one of them, EIA (SERPINB1a), being the mouse
ortholog of MNEI.
In the present study, Western blots indicated up-regulation of LEI in infected HeLa cells
pre-treated with 1,25-(OH)2D3, but not in pre-treated un-infected HeLa cells (confirming the
proteomics results). A greater amount of LEI was detected by immunohistochemistry in the
oviduct and ovary of infected VDR+/+ mice than in the oviduct and ovary of infected
VDR−/− mice, although monocytes and neutrophils (the primary cellular sources of LEI)
were more numerous in the oviduct of infected VDR−/− mice than in the oviduct of infected
VDR+/+ mice. The association of more prolonged chlamydial infection and inflammatory
response with lesser LEI expression in the oviduct and ovary of VDR−/− mice, compared
with VDR+/+ mice, would suggest a role for LEI in countering the inflammatory response
and for vitamin D hormone in LEI expression. Previous studies have demonstrated a role for
1,25-(OH)2D3 in LEI expression. Protiva et al. [38] demonstrated, via global gene array
analysis, activation of the VDR pathway in rectal mucosal biopsies from estradiol treated
postmenopausal women and up-regulation of SERPINB1. Kovalenko et al. [39] identified
numerous transcript changes, including up-regulation of SERPINB1, in response to 1,25-
(OH)2D3 treatment of the immortalized, non-transformed prostate epithelial cell line,
RWPE1. Unlike the anti-microbial proteins, LEI appears to be regulated by 1,25-(OH)2D3 in
mice, as well as in cells of human origin. Wang et al. [40] have demonstrated that multiple
DR3 elements in the serpinB1 gene are conserved between mouse and human.
Consequently, the mouse can serve as a model for examining the relationship between the
vitamin D endocrine system and LEI, a protein with anti-inflammatory effects.
In summary, we demonstrate here that genital chlamydial infectivity was more intense and
prolonged in VDR−/− mice than in VDR+/+ mice. The inflammatory response to chlamydial
infection was also more prolonged in VDR−/− mice than in VDR+/+ mice. The more
prolonged chlamydial infection and inflammatory response in the VDR−/− mice coincided
with lesser expression of LEI (an antiinflammatory protein) in the oviduct and ovary of
VDR−/− mice, compared with VDR+/+ mice, suggesting a role for LEI in countering the
inflammatory response to chlamydial infection and for vitamin D hormone in LEI
expression.
We conclude that the VDR is involved in immunoregulation of chlamydial infection. LEI,
the protein examined in this study, was only one of numerous proteins differentially
expressed in infected HeLa cells pre-treated with 1,25-(OH)2D3. Additional studies are
necessary to determine other mechanisms by which vitamin D hormone attenuates
chlamydial infection and the anti-inflammatory response.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
This work was funded by Georgia Research Alliance Collaboration Planning Grant GRA VAC10.C, the Centers for
Disease Control and Prevention, and Morehouse School of Medicine. Facilities and support services were partially
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funded by National Institutes of Health/National Center for Research Resources grants G12-RR03034, 1 C06
RR18386, 1 U54 RR026137 (to Morehouse School of Medicine) and RR03062 and 08247 (to Clark Atlanta
University).
Abbreviations
1,25-(OH)2D31,25-dihydroxycholecalciferol
1,25-(OH)2D1,25-dihydroxyvitamin D
2D-DIGE two-dimensional differential in gel electrophoresis
HE hematoxylin and eosin
HeLa cells human cervical epithelial cells
IFU inclusion forming units
LEI MNEI or serpinB1, leukocyte elastase inhibitor
MOI multiplicity of infection
SERPINB1 leukocyte elastase inhibitor gene
VDR vitamin D receptor
VDR−/− vitamin D receptor knock out mouse
VDR+/+ C57BL/6J mouse
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Fig. 1.
VDR−/− mice infected intra-vaginally with Chlamydia muridarum exhibit greater bacteria
loads and slower rates of bacterial clearance than VDR+/+ mice. VDR−/− and VDR+/+ mice
were infected intra-vaginally with 103 inclusion forming units (IFU) of C. muridarum as
described in Section 2.3. The course of infection was monitored by isolation of C.
muridarum from cervicovaginal swabs [21]. Values are mean ± SD, n = 6. Overall intensity
different from VDR+/+, P< 0.01. Rate of clearance different from VDR+/+, P< 0.001.
Intensity of infection different from VDR+/+, *P< 0.001.
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Fig. 2.
More monocytes and neutrophils are observed in the uterus of infected VDR−/− mice,
compared with that of infected VDR+/+ mice. The uterus horns of each mouse were removed
at d 45 post-infection with Chlamydia muridarum and stained with hematoxylin and eosin
(HE), as described in Section 2.4. HE-stained sections are at 40× magnification. Open
arrows point to scattered neutrophils; closed arrows indicate monocytes/lymphocytes.
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Fig. 3.
More monocytes and neutrophils are observed in the oviducts of infected VDR−/− mice,
compared with that of infected VDR+/+ mice. The oviducts of each mouse were removed at
d 45 post-infection with Chlamydia muridarum and stained with hematoxylin and eosin
(HE), as described in Section 2.4. HE-stained sections are at 40× magnification. Open
arrows point to scattered neutrophils; closed arrows indicate monocytes/lymphocytes.
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Fig.4.
Chlamydial infectivity of HeLa cells pre-treated with 1,25-(OH)2D3 is significantly less than
chlamydial infectivity of un-treated HeLa cells. HeLa cells were pre-treated with 1,25-
(OH)2D3 at different concentrations for 24h. Treated and un-treated cells were washed and
infected with Chlamydia muridarum for 48 h as described in Section 2.5. The intensity of
infection was measured by staining with a chlamydial antibody and determining the number
of inclusions [23]. Values are mean ± SD, n = 6. Significant effect of 1,25-(OH)2D3
treatment, P< 0.001. Intensity of infection different from un-treated, *P< 0.001.
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Fig. 5.
Leukocyte elastase inhibitor is up-regulated in infected HeLa cells pre-treated with 1,25-
(OH)2D3, but not in pre-treated un-infected HeLa cells. HeLa cells that had been pre-treated
with 1,25-(OH)2D3 (100nM) or un-treated for 24h were pulsed with Chlamydia muridarum
(MOI 5:1) for up to 2 h. Total protein was extracted, subjected to SDS-PAGE, transferred
onto nitrocellulose membrane, probed with primary and secondary antibodies, and imaged
as described in Section 2.7.
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Fig. 6.
Leukocyte elastase inhibitor is detectable in the ovaries of infected VDR+/+ mice, but not in
the ovaries of infected VDR−/− mice. VDR−/− and VDR+/+ mice were infected intra-
vaginally with Chlamydia muridarum as described in Section 2.3. Tissue slides of ovaries
from the mice were incubated with a mouse anti-leukocyte elastase inhibitor primary
antibody and imaged as described in Section 2.4.
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Table 1
Several spots were up-regulated in chlamydial-infected HeLa cells pre-treated with 1,25-dihydroxyvitamin D3.
SpotaProtein name Accession IDbMW(Da)cpIcScoredMatched peptideseSequence coverage (%)
10 Protein S100-A6 S10A6_RABIT 10,147 5.30 54 1 20
13 Tropomyosin alpha-3 chain TPM3_HUMAN 32,799 4.68 113 3 11
18 Tubulin beta chain TBB5_HUMAN 49639 4.78 178 7 17
19 Hsc70-interacting protein F10A1_HUMAN 41,305 5.18 91 2 6
20 ATP synthase subunit beta ATPB_HUMAN 56,525 5.26 384 11 33
21 Vimentin VIME_HUMAN 53,619 5.06 282 7 18
22 60 kDa heat shock protein CH60_HUMAN 61,016 5.70 488 12 31
23 60 kDa heat shock protein CH60_HUMAN 61,016 5.70 111 4 11
24 T-complex protein 1 subunit epsilon TCPE_HUMAN 59,633 5.45 271 10 23
25 Heat shock cognate 71 kDa protein HSP7C_HUMAN 70,854 5.37 393 11 22
26 Serum albumin precursor ALBU_BOVIN 69,248 5.82 58 3 5
30 Protein disulfide-isomerase A3 precursor PDIA3_HUMAN 56,747 5.98 223 8 21
34 T-complex protein 1 subunit beta TCPB_HUMAN 57,452 6.01 183 4 9
36 Proliferation-associated protein 2G4 PA2G4_HUMAN 43,759 6.13 157 4 15
37 Proliferation-associated protein 2G4 PA2G4_HUMAN 43,759 6.13 41 1 7
39 Leukocyte elastase inhibitor ILEU_HUMAN 42,715 5.90 103 3 8
42 60S acidic ribosomal protein P0 RLA0_HUMAN 34,252 5.71 173 4 13
46 Peroxiredoxin-6 PRDX6_HUMAN 25,019 6.00 130 4 20
48 Heat-shock protein beta-1 (HspB1) HSPB1_HUMAN 22,768 5.98 122 3 18
49 Thioredoxin-dependent peroxide reductase PRDX3_HUMAN 27,675 7.67 142 3 13
54 Peroxiredoxin-2 PRDX2_HUMAN 21,878 5.66 122 3 19
57 Tumor protein D54 (hD54) TPD54_HUMAN 22,224 5.26 42 2 11
58 Transitional endoplasmic reticulum ATPase TERA_HUMAN 89,266 5.14 144 7 9
64 Vinculin (Metavinculin) VINC_HUMAN 123,722 5.50 60 3 2
aAs indicated in Supplemental Fig. 1. Down-regulated protein spots were not identified fortechnical reasons.
bSwissProt protein accession number.
cRelative molecular mass of proteins and isoelectric point.
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dProbability based mowse score that indicates the quality of the MS/MS peptide fragment ion matches. Ion score is−10 × log(P),where P is the probability that the observed match is a random event.
Protein scores are derived from ions scores as a non-probabilistic basis for ranking protein hits.
eNumber of peptides that match the theoretical digest of the primary protein identified.
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