Cp

QFa

b

c

d

e

a

ARRA

KC1VHL

1

(mb(

1tIMvm

IG

0h

Journal of Steroid Biochemistry & Molecular Biology 135 (2013) 7– 14

Contents lists available at SciVerse ScienceDirect

Journal of Steroid Biochemistry and Molecular Biology

jo u r n al hom epage: www.elsev ier .com/ locate / j sbmb

hlamydial infection in vitamin D receptor knockout mice is more intense androlonged than in wild-type mice

ing Hea,d,∗, Godwin A. Ananabab, John Patricksonc, Sidney Pittsc, Yeming Yid, Fengxia Yane,rancis O. Ekoa, Deborah Lyna, Carolyn M. Blackd, Joseph U. Igietsemea,d, Myrtle Thierry-Palmera

Department of Microbiology, Biochemistry, and Immunology, Morehouse School of Medicine, Atlanta, GA 30310, United StatesDepartment of Biology, Clark Atlanta University, Atlanta, GA 30314, United StatesDepartment of Anatomy and Neuroscience, Morehouse School of Medicine, Atlanta, GA 30310, United StatesCenters for Disease Control and Prevention, Atlanta, GA 30333, United StatesClinical Research Center, Morehouse School of Medicine, Atlanta, GA 30310, United States

r t i c l e i n f o

rticle history:eceived 26 June 2012eceived in revised form 5 November 2012ccepted 6 November 2012

eywords:hlamydial infection,25-Dihydroxyvitamin D3

itamin D receptor knock out mouseeLa cellseukocyte elastase inhibitor

a b s t r a c t

Vitamin D hormone (1,25-dihydroxyvitamin D) is involved in innate immunity and induces host defensepeptides in epithelial cells, suggesting its involvement in mucosal defense against infections. Chlamydiatrachomatis is a major cause of bacterial sexually transmitted disease worldwide. We tested the hypoth-esis that the vitamin D endocrine system would attenuate chlamydial infection. Vitamin D receptorknock-out mice (VDR−/−) and wild-type mice (VDR+/+) were infected with 103 inclusion forming units ofChlamydia muridarum and cervical epithelial cells (HeLa cells) were infected with C. muridarum at multi-plicity of infection 5:1 in the presence and absence of 1,25-dihydroxyvitamin D3. VDR−/− mice exhibitedsignificantly higher bacterial loading than wild-type VDR+/+ mice (P < 0.01) and cleared the chlamydialinfection in 39 days, compared with 18 days for VDR+/+ mice. Monocytes and neutrophils were morenumerous 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 nM or 100 nM 1,25-dihydroxyvitamin D3 decreased the infectivity

of C. muridarum (P < 0.001). Several differentially expressed protein spots were detected by proteomicanalysis of chlamydial-infected HeLa cells pre-treated with 1,25-dihydroxyvitamin D3. Leukocyte elas-tase inhibitor (LEI), an anti-inflammatory protein, was up-regulated. Expression of LEI in the ovary andoviduct of infected VDR+/+ mice was greater than that of infected VDR−/− mice. We conclude that thevitamin D endocrine system reduces the risk for prolonged chlamydial infections through regulation of

LEI is

several proteins and that

. Introduction

The vitamin D hormone (1,25-dihydroxyvitamin D, 1,25-OH)2D)1 is a fat-soluble secosteroid and a major regulator of

ineral homeostasis through its actions in the kidney, intestines,one, and parathyroid glands. The biological effects of 1,25-OH)2D3 are mediated by the vitamin D receptor (VDR), a member

Abbreviations: 1,25-(OH)2D3, 1,25-dihydroxycholecalciferol; 1,25-(OH)2D,,25-dihydroxyvitamin D; 2D-DIGE, two-dimensional differential in gel elec-rophoresis; HE, hematoxylin and eosin; HeLa cells, human cervical epithelial cells;FU, inclusion forming units; LEI, MNEI or serpinB1, leukocyte elastase inhibitor;

OI, multiplicity of infection; SERPINB1, leukocyte elastase inhibitor gene; VDR,itamin D receptor; VDR−/− , vitamin D receptor knock out mouse; VDR+/+, C57BL/6Jouse.∗ Corresponding author at: Department of Microbiology, Biochemistry &

mmunology, Morehouse School of Medicine, 720 Westview Drive, S.W., Atlanta,A 30310, United States. Tel.: +1 404 756 8959; fax: +1 404 752 1149.

E-mail address: qhe@msm.edu (Q. He).

960-0760/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.jsbmb.2012.11.002

involved in its anti-inflammatory activity.© 2012 Elsevier Ltd. All rights reserved.

of the super family of nuclear hormone receptors that functionas agonist-activated transcription factors. Recent studies indicatethat vitamin D hormone regulates the immune system throughinduction of natural host defense peptides, activation of pat-tern recognition toll-like receptors, and anti-inflammatory activity[1,2]. Liu et al. [3] reported that toll-like receptor activation ofhuman macrophages up-regulated expression of VDR and the25-hydroxyvitamin D-1-hydroxylase (required for synthesis of1,25-(OH)2D), leading to induction of cathelicidin, an antimicro-bial peptide, and killing of intracellular Mycobacterium tuberculosis.Subsequent animal and clinical studies have provided further evi-dence of the role of the vitamin D endocrine system in innateimmunity [4–9]. It has also been reported that 1,25-(OH)2D3/VDRmodulates the capacity of antigen presenting cells to induce Tcell activation, cytokine secretion, and localized antigen-specific

immune responses [10–13].

Chlamydia trachomatis is a major infectious bacterial agent ofsexually transmitted disease (STD) in industrialized and develop-ing nations. An estimated three million new cases of chlamydial

8 mistry

iiciittctes

2

2

ciCo

2

gHllaLewTAM

2

waaoa3wp

2

hira(Atpmmi

mb

Q. He et al. / Journal of Steroid Bioche

nfection occur annually in the United States [14]. Chlamyd-al infection, the most common STD in the United States, canause severe health consequences for women, including pelvicnflammatory disease, ectopic pregnancy, chronic pelvic pain, andnfertility [15]. The local mucosal epithelial cells of the genitalract are important in chlamydial infectivity, acting as sentinelso recognize pathogens and send signals to underlying immuneells [16,17]. We report here on the use of the vitamin D recep-or knock-out (VDR−/−) mouse and HeLa cells (human cervicalpithelial cells) to test the hypothesis that the vitamin D endocrineystem attenuates chlamydial infection.

. Materials and methods

.1. Chemicals

1,25-Dihydroxyvitamin D3 was obtained from MP Biomedi-als (LLC, Solon, OH). Anti-mouse antibody to leukocyte elastasenhibitor (LEI) was purchased from Santa Cruz Biotechnology (Santaruz, CA). An antibody to Chlamydia conjugated with FITC wasbtained from Bio-Rad (Hercules, CA).

.2. Animals

Female VDR−/− [18,19] and VDR+/+ mice on a C57BL/6 J back-round (6 week old) were purchased from Jackson Laboratory (Bararbor, MA), fed food and water ad libitum, and maintained in

aminar flow racks under pathogen-free conditions with a 12 hight and 12 h dark cycle. All VDR−/− and VDR+/+ mice were fed

rescue diet [20] high in calcium, phosphate, and lactose (Harlanaboratories, Madison, WI) to achieve normal plasma calcium lev-ls in the VDR−/− mice. The mice (n = 6/group) were infected at 8eek old and killed on d 45 after infection by cervical dislocation.

he protocols involving mice were approved by the Institutionalnimal Care and Use Committee (IACUC) of Morehouse School ofedicine.

.3. Infectivity assay in VDR−/− and VDR+/+ mice

Female VDR−/− and VDR+/+ mice (n = 6/group, 8 week old)ere subcutaneously administered 2 mg of medroxyprogesterone

cetate (Sigma–Aldrich Co, St Louis, MO) 7 d prior to infectionnd infected intra-vaginally with 103 inclusion forming units (IFU)f Chlamydia muridarum in 20 �l of PBS. Bacterial shedding wasssessed by performing vaginal swabs on d 3, 6, 9, 12, 15, 18, 25,2, and 39 after infection. Tissue culture isolation of C. muridarumas by standard procedures and the number of inclusions per grouper time point was determined [21].

.4. Histology and immunohistochemistry

The entire genital tract was collected and fixed in 4% formalde-yde. The samples were embedded in paraffin, cut longitudinally

nto 4 �m sections, and stained with hematoxylin and eosin. Theight and left uterine horns and oviducts were individually evalu-ted in a blinded method for the presence of acute inflammationneutrophils), chronic inflammation (monocytes), and plasma cells.

four-tiered semiquantitative scoring system was used to quan-itate the inflammation [22]: 0, normal; 1+, rare foci (minimalresence) of inflammatory cells; 2+, scattered (1–4) aggregates orild diffuse increase in parameter; 3+, numerous aggregates (>4) oroderate diffuse or confluent areas of parameter; 4+, severe diffuse

nfiltration or confluence of parameter.Tissue slides from the oviducts were incubated with a

ouse anti-LEI (serpinB1a) primary antibody, followed by incu-ation for 30 min with a horseradish peroxidase-conjugated goat

& Molecular Biology 135 (2013) 7– 14

anti-mouse secondary antibody in accordance with the manufac-turer’s directions. Images of representative fields were obtainedusing an Olympus Provis AX70 microscope equipped with aLeica 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 con-centrations for 24 h. The treated and un-treated cells were washedand infected with C. muridarum at multiplicity of infection (MOI)5:1 for 48 h. The intensity of infection was measured by stainingwith a chlamydial antibody conjugated with fluorescein isothio-cyanate. The number of inclusions was calculated as previouslydescribed [23].

2.6. Proteomic analysis

HeLa cells that had been pre-treated with 1,25-(OH)2D3(100 nM) for 24 h or un-treated were infected with C. muri-darum for 2 h. Proteins were extracted with a Bio-Rad proteinextraction kit and cleaned with 2-D Clean Up Kit, according tothe manufacturer’s protocol. Protein concentration was deter-mined by 2D Quant Kit from GE Healthcare (GE Healthcare,Piscataway, NJ). Samples (80 �g) were labeled with Cy3 or Cy5 flu-orescence dyes, mixed in a rehydration buffer, and subjected totwo-dimensional fluorescence differential gel electrophoresis anal-ysis (2D-DIGE) [24,25]. The spots corresponding to differentiallyexpressed proteins were digested and analyzed by nanocapillaryLC–MS/MS (Xevo G2 Tof, Waters, Milford, MA). Protein candi-dates were identified with automated database searching against aNCBI 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 muri-darum agent (MOI 5:1) for up to 2 h. Protein expression of LEI at 0.5,1, and 2 h after infection with Chlamydia was determined by West-ern blot analysis. The cell lysates were subjected to electrophoresisfor protein separation on 4–20% Mini-PROTEAN TGX Precast Gels(Bio-Rad, Hercules, CA) in running buffer (Tris/glycine/SDS). Pro-teins were then transferred for 1 h onto nitrocellulose membranesin transfer buffer (Tris/glycine/methanol). Non-specific bindingwas blocked by incubating the membranes in 5% non-fat driedmilk, 0.05% (v/v) Tween 20 in 1X TBS for 1 h at room temperature.After several washes with buffer (TBS Tween 0.05%), membraneswere incubated with the primary antibodies overnight at 4 ◦C.The polyclonal antibodies used were: serpinB1 (1:200) (SantaCruz Biotech. Inc., Santa Cruz, CA); glyceraldehide-3-phosphatedehydrogenase (GAPDH) (1:200) (R&D Systems, Minneapolis, MN).Horseradish peroxidase-conjugated secondary antibodies wereutilized in accordance with the manufacturer’s directions (SantaCruz Biotechnology, Santa Cruz, CA).

2.8. Statistical analysis

A mean ± SD was calculated for each group and statistical sig-nificance (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 wayANOVA to assess the effect of 1,25-(OH)2D3 on intensity of infectionin HeLa cells. A t-test was used to compare the pathology scores ofVDR−/− and VDR+/+ uterus and oviducts.

mistry & Molecular Biology 135 (2013) 7– 14 9

3

3a

stVihtrlc00mtmssom

3

o

Fig. 1. VDR−/− mice infected intra-vaginally with Chlamydia muridarum exhibitgreater bacteria loads and slower rates of bacterial clearance than VDR+/+ mice.VDR−/− and VDR+/+ mice were infected intra-vaginally with 103 inclusion form-ing units (IFU) of C. muridarum as described in Section 2.3. The course of infectionwas monitored by isolation of C. muridarum from cervicovaginal swabs [21]. Valuesare mean ± SD, n = 6. Overall intensity different from VDR+/+, P < 0.01. Rate of clear-

Fwm

Q. He et al. / Journal of Steroid Bioche

. Results

.1. Chlamydial infection and inflammatory response in VDR−/−

nd VDR+/+ mice

VDR−/− mice that were genitally infected with C. muridarum hadignificantly higher bacteria loads at d 6, 9, 12, 15 post-infectionhan VDR+/+ mice, indicating a greater intensity of infection (Fig. 1).DR−/− mice also exhibited a slower rate of clearance of the gen-

tal chlamydial infection than VDR+/+ mice. Whereas VDR+/+ micead cleared the infection by d 18, VDR−/− mice cleared the infec-ion by d 39. The uterus of uninfected VDR+/+ and VDR−/− miceevealed normally observed branched folia with intact epithelialining composed of both secretory and ciliated cells (Fig. 2). Signifi-antly higher numbers of monocytes (pathology scores: 2.5 ± 0.3 vs..7 ± 0.7, P < 0.01) and neutrophils (pathology scores: 1.2 ± 0.6 vs..2 ± 0.2, P < 0.05) were observed in the infected uterus of VDR−/−

ice, compared with that of infected VDR+/+ mice. No inflamma-ion was observed in the oviduct of un-infected VDR−/− and VDR+/+

ice (Fig. 3). Significantly higher numbers of monocytes (pathologycores: 3.2 ± 0.6 vs. 1.7 ± 0.7, P < 0.01) and neutrophils (pathologycores: 1.5 ± 0.3 vs. 0.3 ± 0.3, P < 0.01) were observed in the oviductf infected VDR−/− mice, compared with that of infected VDR+/+

ice.

.2. Effect of 1,25-(OH)2D3 on chlamydial infection in HeLa cells

Chlamydial infectivity of HeLa cells pre-treated with 10 nMr 100 nM 1,25-(OH)2D3 for 24 h was significantly less than

ig. 2. More monocytes and neutrophils are observed in the uterus of infected VDR−/− mere removed at d 45 post-infection with Chlamydia muridarum and stained with hemaagnification. Open arrows point to scattered neutrophils; closed arrows indicate monoc

ance different from VDR+/+, P < 0.001. Intensity of infection different from VDR+/+,*P < 0.001.

chlamydial infectivity of un-treated HeLa cells or HeLa cells

pre-treated with 1.0 nM 1,25-(OH)2D3 (Fig. 4). Vitamin D hor-mone treatment of chlamydial-infected HeLa cells resulted innumerous differentially expressed protein spots, of which 25

ice, compared with that of infected VDR+/+ mice. The uterus horns of each mousetoxylin and eosin (HE), as described in Section 2.4. HE-stained sections are at 40×ytes/lymphocytes.

10 Q. He et al. / Journal of Steroid Biochemistry & Molecular Biology 135 (2013) 7– 14

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 mousewere 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.

Table 1Several spots were up-regulated in chlamydial-infected HeLa cells pre-treated with 1,25-dihydroxyvitamin D3.

Spota Protein name Accession IDb MW (Da)c pIc Scored Matched peptidese Sequence coverage (%)

10 Protein S100-A6 S10A6 RABIT 10,147 5.30 54 1 2013 Tropomyosin alpha-3 chain TPM3 HUMAN 32,799 4.68 113 3 1118 Tubulin beta chain TBB5 HUMAN 49639 4.78 178 7 1719 Hsc70-interacting protein F10A1 HUMAN 41,305 5.18 91 2 620 ATP synthase subunit beta ATPB HUMAN 56,525 5.26 384 11 3321 Vimentin VIME HUMAN 53,619 5.06 282 7 1822 60 kDa heat shock protein CH60 HUMAN 61,016 5.70 488 12 3123 60 kDa heat shock protein CH60 HUMAN 61,016 5.70 111 4 1124 T-complex protein 1 subunit epsilon TCPE HUMAN 59,633 5.45 271 10 2325 Heat shock cognate 71 kDa protein HSP7C HUMAN 70,854 5.37 393 11 2226 Serum albumin precursor ALBU BOVIN 69,248 5.82 58 3 530 Protein disulfide-isomerase A3 precursor PDIA3 HUMAN 56,747 5.98 223 8 2134 T-complex protein 1 subunit beta TCPB HUMAN 57,452 6.01 183 4 936 Proliferation-associated protein 2G4 PA2G4 HUMAN 43,759 6.13 157 4 1537 Proliferation-associated protein 2G4 PA2G4 HUMAN 43,759 6.13 41 1 739 Leukocyte elastase inhibitor ILEU HUMAN 42,715 5.90 103 3 842 60S acidic ribosomal protein P0 RLA0 HUMAN 34,252 5.71 173 4 1346 Peroxiredoxin-6 PRDX6 HUMAN 25,019 6.00 130 4 2048 Heat-shock protein beta-1 (HspB1) HSPB1 HUMAN 22,768 5.98 122 3 1849 Thioredoxin-dependent peroxide reductase PRDX3 HUMAN 27,675 7.67 142 3 1354 Peroxiredoxin-2 PRDX2 HUMAN 21,878 5.66 122 3 1957 Tumor protein D54 (hD54) TPD54 HUMAN 22,224 5.26 42 2 1158 Transitional endoplasmic reticulum ATPase TERA HUMAN 89,266 5.14 144 7 964 Vinculin (Metavinculin) VINC HUMAN 123,722 5.50 60 3 2

a As indicated in Supplemental Fig. 1. Down-regulated protein spots were not identified for technical reasons.b SwissProt protein accession number.c Relative molecular mass of proteins and isoelectric point.d Probability 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.e Number of peptides that match the theoretical digest of the primary protein identified.

Q. He et al. / Journal of Steroid Biochemistry & Molecular Biology 135 (2013) 7– 14 11

Fig. 4. Chlamydial infectivity of HeLa cells pre-treated with 1,25-(OH)2D3 is signif-icantly less than chlamydial infectivity of un-treated HeLa cells. HeLa cells werepre-treated with 1,25-(OH)2D3 at different concentrations for 24 h. Treated andun-treated cells were washed and infected with Chlamydia muridarum for 48 h asdescribed in Section 2.5. The intensity of infection was measured by staining withami

hap(

o

3

cHediV

4

mop[escFl[fccof

trtuc

Fig. 5. Leukocyte elastase inhibitor is up-regulated in infected HeLa cells pre-treatedwith 1,25-(OH)2D3, but not in pre-treated un-infected HeLa cells. HeLa cells that hadbeen pre-treated with 1,25-(OH)2D3 (100 nM) or un-treated for 24 h were pulsed

chlamydial antibody and determining the number of inclusions [23]. Values areean ± SD, n = 6. Significant effect of 1,25-(OH)2D3 treatment, P < 0.001. Intensity of

nfection different from un-treated, *P < 0.001.

ave been identified, using LC–MS/MS ion search software andssociated databases (Table 1). Fewer differentially expressedrotein spots were observed for pre-treated un-infected HeLa cellsSupplemental Fig. 1).

Supplementary material related to this article found, in thenline version, at http://dx.doi.org/10.1016/j.jsbmb.2012.11.002.

.3. Leukocyte elastase inhibitor expression

Western blots indicated significantly more LEI in infected HeLaells pre-treated with 1,25-(OH)2D3 than in un-treated infectedeLa cells (Fig. 5). Pre-treatment with 1,25-(OH)2D3 did not, how-ver, significantly increase LEI in un-infected HeLa cells. LEI wasetected in the oviduct and ovary of infected VDR+/+ mice by

mmunohistochemistry, but not in the oviduct and ovary of infectedDR−/− mice (Fig. 6).

. Discussion

In this study, vitamin D receptor knock-out mice and controlice (VDR+/+) were used to evaluate the 1,25-(OH)2D3/VDR effect

n genital chlamydial infection. Homozygous VDR−/− mice arehenotypically normal at birth and survive for at least six months18]. These mice exhibit hypocalcemia, hyperparathyroidism, rick-ts and osteomalacia. One feature seen in VDR−/− mice, but noteen in mice made vitamin D deficient by dietary means, is alope-ia, which develops progressively from the age of four weeks [18].eeding VDR−/− mice a rescue diet high in calcium, phosphorus, andactose [20] normalized all of the symptoms above except alopecia19]. In the present study, VDR−/− mice fed the rescue diet suf-ered a more intense and prolonged genital chlamydial infectionompared with VDR+/+ mice, suggesting that attenuation of genitalhlamydial infectivity in the VDR+/+ mice is not an indirect effectf maintaining normal calcium, but is a process which requires aunctional VDR.

Intensive chlamydial infection and a prolonged infection courserigger inflammatory responses that are thought to be largely

esponsible for the chlamydial induced pathology of the genitalract. Inflammation was not observed in the uterus and oviduct ofn-infected VDR−/− and VDR+/+ mice, but higher numbers of mono-ytes and neutrophils were observed in the infected uterus and

with Chlamydia muridarum (MOI 5:1) for up to 2 h. Total protein was extracted, sub-jected to SDS-PAGE, transferred onto nitrocellulose membrane, probed with primaryand secondary antibodies, and imaged as described in Section 2.7.

oviduct of VDR−/− mice, compared with that of VDR+/+ mice. Themore intense and prolonged chlamydial infection in the VDR−/−

mice was thus accompanied by a more prolonged inflammatoryresponse.

Genital epithelial cells are the first line of defense againstmicrobial invasion, so we conducted chlamydial infection assayswith HeLa cells, a genital epithelial cell line. Pre-treatment ofHeLa cells with 1,25-(OH)2D3 dramatically inhibited intracellu-lar replication of C. muridarum and inclusion development. It ispossible that 1,25-(OH)2D3 mediates the protective response ofepithelial cells to bacterial replication [26]. HeLa cells pre-treatedwith 1,25-(OH)2D3 and infected with C. muridarum exhibiteddifferential expression of 65 protein spots, whereas fewer dif-ferentially expressed protein spots were detected in un-infectedHeLa cells pre-treated with 1,25-(OH)2D3. Several categories ofproteins were differentially expressed: cytoskeleton proteins thataffect microfilament network and cell–cell junctions (tubulin,vimentin, tropomyosin chain, vinculin); stress response pro-teins related to protein folding and chaperones (heat shockproteins, protein disulfide isomerase, T-complex protein-1); pro-teins involved in oxidation/reduction (peroxiredoxin-2 and -6,thioredoxin-dependent peroxide reductase); proteins involved ingrowth and regulation of cell proliferation (tumor protein D54,60S acidic ribosomal protein P0, proliferation-associated protein2G4); proteins involved with ATP (ATP synthase subunit beta,transitional endoplasmic reticulum ATPase); calcium binding pro-tein (protein S100-A7); anti-inflammatory protein (LEI); serumalbumin precursor. A caveat to be considered in conducting com-plementary experiments with HeLa cells is that human monocytesand macrophages show vitamin D hormone induced expression of

antimicrobial proteins (e.g. cathelicidin, beta-defensin), but thesegenes are not regulated by 1,25-(OH)2D3 in mice.

We selected for further study LEI, an anti-inflammatory pro-tein that was up-regulated in chlamydial infected HeLa cells

12 Q. He et al. / Journal of Steroid Biochemistry & Molecular Biology 135 (2013) 7– 14

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 infectedi of ovap

pLst(ppnrpptdoewom[nfTittLda

ntra-vaginally with Chlamydia muridarum as described in Section 2.3. Tissue slidesrimary antibody and imaged as described in Section 2.4.

re-treated with 1,25-(OH)2D3, compared with un-treated cells.EI is a 42 kDa member of the serine protease inhibitor (serpin)uperfamily [27] and is also known as monocyte/neutrophil elas-ase inhibitor (MNEI) and serpinB1. It inhibits neutrophil proteaseselastase, cathepsin G, proteinase-3) that are involved in killinghagocytosed microbes by forming irreversible covalent com-lexes with them. LEI is abundant in the cytoplasm of monocytes,eutrophils, and macrophages and attenuates the inflammatoryesponse to microbial infection by limiting the activity of theroteases, thus limiting degradation of host defense and matrixroteins [28]. Recombinant MNEI administered daily by aerosoliza-ion to rats previously inoculated with Pseudomonas aeruginosaecreased the inflammatory injury and enhanced the clearancef bacteria from the infected rat lungs [29]. SerpinB1−/− micexhibited higher mortality relative to wild type mice, associatedith late-onset failed clearance of P. aeruginosa. Co-administration

f recombinant serpinB1 with the P. aeruginosa inoculum nor-alized bacterial clearance in serpinB1−/− mice [30]. Gong et al.

31] reported that serpinB1−/− mice died earlier and in greaterumbers than wild-type mice when infected with high-dose sur-

actant protein-D-sensitive influenza A/Philadelphia/82(H3N2).hey concluded that serpinB1 plays a critical role in mitigatingnflammation and restricting pro-inflammatory cytokine produc-ion in influenza infection. L-DNase II, an endonuclease involved in

he degradation of genomic DNA during apoptosis, is derived fromEI by an acidic-dependent post-translational modification or byigestion with elastase [32–36]. Appearance of the endonucleasectivity results in loss of the anti-protease activity [32]. Benarafa

ries from the mice were incubated with a mouse anti-leukocyte elastase inhibitor

et al. [37] have indicated that human MNEI is encoded by a singleSERPINB1 gene, whereas four murine genes have been identifiedand fully sequenced, with one of them, EIA (SERPINB1a), being themouse ortholog of MNEI.

In the present study, Western blots indicated up-regulation ofLEI in infected HeLa cells pre-treated with 1,25-(OH)2D3, but notin pre-treated un-infected HeLa cells (confirming the proteomicsresults). A greater amount of LEI was detected by immunohisto-chemistry in the oviduct and ovary of infected VDR+/+ mice thanin the oviduct and ovary of infected VDR−/− mice, although mono-cytes and neutrophils (the primary cellular sources of LEI) weremore numerous in the oviduct of infected VDR−/− mice than in theoviduct of infected VDR+/+ mice. The association of more prolongedchlamydial infection and inflammatory response with lesser LEIexpression in the oviduct and ovary of VDR−/− mice, compared withVDR+/+ mice, would suggest a role for LEI in countering the inflam-matory response and for vitamin D hormone in LEI expression.Previous studies have demonstrated a role for 1,25-(OH)2D3 in LEIexpression. Protiva et al. [38] demonstrated, via global gene arrayanalysis, activation of the VDR pathway in rectal mucosal biopsiesfrom estradiol treated postmenopausal women and up-regulationof SERPINB1. Kovalenko et al. [39] identified numerous transcriptchanges, 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, LEIappears to be regulated by 1,25-(OH)2D3 in mice, as well as in cellsof human origin. Wang et al. [40] have demonstrated that mul-tiple DR3 elements in the serpinB1 gene are conserved between

mistry

mfs

tVwmiictD

coHnmr

A

taaoRo

R

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

Q. He et al. / Journal of Steroid Bioche

ouse and human. Consequently, the mouse can serve as a modelor examining the relationship between the vitamin D endocrineystem and LEI, a protein with anti-inflammatory effects.

In summary, we demonstrate here that genital chlamydial infec-ivity was more intense and prolonged in VDR−/− mice than inDR+/+ mice. The inflammatory response to chlamydial infectionas also more prolonged in VDR−/− mice than in VDR+/+ mice. Theore prolonged chlamydial infection and inflammatory response

n the VDR−/− mice coincided with lesser expression of LEI (an anti-nflammatory protein) in the oviduct and ovary of VDR−/− mice,ompared with VDR+/+ mice, suggesting a role for LEI in counteringhe inflammatory response to chlamydial infection and for vitamin

hormone in LEI expression.We conclude that the VDR is involved in immunoregulation of

hlamydial infection. LEI, the protein examined in this study, wasnly one of numerous proteins differentially expressed in infectedeLa cells pre-treated with 1,25-(OH)2D3. Additional studies areecessary to determine other mechanisms by which vitamin D hor-one attenuates chlamydial infection and the anti-inflammatory

esponse.

cknowledgements

This work was funded by Georgia Research Alliance Collabora-ion Planning Grant GRA VAC10.C, the Centers for Disease Controlnd Prevention, and Morehouse School of Medicine. Facilitiesnd support services were partially funded by National Institutesf Health/National Center for Research Resources grants G12-R03034, 1 C06 RR18386, 1 U54 RR026137 (to Morehouse Schoolf Medicine) and RR03062 and 08247 (to Clark Atlanta University).

eferences

[1] J.H. White, Vitamin D signaling, infectious diseases, and regulation of innateimmunity, Infection and Immunity 76 (2008) 3837–3843.

[2] M. Hewison, Antibacterial effects of vitamin D, Nature Reviews Endocrinology7 (6) (2011) 337–345.

[3] P.T. Liu, S. Stenger, H. Li, L. Wenzel, B.H. Tan, S.R. Krutzik, M.T. Ochoa, J. Schauber,K. Wu, C. Meinken, D.L. Kamen, M. Wagner, R. Bals, A. Steinmeyer, U. Zügel, R.L.Gallo, D. Eisenberg, M. Hewison, B.W. Hollis, J.S. Adams, B.R. Bloom, R.L. Mod-lin, Toll-like receptor triggering of a vitamin D-mediated human antimicrobialresponse, Science 311 (2006) 1770–1773.

[4] J. Schauber, R.A. Dorschner, A.B. Coda, A.S. Büchau, P.T. Liu, D. Kiken, Y.R. Hel-frich, S. Kang, H.Z. Elalieh, A. Steinmeyer, U. Zügel, D.D. Bikle, R.L. Modlin, R.L.Gallo, Injury enhances TLR2 function and antimicrobial peptide expressionthrough a vitamin D-dependent mechanism, Journal of Clinical Investigation117 (2007) 803–811.

[5] P.T. Liu, M. Schenk, V.P. Walker, P.W. Dempsey, M. Kanchanapoomi, M. Wheel-wright, A. Vazirnia, X. Zhang, A. Steinmeyer, U. Zügel, B.W. Hollis, G. Cheng,R.L. Modlin, Convergence of IL-1� and VDR activation pathways in humanTLR2/1-induced antimicrobial responses, PLoS ONE 4 (2009) e5810.

[6] V. Lagishetty, A.V. Misharin, N.Q. Liu, T.S. Lisse, R.F. Chun, Y. Ouyang, S.M.McLachlan, J.S. Adams, M. Hewison, Vitamin D deficiency in mice impairscolonic antibacterial activity and predisposes to colitis, Endocrinology 151(2010) 2423–2432.

[7] A.R. Martineau, R.J. Wilkinson, K.A. Wilkinson, S.M. Newton, B. Kampmann, B.M.Hall, G.E. Packe, R.N. Davidson, S.M. Eldridge, Z.J. Maunsell, S.J. Rainbow, J.L.Berry, C.J. Griffiths, A single dose of vitamin D enhances immunity to mycobac-teria, American Journal of Respiratory and Critical Care Medicine 176 (2007)208–213.

[8] M. Urashima, T. Segawa, M. Okazaki, M. Kurihara, Y. Wada, H. Ida, Randomizedtrial of vitamin D supplementation to prevent seasonal influenza A in schoolchildren, American Journal of Clinical Nutrition 91 (2010) 1255–1260.

[9] A.R. Martineau, F.U. Honecker, R.J. Wilkinson, C.J. Giffiths, Vitamin D in thetreatment of pulmonary tuberculosis, The Journal of Steroid Biochemistry andMolecular Biology 103 (2007) 793–798.

10] E. van Etten, C. Mathieu, Immunoregulation by 1,25-dihydroxyvitamin D3:basic concepts, Journal of Steroid Biochemistry and Molecular Biology 97 (2005)93–101.

11] I. Imazeki, J. Matsuzaki, K. Tsuji, T. Nishimura, Immunomodulating effect of

vitamin D3 derivatives on type-1 cellular immunity, Biomedical Research 27(2006) 1–9.

12] J.E. Do, S.Y. Kwon, S. Park, E.-S. Lee, Effects of vitamin D on expression of toll-likereceptors of monocytes from patients with Behc et’s disease, Rheumatology 47(2008) 840–848.

[

& Molecular Biology 135 (2013) 7– 14 13

13] J.R. Stubbs, A. Idiculla, J. Slusser, R. Menard, L.D. Quarles, Cholecalciferolsupplementation alters calcitriol-responsive monocyte proteins and decreasesinflammatory cytokines in ESRD, Journal of the American Society of Nephrology21 (2010) 353–361.

14] D.S. Meyers, H. Halvorson, S. Luckhaupt, Screening for chlamydial infection: anevidence update for the U.S. Preventive Services Task Force, Annals of InternalMedicine 147 (2007) 135–142.

15] C.R. Cohen, J. Gichui, R. Rukaria, S.S. Sinei, L.K. Gaur, R.C. Brunham, Immuno-genetic correlates for Chlamydia trachomatis-associated tubal infertility,Obstetrics and Gynecology 101 (2003) 438–444.

16] A.J. Quayle, The innate and early immune response to pathogen challenge inthe female genital tract and the pivotal role of epithelial cells, Journal of Repro-ductive Immunology 57 (2002) 61–79.

17] A.W. Horne, S.J. Stock, A.E. King, Innate immunity and disorders of the femalereproductive tract, Reproduction 135 (2008) 739–749.

18] Y.C. Li, A.E. Pirro, M. Amling, G. Delling, R. Baron, R. Bronson, M.B. Demay,Targeted ablation of the vitamin D receptor: an animal model of vitamin D-dependent rickets type II with alopecia, Proceedings of the National Academyof Sciences of the United States of America 94 (1997) 9831–9835.

19] M. Amling, M. Priemel, T. Holzmann, K. Chapin, J.M. Rueger, R. Baron, M.B.Demay, Rescue of the skeletal phenotype of vitamin D receptor-ablated micein the setting of normal mineral ion homeostasis: formal histomorphometricand biomechanical analyses, Endocrinology 140 (1999) 4982–4987.

20] M.J. Rowling, C. Gliniak, J. Welsh, J.C. Fleet, High dietary vitamin D preventshypocalcemia and osteomalacia in CYP27B1 knockout mice, Journal of Nutri-tion 137 (2007) 2608–2615.

21] Q. He, F.O. Eko, D. Lyn, G.A. Ananaba, C. Bandea, J. Martinez, K. Joseph, K.Kellar, C.M. Black, J.U. Igietseme, Involvement of LEK1 in dendritic cell regula-tion of T cell immunity against Chlamydia, Journal of Immunology 181 (2008)4037–4042.

22] A.M. Scurlock, L.C. Frazer, C.W. Andrews Jr., C.M. O’Connell, I.P. Foote, S.L. Bailey,K. Chandra-Kuntal, J.K. Kolls, T. Darville, Interleukin-17 contributes to genera-tion of Th1 immunity and neutrophil recruitment during Chlamydia muridarumgenital tract infection but is not required for macrophage influx or normalresolution of infection, Infection and Immunity 79 (2011) 1349–1362.

23] Q. He, T.T. Moore, F.O. Eko, D. Lyn, G.A. Ananaba, A. Martin, S. Singh, L. James,J. Stiles, C.M. Black, J.U. Igietseme, Molecular basis for the potency of IL-10-deficient dendritic cells as a highly efficient APC system for activating Th1response, Journal of Immunology 174 (2005) 4860–4869.

24] Y. Ye, E.C. Mar, S. Tong, S. Sammons, S. Fang, L.J. Anderson, D. Wang, Applicationof proteomics methods for pathogen discovery, Journal of Virological Methods163 (1) (2010) 87–95.

25] S.C. Arruda, S. Barbosa Hde, R.A. Azevedo, M.A. Arruda, Two-dimensional dif-ference gel electrophoresis applied for analytical proteomics: fundamentalsand applications to the study of plant proteomics, Analyst 136 (20) (2011)4119–4126.

26] J. Sun, Vitamin D and mucosal immune function, Current Opinion in Gastroen-terology 26 (2010) 591–595.

27] E. Remold-O’Donnell, J. Chin, M. Alberts, Sequence and molecular character-ization of human monocyte/neutrophil elastase inhibitor, Proceedings of theNational Academy of Sciences 89 (1992) 5635–5639.

28] P.M. Fitch, A. Roghanian, S.E. Howie, J.M. Sallenave, Human neutrophil elastaseinhibitors in innate and adaptive immunity, Biochemical Society Transactions34 (2006) 279–282.

29] D.E. Woods, A. Cantin, J. Cooley, D.M. Kenney, E. Remold-O’Donnell, Aerosoltreatment with MNEI suppresses bacterial proliferation in a model of chronicPseudomonas aeruginosa lung infection, Pediatric Pulmonology 39 (2005)141–149.

30] C. Benarafa, G.P. Priebe, E. Remold-O’Donnell, The neutrophil serine proteaseinhibitor serpinB1 preserves lung defense functions in Pseudomonas aeruginosainfection, Journal of Experimental Medicine 204 (2007) 1901–1909.

31] D. Gong, K. Farley, M. White, K.L. Hartshorn, C. Benarfa, E. Remold-O’Donnell,Critical role of serpinB1 in regulating inflammatory responses in pulmonaryinfluenza infection, Journal of Infectious Diseases 204 (2011) 592–600.

32] A. Torriglia, P. Perani, J.Y. Brossas, E. Chaudun, J. Treton, Y. Courtois, M.-F. Counis,L-DNase II, a molecule that links proteases and endonucleases in apoptosis,derives from the ubiquitous serpin leukocyte elastase inhibitor, Molecular andCellular Biology 18 (1998) 3612–3619.

33] C. Leprêtre, Y. Fleurier, E. Martin, A. Torriglia, Nuclear export of LEI/L-DNase IIby Crm1 is essential for cell survival, Biochimica et Biophysica Acta 1783 (2008)1068–1075.

34] A. Torriglia, C. Leprêtre, L. Padrón-Barthe, S. Chahory, E. Martin, Molecularmechanisms of L-DNase II activation and function as a molecular switch inapoptosis, Biochemical Pharmacology 76 (2008) 1490–1502.

35] L. Padrón-Barthe, J. Courta, C. Leprêtre, A. Nagbou, A. Torriglia, Leukocyte elas-tase inhibitor, the precursor of L-DNase II, inhibits apoptosis by interfering withcaspase-8 activation, Biochimica et Biophysica Acta 1783 (2008) 1755–1766.

36] C. Leprêtre, G. Sidoli, A.I. Scovassi, A. Torriglia, Leukocyte elastase inhibitor: anew regulator of PARP-1, Annals of the New York Academy of Sciences 1171(2009) 25–31.

37] C. Benarafa, J. Cooley, W. Zeng, P.I. Bird, E. Remold-O’Donnell, Characteriza-

tion of four murine homologs of the human ov-serpin monocyte neutrophilelastase inhibitor MNEI (SERPINB1), Journal of Biological Chemistry 277 (2002)42028–44233.

38] P. Protiva, H.S. Cross, M.E. Hopkins, E. Kallay, G. Bises, E. Dreyhaupt, L. Augen-licht, M. Lipkin, M. Lesser, E. Livote, P.R. Holt, Chemoprevention of colorectal

1 mistry

[

[40] T.-T Wang, L.E. Travera-Mendoza, D. Laperriere, E. Libby, N.B. MacLeod, Y.

4 Q. He et al. / Journal of Steroid Bioche

neoplasia by estrogen: potential role of vitamin D activity, Cancer Prevention

Research 2 (1) (2009) 43–51.

39] P.L. Kovalenko, Z. Zhang, M. Cui, S.K. Clinton, J.C. Fleet, 1,25 dihydroxyvitaminD-mediated orchestration of anticancer, transcript-level effects in the immor-talized, non-transformed prostate epithelial cell line, RWPE1, BMC Genomics11 (2010) 26.

& Molecular Biology 135 (2013) 7– 14

Nagai, V. Bourdeau, A. Konstorum, B. Lallemant, R. Zhang, S. Mader, J.H.White, Large-scale in silico and microarray-based identification of direct1,25-dihydroxyvitamin D3 target genes, Molecular Endocrinology 19 (2005)2685–2695.