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REPRODUCTION-DEVELOPMENT

Hormonal Contraception and HIV-1 Infection:


Medroxyprogesterone Acetate Suppresses Innate and
Adaptive Immune Mechanisms

Richard P. H. Huijbregts, E. Scott Helton, Katherine G. Michel, Steffanie Sabbaj,


Holly E. Richter, Paul A. Goepfert, and Zdenek Hel
Departments of Pathology (R.P.H.H., E.S.H., K.G.M., Z.H.), Microbiology (P.A.G., Z.H.), Obstetrics and
Gynecology (H.E.R.), and Medicine (S.S., P.A.G.); Center for AIDS Research (S.S., P.A.G., Z.H.); and
Mucosal HIV and Immunobiology Center (S.S., P.A.G., Z.H.), University of Alabama at Birmingham,
Birmingham, Alabama 35294

Recent observational studies indicate an association between the use of hormonal contraceptives
and acquisition and transmission of HIV-1. The biological and immunological mechanisms under-
lying the observed association are unknown. Depot medroxyprogesterone acetate (DMPA) is a
progestin-only injectable contraceptive that is commonly used in regions with high HIV-1 preva-
lence. Here we show that medroxyprogesterone acetate (MPA) suppresses the production of key
regulators of cellular and humoral immunity involved in orchestrating the immune response to
invading pathogens. MPA inhibited the production of interferon (IFN)-, IL-2, IL-4, IL-6, IL-12, TNF,
macrophage inflammatory protein-1 (MIP-1), and other cytokines and chemokines by peripheral
blood cells and activated T cells and reduced the production of IFN and TNF by plasmacytoid
dendritic cells in response to Toll-like receptor-7, -8, and -9 ligands. Women using DMPA displayed
lower levels of IFN in plasma and genital secretions compared with controls with no hormonal
contraception. In addition, MPA prevented the down-regulation of HIV-1 coreceptors CXCR4 and
CCR5 on the surface of T cells after activation and increased HIV-1 replication in activated periph-
eral blood mononuclear cell cultures. The presented results suggest that MPA suppresses both
innate and adaptive arms of the immune system resulting in a reduction of host resistance to
invading pathogens. (Endocrinology 154: 12821295, 2013)

afe and effective methods of contraception represent a most commonly used contraceptives in sub-Saharan Af-
S critical component of preventive health care. Contra-
ception provides women with a control over their repro-
rica and other areas with high HIV-1 prevalence. It is es-
timated that over 14 million women worldwide use
ductive health, reduces the number of unwanted pregnan- DMPA; in some countries, DMPA is the method of choice
cies, decreases maternal and infant mortality and for over 50% of women using modern methods of con-
morbidity, and lowers the risk of mother-to-child trans- traception (2, 3). Unfortunately, multiple observational
mission of HIV-1 (1). Injectable hormonal contraception studies suggest an association between the use of hor-
is effective, affordable, and increasingly popular especially monal contraception and increased risk of HIV-1 acqui-
in countries with limited resources. Depot medroxypro- sition and transmission (4 11). In most studies, the ad-
gesterone acetate (DMPA) (Depo-Provera), a progestin- justed hazard ratio of HIV-1 acquisition associated with
only based contraceptive typically administered as a the use of injectable contraception, in particular DMPA,
3-monthly intramuscular (I.M.) injection, is one of the was higher than that associated with oral contraception

ISSN Print 0013-7227 ISSN Online 1945-7170 Abbreviations: CVL, cervicovaginal lavage; DC, dendritic cell; DEX, dexamethasone; DMPA,
Printed in U.S.A. depot medroxyprogesterone acetate; E2, 17-estradiol; FBS, fetal bovine serum; GR, glu-
Copyright 2013 by The Endocrine Society cocorticoid receptor; IFN, interferon; LPS, lipopolysaccharide; MDC, monocyte-derived
doi: 10.1210/en.2012-1850 Received August 13, 2012. Accepted December 18, 2012. cytokine; NFB, nuclear factor B; OVA, ovalbumin; P4, progesterone; PBMC, peripheral
First Published Online January 25, 2013 blood mononuclear cell; pDC, plasmacytoid dendritic cell; PHA-L, phytohaemagglutinin-L;
PR, P4 receptor; TCR, T cell receptor; TLR, Toll-like receptor; VMMC, vaginal mucosal
For editorial see page 985 mononuclear cell.

1282 endo.endojournals.org Endocrinology, March 2013, 154(3):12821295

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Endocrinology, March 2013, 154(3):12821295 endo.endojournals.org 1283

(510, 12). Hormonal contraception was also shown to be Materials and Methods
associated with increased rate of HIV-1 replication (13,
14), accelerated disease progression (15), increased cervi- Study participants and sample collection
All procedures involving the use of human subjects were ap-
covaginal shedding (16, 17), and elevated risk of acquisi-
proved by the Institutional Review Board of the University of
tion of cervical candidiasis, chlamydial, gonococcal, and Alabama at Birmingham. Informed consent was obtained from
Mycoplasma genitalium infections (4, 6, 18 21). Studies all participants. Acid citrate dextrose-treated blood (100 ml) was
using nonhuman primate models of infection demon- collected from healthy premenopausal women by personnel
strated that administration of DMPA enhanced the risk of trained in phlebotomy. Women volunteers were of a good gen-
simian immunodeficiency virus (SIV) acquisition via vag- eral health, age 19 40 years, mixed race, not pregnant, not using
steroids, and with no signs of vaginitis, sores, or ulcers at the time
inal exposure, increased viral levels in the acute phase of
of collection. History of vaginal infections, medication, and men-
infection, and reduced the protective effect of prior im- strual cycle information was collected. Women volunteers using
munization (2225). However, the results of observa- hormonal contraception were using either DMPA delivered by
tional studies in humans have been inconsistent with some 3-monthly im injections (150 mg; Depo-Provera) for a median of
studies reporting no effect of hormonal contraception on 7 months (range, 196 months) or several different forms of
HIV-1 acquisition or disease progression (26 30) or find- combined oral contraception for a median of 3.5 months (range,
1-17 months). Cervicovaginal lavage (CVL) was obtained from
ing increased risk only in subgroups of subjects differing in
women volunteers, by vigorous flushing of the cervix and vagina
age and herpes simplex virus 2 status (3133). It was ar- with 5 ml sterile saline; the sample was mixed with protease
gued that the elevated risk of HIV-1 acquisition observed inhibitors (5 g/ml aprotinin, 1 g/ml leupeptin, 1 g/ml anti-
in hormonal contraceptive users may be due to confound- pain, 1 g/ml pepstatin, 200 g/ml sodium azide, and 1mM
ing behavioral factors that are difficult to be accounted for phenylmethylsulfonyl fluoride; all from Sigma, St Louis, Mis-
souri) and subsequently cleared by centrifugation at 14,000g for
due to methodological limitations (34, 35). It is therefore
5 minutes before storage at 80C. Vaginal tissue was obtained
critical to establish whether the observed associations re- from postmenopausal women undergoing pelvic reconstructive
flect a direct biological effect of hormonal contraceptives surgery as anonymous remnant surgical material.
on immune system and determine the mechanisms exerted
by specific hormones and dosages. Materials
The potential biological mechanisms of the effect of All cell culture reagents were obtained from Mediatech Inc
MPA on viral and bacterial infections are unknown. In (Manassas, Virginia), unless indicated otherwise. Chem-
addition to binding to the progesterone (P4) receptor (PR), icals, hormones, and enzymes were purchased from Sigma
MPA binds with high affinity and activates the glucocor- (St Louis, Missouri). Antibodies, beads, and columns for
ticoid receptor (GR) expressed at high levels by multiple cell purification were obtained from Miltenyi Biotec (Au-
immune cell types (36 40). The affinity of MPA for GR is burn, California). Antibodies for flow cytometry were
significantly higher than that of its endogenous ligand cor- purchased from eBioscience (San Diego, California), un-
tisol (38). The MPA-GR complex suppresses the transcrip- less listed otherwise. 17-Estradiol (E2), P4, MPA, dexa-
tion of GR-regulated genes, including IL-2, IL-6, and IL-8, methasone (DEX), and mifepristone (RU486) (all from
via transrepression (36, 42, 43). Due to its glucocorticoid Sigma) were dissolved in 200-proof ethanol at a concen-
activity, it is feasible that MPA exhibits significant immune tration of 10mM and stored at 80C before use.
regulatory effects that may impact the hosts susceptibility to
pathogens. Recently, it was reported that the use of DMPA Mice
is associated with decreased systemic responses to Mycobac- Female C57BL/6 (B6) mice and ovalbumin (OVA)-spe-
terium bovis Bacillus CalmetteGurin in household con- cific T cell receptor (TCR)-transgenic OT2 mice on a B6
tacts of active tuberculosis patients (44). background were obtained from The Jackson Laboratory
In this study, we characterized the effect of MPA on (Bar Harbor, Maine). All experimental procedures involv-
innate and adaptive immune mechanisms and compared it ing animals were approved by the University of Alabama
with the effect of the natural hormones P4 and estrogen Institutional Animal Care and Use Committee.
and a commonly used synthetic glucocorticoid, dexameth-
asone. We show that MPA inhibits the secretion of key Isolation of peripheral blood mononuclear cells, T
regulators of cellular and humoral immunity by activated cells, and monocytes
T cells and plasmacytoid dendritic cells (pDCs). Further- Peripheral blood mononuclear cells (PBMCs) were iso-
more, MPA prevents the down-regulation of HIV core- lated by density gradient centrifugation using lymphocyte
ceptors CXCR4 and CCR5 on activated T cells and in- separation medium (MP Biomedicals, Solon, Ohio). T
creases the rate of HIV-1 replication in peripheral blood cells were purified from PBMCs by positive selection for
cell culture. CD3 cells on a MACS column according to manufac-

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1284 Huijbregts et al Hormonal Contraception and HIV-1 Endocrinology, March 2013, 154(3):12821295

turers directions (Miltenyi). Macrophages were purified saccharide (LPS) (Escherichia coli 0111:B4; Sigma). Cells
by positive selection for CD14 cells from the CD3-de- were activated for 24 hours, cell-free media was collected
pleted population. PBMCs and macrophages were cul- and filtered through 0.45-m polyvinylidene difluoride
tured in phenol red-free RPMI containing 10% heat-in- filter plates (Millipore, Billerica, Massachusetts). Concen-
activated charcoal dextran-scrubbed fetal bovine serum trations of interferon (IFN)- and TNF were determined
(FBS) (Atlanta Biologicals, Lawrenceville, Georgia), 2mM with Ready-Set-Go ELISA kits (eBioscience). Concentra-
GlutaMAX (Invitrogen, Carlsbad California), 100 IU/ml tions of 39 cytokines and chemokines were determined
penicillin, and 100 g/ml streptomycin. Purified T cells using the 39-plex MILLIPLEX human cytokine/chemo-
were cultured in identical media except that regular FBS kine panel kit (Millipore), and samples were analyzed
(Hyclone, Logan, Utah) was used. undiluted on a Bioplex 100 system with Bioplex Man-
ager Software version 5.0 (Bio-Rad, Hercules, Califor-
Isolation of human vaginal mucosal mononuclear nia). GRO stands for CXCL1, -2, and -3; eotaxin stands
cells for CCL-11. Heat maps were created using matrix2png
Vaginal mucosal mononuclear cells (VMMCs) were (http://www.bioinformatics.ubc.ca/matrix2png/).
isolated from remnant vaginal tissue from volunteers un-
dergoing pelvic reconstructive surgery. In brief, vaginal Antigen presentation and T cell proliferation assay
tissue was collected immediately after the surgery in Bone marrow-derived dendritic cells (DCs) were pre-
Hanks Balanced Salt Solution supplemented with 25mM pared from bone marrow cells harvested from B6 mice by
HEPES, 2mM GlutaMAX, 2mM pyruvate, 200 IU/ml 8 days incubation in the presence of 50 ng/ml granulocyte-
penicillin, 200 g/ml streptomycin, 62.5 pg/ml Fungizone, macrophage colony-stimulating factor (Peprotech, Rocky
and 50 g/ml gentamicin (solution 1). The mucosa was Hill, New Jersey) in phenol red-free RPMI containing
mechanically removed from the vaginal tissue, dissected 10% FBS, 2mM GlutaMAX, 100 IU/ml penicillin, and
into 3 3 mm blocks and incubated in solution 1 sup- 100 g/ml streptomycin as described (45). Purified DCs
plemented with 1mM EDTA and 0.2 mg/ml dithiothreitol were incubated with OVA protein (100 g/ml) in the ab-
for 20 minutes with gentle agitation at 37C to remove sence or presence of hormones for 24 hours followed by
mucus. After two consecutive 30-minute incubations in irradiation (3000 rads). Cells were washed with media
solution 1 with 1mM EDTA, the tissue was digested with without antigen or hormones before co-incubation with T
0.6 mg/ml collagenase (type IV from C. histolyticum; cells. OVA-specific TCR-transgenic T lymphocytes were
Sigma) and 0.1 mg/ml deoxyribonuclease in phenol red- purified from spleens and lymph nodes of OT-2 mice using
free RPMI supplemented with 25mM HEPES, 2mM Glu- the Pan-T cell kit (Miltenyi). OT-2 T cells (5 104) were
taMAX, 2mM pyruvate, 200 IU/ml penicillin, 200 g/ml co-incubated with sequential dilutions of DCs in the pres-
streptomycin, 62.5 pg/ml Fungizone, and 50 g/ml gen- ence of 100 g/ml OVA and indicated hormonal regula-
tamicin for 30 minutes with agitation at 37C. Media were tors for 96 hours; T cell proliferation was determined by
collected, and the tissue was minced and incubated for an [3H]thymidine incorporation during the last 24 hours of
additional 45 minutes in collagenase/deoxyribonuclease- culture.
containing media. Mononuclear cells were purified by
density gradient centrifugation. The yield of VMMCs ob- pDC activation and intracellular cytokine staining
tained from remnant vaginal tissue typically ranged from assay
4 to 15 106. PBMCs (1.5 106 cells/ml) were preincubated for 6
hours in the presence or absence of hormones before the
Cytokine production in vitro stimulation with respective Toll-like receptor (TLR) li-
PBMCs, VMMCs, or purified CD3 T cells at 5 105 gands (5 g/ml TLR7/8 ligand R848 [Invivogen, San Di-
cells/ml or purified CD14 monocytes at 2.5 105 ego, California] or 2M TLR9 ligand CpG ODN2216
cells/ml were incubated for 24 hours in 200 l medium [Hycult Biotech, Uden, The Netherlands]). Immediately
containing hormones. Medium containing the final per- after the addition of the stimulant, 1 l of GolgiPlug
centage of ethanol of the highest hormone concentration (BD Biosciences, San Diego, California) was added. Af-
(0.1%) was used as vehicle control. PBMCs, VMMCs, and ter 20 hours, cells were collected and stained for pDC
purified T cells were activated with the T cell activation/ markers (CD123-PE-Cy7 [eBioscience] and CD303-
expansion kit MACSi beads coated with antibodies APC [BCDA-2; Miltenyi]). Cells were permeabilized using
against CD2/CD3/CD28 (Miltenyi) at a bead to cell ratio the Cytofix/Cytoperm kit (BD Biosciences) and stained
of 1:2 (PBMCs and VMMCs) or 1:1 (T cells). Blood-de- intracellularly with IFN-PE (BD Biosciences) and TNF-
rived monocytes were stimulated with 100 ng/ml lipopoly- fluorescein isothiocyanate monoclonal antibodies. Sam-

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Endocrinology, March 2013, 154(3):12821295 endo.endojournals.org 1285

ples were analyzed on the LSR-II flow cytometer (BD Bio- GraphPad Prism version 5 (GraphPad Software Inc, La
sciences), and data analysis was performed using the Jolla, California) statistical and graphing software pack-
FACSDiva (BD Biosciences) and Flowjo (Treestar, Inc, ages were used. The adjustments for multiple comparisons
Ashland, Oregon) software. of cytokine expression analysis were not applied because
that would result in an increase in the frequency of the null
Analysis of coreceptor expression on T cells hypothesis being valid (type II error) when the association
PBMCs (1.25 106 cells/ml) were incubated for 24 in the data is not a result of chance (47). The correction for
hours in media in the absence or presence of hormonal the type I error applies only to the universal null hypoth-
regulators at the indicated concentrations. Cells were ac- esis, that is, that the two groups are identical on all vari-
tivated with MACSi beads at a 1:2 bead to cell ratio for an ables analyzed (48). The concentration of IFN at each
additional 24 hours. Nonadherent cells were collected and assay condition was determined by independent assays
stained with anti-CD3-eFluor450, CD4-APC-eFluor780, (ELISA and Luminex), therefore, a valid association that
CD8-PerCP-CY5.5, CD27-PE-Cy7, CD45RO-PE (eBio- is not null. Furthermore, there is no empirical justification
science), CCR5-AlexaFluor 647, and CXCR4-APC anti- for a hypothesis that all the associations observed are un-
bodies (Biolegend, San Diego, California). Appropriate predictable manifestations of random processes (47).
isotype controls were used to determine the percentages of MPA causes systemic interdependent effects on cytokine
cells expressing the respective HIV-1 coreceptors. and chemokine expression and is likely to simultaneously
inhibit the production of multiple factors. Therefore, the
Virus infection in vitro null hypothesis does not apply, and an application of Bon-
The construction of the Renilla luciferase-expressing, ferroni adjustment would result in an amplification of the
replication-competent HIV-1 CXCR4 tropic virus NL- type II error (47).
LucR-T2A and CCR5 tropic virus NL-LucR-T2A
Bal.ecto was described previously (46). Viruses were
kindly provided by the University of Alabama Center for Results
AIDS Research virology core facility directed by Dr. J.
Kappes. Target cells were cultured in media supplemented MPA inhibits cytokine production by activated T
with 20 U/ml IL-2 (National Institutes of Health AIDS cells
Research and Reference Reagent Program; Germantown, To address whether MPA affects cytokine production
Maryland). 5 105 cells/ml of CD8-depleted PBMCs or by activated T cells, PBMCs from healthy women volun-
purified CD4 T cells (depleted of CD14 cells using teers were incubated with T cell-activating microbeads
CD14-specific beads; Miltenyi) were incubated for 1 or 5 coated with antibodies against CD2/CD3/CD28 (MACSi
days in media containing hormones. Cells were activated beads) in the presence of increasing concentrations of
with either MACSi beads at a bead to cell ratio of 1:2 for MPA, E2, P4, or DEX. As demonstrated in Figure 1A and
CD8-depleted PBMCs and 1:1 for purified CD4 T cells B, MPA exerts a significant inhibitory effect on the pro-
or with 2 g/ml phytohaemagglutinin-L (PHA-L). Virus duction of IFN by CD2/CD3/CD28-stimulated PBMCs
was added 24 hours after activation at a multiplicity of and VMMCs at concentrations 107M and higher. Inhi-
infection of 0.5. At day 4 after infection, fresh media were bition of IFN production was observed after stimulation
supplemented. At day 7, the cells were lysed in 1 lysis of purified blood CD3 cells, suggesting that MPA acts
buffer (Renilla luciferase kit; Promega, Madison, Wiscon- directly on T cells (Figure 1C). The suppressive effect of
sin). Lysates were subjected to 1 freeze-thaw cycle to aid MPA is not restricted to T cells because MPA also reduced
the lysis and stored at 80C. The level of viral infection the production of TNF by purified CD14 monocytes
and replication was determined as relative light units using after stimulation with TLR4 ligand bacterial LPS (Figure
Renilla luciferase assay and Victor 3V luminometer 1D). In contrast, E2 and P4 did not have a significant
(PerkinElmer, Waltham, Massachusetts). inhibitory effect on T cells or monocytes at concentrations
up to 106M. DEX displayed an inhibitory effect on cy-
Statistical analysis tokine production by PBMCs, VMMCs, and monocytes at
Data were analyzed using Students t test, Mann-Whit- concentration as low as 109M. To minimize the potential
ney rank sum test, and repeated-measures ANOVA test as influence of hormones present in serum and to avoid es-
appropriate. Correlations were performed using Spear- trogen receptor activation by phenol red in culture media,
man rank order test. A standard level of statistical signif- all experiments were performed in phenol red-free me-
icance .05 was used; all reported P values are two- dium supplemented with charcoal-dextrantreated FBS.
sided. SigmaStat version 3.1 (SPSS, Chicago, Illinois) and However, the inhibition by MPA was independent of the

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1286 Huijbregts et al Hormonal Contraception and HIV-1 Endocrinology, March 2013, 154(3):12821295

A PBMC
B VMMC
E PBMC F VMMC
1.5 1.5 < 0.0001 0.0008 0.006 0.014
1.0 1.0

1.0 1.0

IFN
*

IFN
* * *
IFN

IFN
* *
0.5 0.5
0.5
* * 0.5
*
* * * * 0.0 0.0
0.0 0.0 RU MPA MPA DEX DEX RU MPA MPA DEX DEX
Veh -9 -8 -7 -6 Veh -9 -8 -7 -6 + RU + RU + RU + RU
x x Veh
Hormone [10 M] Hormone [10 M]
E2
P4
C CD3+ cells
D CD14+ cells MPA
G CD3+ cells H CD14+ cells
1.5 1.5 n.s. 0.018 0.017 0.015
DEX 1.0 1.0

TNF
1.0 1.0

IFN
TNF
IFN

0.5 0.5
0.5 * * 0.5

* *
0.0
* 0.0
* * 0.0
RU MPA MPA DEX DEX
0.0
RU MPA MPA DEX DEX
Veh -9 -8 -7 -6 Veh -9 -8 -7 -6 + RU + RU + RU + RU
Hormone [10 x M] Hormone [10 x M]

I IFN IL-2 IL-4 IL-6 IL-12(p40)


1.5

**

**
**

**
1.0

*
*

***
*
***

*
***
**
Relave concentraon

***
***

0.5

0.0
-8 -7 -6 -8 -7 -6 -8 -7 -6 -8 -7 -6 -8 -7 -6 -8 -7 -6 -8 -7 -6 -8 -7 -6 -8 -7 -6 -8 -7 -6 -8 -7 -6 -8 -7 -6 -8 -7 -6 -8 -7 -6 -8 -7 -6

IL-12(p70) IL-13 IL-17 MIP-1 TNF


1.5

1.0
**
*

***
**
*

***
*
***

***

0.5

0.0
-8 -7 -6 -8 -7 -6 -8 -7 -6 -8 -7 -6 -8 -7 -6 -8 -7 -6 -8 -7 -6 -8 -7 -6 -8 -7 -6 -8 -7 -6 -8 -7 -6 -8 -7 -6 -8 -7 -6 -8 -7 -6 -8 -7 -6

Hormone [10x M] E2 P4 MPA


Figure 1. The effect of estrogen (E2), P4, and MPA on cytokine production by activated mononuclear cells. AD, PBMCs (A), VMMCs (B), purified
CD3 T cells (C), or CD14 monocytes (D) obtained from healthy female volunteers were incubated for 24 hours in the presence of a vehicle (Veh)
or indicated concentrations of E2, P4, MPA, or DEX and activated for an additional 24 hours with microbeads (MACSi) coated with antibodies
against CD2/CD3/CD28 (AC) or with 100 ng/ml LPS (D). Graphs represent the concentration of IFN or TNF in the culture media. Data are
normalized to vehicle control (Veh); mean SEM of 5 (A) or 3 (BD) donors is shown. *, Statistically significant difference of MPA- and DEX-
treated samples compared with the P4-treated samples. EH, Inhibitory effect of MPA is reversed by RU486 (RU). Experimental conditions and
analysis are as described in A except that the cells were incubated with MPA or DEX in the presence of RU486 (all reagents at 106M). Mean
SEM of normalized data is presented. I, Effect of sex steroid hormones and MPA on the production of cytokines by CD2/CD3/CD28-stimulated
PBMCs. Data are presented as normalized values relative to control of 5 independent experiments; error bars depict SEM. Statistically significant
differences compared with vehicle control are indicated: * P .05; ** P .01; *** P .001.

type of serum or medium, and a similar effect was observed (Figure 1, EH). In contrast, the inhibition mediated by DEX
in the presence of normal human serum (Supplemental Fig- was only partially reversed by RU486.
ure 1, A and B, published on The Endocrine Societys Jour- Importantly, the inhibitory effect of MPA on cytokine
nals Online web site at http://endo.endojournals.org). An im- production is not limited to IFN and TNF. As shown on
munosuppressive effect of MPA but not P4 was also specific examples in Figure 1I and in the heat map repre-
observed in experiments using antigen-specific stimulation sentation in Figure 2, a significant reduction of the pro-
withHIV-1GagpeptidepoolandPBMCsfromHIV-1-infected duction of IL-2, IL-3, IL-4, IL-5, IL-9, IL-12 (p40 and
individuals (Supplemental Figure 1C). The MPA-mediated in- p70), IL-13, sIL-2ra, and CD154 (sCD40L) by PBMCs
hibition of cytokine production by T cells and monocytes was incubated with T cell-activating microbeads was observed
fully reversed in the presence of equimolar concentrations of in the presence of MPA at concentration 107M and
RU486, an antagonist of GR and progesterone receptor (PR) higher. Other cytokines and chemokines including

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Endocrinology, March 2013, 154(3):12821295 endo.endojournals.org 1287

PBMC VMMC
EGF 1.00 0.91 1.08 1.05 0.99 1.09 0.87 0.97 0.97 0.68 EGF 1.00 1.10 1.74 1.49 2.07 1.93 1.92 1.31 1.72 2.38
Eotaxin 1.00 0.97 1.13 1.09 0.99 1.11 1.08 1.00 1.08 0.98 Eotaxin 1.00 1.09 1.13 1.29 1.32 1.13 1.20 1.10 1.24 1.21
FGF-2 1.00 0.97 1.12 0.96 0.98 1.03 0.85 0.96 0.89 0.82 FGF-2 1.00 1.00 1.04 1.03 1.09 0.98 1.02 1.01 1.01 1.09
Flt Ligand 1.00 1.00 1.01 0.99 0.96 1.03 0.97 0.98 0.97 0.84 Flt Ligand 1.00 0.99 1.09 1.13 *1.21 1.07 1.05 1.06 1.08 0.98
Fractalkine 1.00 0.89 1.08 1.00 0.86 1.00 0.98 0.98 0.95 0.80 Fractalkine 1.00 1.16 1.23 1.31 1.51 1.20 1.23 1.17 *1.39 1.10
G-CSF 1.00 0.84 1.16 0.97 1.00 0.94 1.02 1.19 1.05 0.61 G-CSF 1.00 1.06 1.22 1.03 1.24 1.01 0.96 0.92 0.81 0.79
GM-CSF 1.00 1.05 1.14 1.00 0.90 1.04 0.87 0.98 *0.69 *0.45 GM-CSF 1.00 1.34 1.52 *1.23 1.38 1.21 1.09 1.09 0.83 *0.63
GRO 1.00 0.92 0.90 0.88 0.91 0.90 0.89 0.90 0.99 1.01 GRO 1.00 *1.20 1.57 1.16 1.36 1.12 1.07 1.15 1.14 1.04
IFN 2 1.00 0.95 1.01 0.94 0.88 1.05 0.95 1.08 0.96 0.72 IFN 2 1.00 1.08 1.45 1.27 1.88 1.23 1.36 *1.17 1.30 1.42
IFN 1.00 1.12 1.07 0.94 0.90 0.98 1.07 0.96 *0.66 *0.39 IFN 1.00 1.15 1.24 1.04 1.20 0.92 0.94 0.92 0.65 *0.42
IL-1 1.00 1.01 1.02 1.03 0.85 1.04 0.89 0.92 0.69 *0.54 IL-1 1.00 0.99 0.98 0.91 1.18 0.84 0.82 1.00 0.83 0.84
IL-1 1.00 1.12 1.13 1.08 1.28 1.30 1.16 1.35 0.83 *0.51 IL-1 1.00 1.04 1.21 1.11 0.87 1.13 1.14 1.01 0.82 0.76
IL-1ra 1.00 0.93 1.06 0.93 0.86 1.10 0.89 0.97 0.81 0.66 IL-1ra 1.00 1.35 1.43 1.47 1.55 1.38 1.25 1.22 1.44 1.16
IL-2
IL 2 1.00 1.07 1.00 0.85 0.90 0.92 *0.72
0.72 0.89 *0.48
0.48 *0.20
0.20 IL-2 1 00
1.00 1 10
1.10 1 23
1.23 1 18
1.18 1 16
1.16 1 23
1.23 1 03
1.03 0 96
0.96 *0
0.74
74 *0
0.37
37
IL-3 1.00 1.04 1.13 1.09 1.02 0.89 0.73 0.86 *0.50 *0.25 IL-3 ND ND ND ND ND ND ND ND ND ND
IL-4 1.00 0.93 0.93 0.86 0.89 *0.78 *0.68 *0.80 *0.63 *0.29 IL-4 ND ND ND ND ND ND ND ND ND ND
IL-5 1.00 0.85 0.78 *0.52 0.82 0.77 *0.61 *0.55 *0.35 *0.14 IL-5 1.00 1.14 1.40 1.24 0.80 1.09 0.88 0.86 *0.61 *0.61
IL-6 1.00 1.01 1.21 0.99 0.95 0.96 0.91 1.16 0.87 *0.43 IL-6 1.00 1.05 1.25 1.09 1.27 1.13 0.93 0.87 0.75 0.65
IL-7 1.00 0.96 1.04 1.03 0.89 0.98 0.91 1.03 0.88 *0.73 IL-7 1.00 1.14 1.14 1.30 1.59 1.25 1.19 1.12 1.20 1.04
IL-8 1.00 1.02 0.96 1.02 1.09 1.13 0.94 1.00 1.01 0.80 IL-8 1.00 1.00 1.26 1.22 1.41 *1.30 1.66 0.95 1.08 0.89
IL-9 1.00 1.05 1.11 0.90 0.82 0.95 0.77 0.79 *0.47 *0.35 IL-9 1.00 1.18 1.16 1.01 0.96 1.01 0.90 0.98 *0.80 0.83
IL-10 1.00 1.24 1.16 1.11 1.15 1.13 1.20 1.37 1.09 0.78 IL-10 1.00 0.94 1.03 1.00 0.97 0.97 0.97 0.96 0.93 0.84
Il-12(p40) 1.00 *0.80 1.00 0.88 0.92 0.91 0.84 0.96 *0.68 *0.24 Il-12(p40) ND ND ND ND ND ND ND ND ND ND
IL-12(p70) 1.00 0.72 1.00 *0.74 0.84 *0.75 1.15 0.95 *0.62 *0.23 IL-12(p70) ND ND ND ND ND ND ND ND ND ND
IL-13 1.00 1.10 1.01 0.87 1.03 1.01 0.87 0.88 *0.58 *0.21 IL-13 1.00 1.24 1.22 *0.82 1.07 0.83 *0.54 0.98 0.52 *0.42
IL-17 1.00 1.26 1.21 1.15 1.08 1.14 0.94 1.14 0.78 *0.54 IL-17 1.00 1.17 1.15 1.09 1.20 1.18 1.17 1.00 0.96 0.76
IP-10 1.00 0.88 0.78 0.88 *0.69 *0.75 0.76 *0.72 0.88 1.10 IP-10 1.00 0.96 0.84 0.77 1.15 *0.66 0.75 1.13 0.87 0.82
MCP-1 1.00 0.97 0.93 1.02 0.84 0.84 0.80 0.83 0.96 1.06 MCP-1 1.00 1.14 1.05 1.37 1.37 1.22 1.13 1.14 1.11 1.19
MCP-3 1.00 0.98 1.19 1.04 1.01 1.05 0.96 0.89 1.13 0.95 MCP-3 1.00 0.95 *0.86 0.92 0.97 0.79 0.83 0.95 *0.84 *0.86
MDC 1.00 0.98 0.99 0.97 *0.65 *0.66 *0.69 *0.66 *0.42 *0.23 MDC 1.00 1.13 1.22 1.14 1.27 1.08 1.08 *1.18 0.94 0.79
MIP-1 1.00 0.96 0.96 0.86 0.76 0.87 0.80 1.17 0.74 *0.44 MIP-1 1.00 0.97 1.05 1.16 1.05 1.09 1.08 1.04 0.93 0.85
MIP-1 1.00 0.92 0.90 0.89 0.81 0.84 0.78 0.88 0.81 *0.66 MIP-1 1.00 0.96 1.22 *1.05 *1.10 1.06 1.11 1.00 1.02 0.93
sCD40L 1.00 0.90 0.98 0.85 0.83 0.87 *0.70 0.83 *0.61 *0.48 sCD40L 1.00 1.38 1.48 1.40 1.51 1.13 1.16 *1.28 1.13 1.05
sIL-2R 1.00 0.98 0.98 0.92 0.90 0.92 0.86 0.93 0.84 *0.56 sIL-2R ND ND ND ND ND ND ND ND ND ND
TGF 1.00 1.04 1.16 1.04 1.09 1.12 1.05 1.15 0.91 0.73 TGF 1.00 *1.13 1.24 1.31 1.28 1.10 1.10 1.00 1.14 1.07
TNF 1.00 1.21 1.07 1.02 0.96 1.09 0.85 0.97 *0.68 *0.41 TNF 1.00 1.09 1.21 1.18 1.12 1.10 1.08 1.00 0.88 0.61
TNF 1.00 0.92 1.03 *0.81 0.88 0.97 0.86 0.93 *0.66 *0.37 TNF 1.00 1.18 1.31 1.27 1.42 1.14 1.18 1.03 1.10 0.90
VEGF 1.00 0.97 1.14 1.07 0.92 1.09 1.02 1.06 1.00 0.86 VEGF 1.00 1.05 1.10 1.22 1.21 1.24 1.18 1.11 1.22 1.07
Veh 10-88 10-77 10-66 10-88 10-77 10-66 10-88 10-77 10-66 Veh 10-8 10-7 10-6 10-8 10-7 10-6 10-8 10-7 10-6

E2 P4 MPA E2 P4 MPA

0 1 2
Figure 2. Heat map representation of the effect of MPA and sex steroid hormones on the production of cytokines and chemokines by PBMCs
activated with a T cell-activating stimulus. PBMCs or VMMCs were activated with antiCD2/CD3/CD28-coated microbeads, and the accumulation
of cytokines and chemokines in the medium was analyzed as described in Figure 1. The concentration of factors in the media was determined
using 39-plex MILLIPLEX MAP assay. Data are presented as normalized values relative to vehicle; means of 5 and 3 independent experiments for
PBMCs and VMMCs, respectively, are presented. *, Statistically significant difference compared with vehicle control.

MIP-1 and -, IL-1 and -, and IL-6 were inhibited in by P4 and synthetic progestins (49 51). To characterize
the presence of 106M MPA. Incubation with P4 at the effect of MPA on pDC function, intracellular cytokine
107M and higher led to a reduced production of IL-4, staining by multiparameter flow cytometry was used to
IL-12 (p70), interferon gamma-induced protein 10 (IP-10; quantify the percentage of IFN- and TNF-producing
CXCL10), and monocyte-derived cytokine (MDC), pDCs after stimulation with TLR7/8 and TLR9 ligands.
whereas estrogen did not exert any discernible effect. PBMCs were preincubated with E2, P4, MPA, or DEX for
MPA inhibited the production of IL-2, IL-13, and other 6 hours before stimulation with TLR7/8-specific ligand
cytokines by VMMCs; however, a greater variability was R848 or TLR9-specific ligand CpG in the presence of brefel-
observed in assays employing the cells isolated from genital din A as described (50). After 20 hours of stimulation, the
tissues (Figure 2). intracellular production of IFN and TNF in pDCs defined as
CD123CD303 cells was determined (Figure 3A). As dem-
MPA inhibits cytokine production by activated pDCs onstrated in Figure 3B, both MPA and DEX inhibit R848- and
Several reports have suggested that the activation of CpG-induced IFN and TNF production by stimulated
pDCs, a cell population playing a key role in the early pDCs. In contrast, P4 or E2 did not exert any effect on cytokine
recognition of viral and bacterial infections, is modulated production by pDCs after TLR stimulation.

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1288 Huijbregts et al Hormonal Contraception and HIV-1 Endocrinology, March 2013, 154(3):12821295

A Ctrl R848 R848/MPA


B 1.5
R848
1.5
R848
10-8 M
1.25% 41.9% 17.6%

TNF + pDCs
IFN + pDCs
10-6 M

*
1.0 1.0
SSC-A

**
*
***
0.5 0.5

*
**
***
0.0 0.0
FSC-A Veh E2 P4 MPA DEX Veh E2 P4 MPA DEX
IFN
4 1.5
2.16% 74.9% 40.5% CpG CpG
3
CD123

+ pDCs
IFN + pDCs
1.0

TNF

**
0.5

**
*
1
CD303

*
TNF 0 0.0
Veh E2 P4 MPA DEX Veh E2 P4 MPA DEX

C Plasma CVL D
200 30 p = 0.034
DC:T cell ratio 1:2 DC:T cell ratio 1:8
p = 0.032 150000 150000
150 10-8 M
IFN (pg/ml)
IFN (pg/ml)

20
10-6 M

***
100000 100000

***
100

CPM

CPM

*
10

*
***
***
50 50000 50000

***

***
0 0
0 0
Control DMPA COC Control DMPA COC Veh E2 P4 MPA DEX Veh E2 P4 MPA DEX

Figure 3. MPA inhibits cytokine production by activated pDCs. A, Gating strategy for the analysis of intracellular production of IFN and TNF by pDCs
that were either unstimulated (control [Ctrl]), stimulated with R848 (5 g/ml) or a combination of R848 and MPA (106M). B, Effect of E2, P4, MPA, and
DEX at 108M (black bars) or 106M (white bars) on intracellular production of IFN and TNF in pDCs. Data from 3 independent donors normalized to
vehicle (Veh) control SEM are presented. C, IFN levels in plasma and CVL of women without any form of hormonal contraception (Control), women
using DMPA, and women using combined oral contraceptives (COC). D, Effect of E2, P4, MPA, and DEX on antigen-induced T cell proliferation. Purified
mouse DCs were incubated with 100 g/ml OVA in the presence of hormonal regulators for 24 hours followed by 4 days incubation with OVA and
purified transgenic OVA-specific OT-2 T cells in the presence of hormonal regulators at the indicated DC to T cell ratios. Cell proliferation was determined
by incorporation of [3H]thymidine during the last 24 hours of culture. Data are shown as mean SEM. Statistically significant differences compared with
vehicle control are indicated: * P .05; ** P .01; *** P .001 (analyzed using paired t test [B], Mann-Whitney U test [C], and unpaired t test [D]).

|DMPA use is associated with lower levels of IFN OVA-specific TCR-transgenic OT-2 T cells at indicated DC
in plasma and CVL to T cell ratios in the presence or absence of hormonal reg-
pDCs represent a major source of IFN in vivo (52, 53). ulators. Both MPA and DEX significantly inhibited T cell
To assess whether the use of DMPA is associated with proliferation after antigen presentation by DCs (Figure 3D).
changes in the systemic and local levels of IFN, plasma Preincubation of DCs with MPA and DEX followed by care-
and CVL from 21 women not using any form of hormonal ful washing and antigen presentation in the absence of hor-
contraception, 19 women using DMPA, and 14 women monal regulators in the culture medium resulted in a lower but
using combined oral contraception were analyzed. As significant inhibition of T cell proliferation (Supplemental Fig-
shown in Figure 3C, use of DMPA was associated with ure 2). These results indicate that MPA inhibits the level of T cell
significantly lower levels of IFN in plasma (P .032) and proliferation after antigen presentation both directly and indi-
CVL (P .034). In the control group without any form of rectly via an effect on antigen-presenting cells.
hormonal contraception, no significant difference in IFN
levels in plasma or CVL was detected between women in MPA prevents activation-induced down-regulation
the follicular vs luteal phase of the ovarian cycle. of surface expression of HIV-1 coreceptors CXCR4
and CCR5 on T cells
MPA suppresses T cell proliferation after antigen It was previously reported that the expression of HIV-1
presentation coreceptor CCR5 on the surface of T cells is modulated by
To address whether MPA or sex steroid hormones affect endogenous levels of P4 as well as by progestins delivered
the capacity of antigen-presenting cells to induce antigen- as a component of hormonal contraception (54, 55). To
specific T cell proliferation, a TCR-transgenic mouse model address whether MPA affects the levels of HIV-1 corecep-
was employed. Bone marrow-derived DCs from B6 mice tors on T cells, PBMCs were incubated with MPA or sex
were preloaded with OVA in the presence of E2, P4, MPA, steroid hormones for 24 hours followed by incubation
or DEX for 24 hours and then co-incubated with purified with T cell-activating antiCD2/CD3/CD28-coated mi-

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A CD4+ T cells B CD8+ T cells

unstim -CD2/CD3/CD28 unstim -CD2/CD3/CD28


Veh Veh P4 MPA DEX Veh Veh P4 MPA DEX
16741 5376 6334 31103 36378 26310 4260 4975 24079 38789
Tcm

26326 4841 5658 29771 36096 22591 2170 2747 17771 30995
Tem

CXCR4 CXCR4
476 180 167 266 325 1142 259 244 505 650
Tcm

2216 861 804 1187 1188 2126 552 522 1113 1311
Tem

CCR5 CCR5
Figure 4. MPA prevents activation-induced down-regulation of HIV-1 coreceptors on the surface of T cells. PBMCs were incubated for 24 hours with
vehicle (Veh), P4, MPA, or DEX at 106M before activation with antiCD2/CD3/CD28-coated microbeads at a bead to cell ratio 1:2 for 24 hours. A and B,
Expression of HIV-1 coreceptors CXCR4 and CCR5 on central memory (Tcm) and effector memory (Tem) subsets of CD4 (A) and CD8 (B) T cells is
shown. Histogram overlays indicate the staining with isotype control (shaded) and CXCR4 or CCR5 antibody (thick line) on unstimulated (unstim) T cells
or T cells stimulated with antiCD2/CD3/CD28-coated microbeads. Results of one of three similar experiments are shown.

crobeads for an additional 24 hours. Expression of CCR5 PHA-L for 24 hours. Activated cells were infected with
and CXCR4 was analyzed on subsets of naive, central either a CCR5-tropic (BaL-LucR) or a CXCR4-tropic
memory, effector memory, and late effector T cells defined (NL4.3-LucR) recombinant HIV-1 virus encoding Renilla
by surface expression of CD27 and CD45RO (gating luciferase, a sensitive indicator of HIV-1 replication. Lu-
strategy is depicted in Supplemental Figure 3). As demon- ciferase activity in cell lysate was determined at 4 and 7
strated in Figure 4 and previously reported (56, 57), ac- days after infection. The presence of MPA at 106M re-
tivation of T cells via CD3 resulted in a significant down- sults in a significant increase in luciferase activity in both
regulation of CXCR4 on the surface of both CD4 and CD8-depleted PBMCs and purified CD4 T cells infected
CD8 T cells. Importantly, the down-regulation was fully with CCR5- or CXCR4-tropic viruses (Figure 5 and Sup-
reversed in the presence of MPA during activation. A sim- plemental Figure 5). In contrast, treatment with E2 or P4
ilar trend was observed for CCR5, although the effect of did not result in an altered rate of viral proliferation.
MPA on CCR5 expression after in vitro activation was less Treatment with DEX increased HIV-1 proliferation at
pronounced (Figure 4 and Supplemental Figure 4). 108M but led to a decreased viral proliferation at 106M
likely due to the suppression of T cell proliferation.
MPA enhances HIV-1 proliferation in in vitro culture
Previous studies suggested that E2 and P4 regulate the
rate of HIV-1 replication in peripheral blood cells (58) and Discussion
that glucocorticoid agonists accelerate the rate of HIV-1
transcription (59-62). To address whether MPA exerts an The effect of hormonal contraception on HIV-1 acquisi-
effect on HIV-1 infection and replication in vitro, CD8- tion and transmission represents an important global pub-
depleted PBMCs or purified CD4 T cells depleted of lic health issue with profound implications for policies on
CD14 monocytes were incubated in the presence of ste- family planning in countries with high HIV-1 risk. In this
roid hormones or MPA for 1 or 5 days before activation study, we addressed the mechanisms by which MPA may
with either antiCD2/CD3/CD28-coated microbeads or affect the acquisition of HIV-1 and other infections. Char-

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1290 Huijbregts et al Hormonal Contraception and HIV-1 Endocrinology, March 2013, 154(3):12821295

***
A BaL / MACSi NL4.3 / MACSi BaL / PHA-L NL4.3 / PHA-L

***
**

**
Luciferase activity (RLU)

Luciferase activity (RLU)


Luciferase activity (RLU)
800000

Luciferase activity (RLU)


600000 400000 1.5 10
1500000 06

*
***
*
600000 300000
400000 1000000

***
*
400000

*
200000

**
***
***

*
200000 500000

*
200000 100000

0 0 0 0
Veh -8 -6 -8 -6 -8 -6 -8 -6 Veh -8 -6 -8 -6 -8 -6 -8 -6 Veh -8 -6 -8 -6 -8 -6 -8 -6 Veh -8 -6 -8 -6 -8 -6 -8 -6
E2 P4 MPA DEX E2 P4 MPA DEX E2 P4 MPA DEX E2 P4 MPA DEX

B BaL / MACSi NL4.3 / MACSi BaL / PHA-L NL4.3 / PHA-L

***
**
Luciferase activity (RLU)
1.5
1500000
10
06 400000 800000

Luciferase activity (RLU)

Luciferase activity (RLU)


Luciferase activity (RLU)

150000
**

**
**

*
300000 600000
100000 1000000
*

**
200000 400000

***
50000 500000
100000 200000

0 0 0 0
Veh -8 -6 -8 -6 -8 -6 -8 -6 Veh -8 -6 -8 -6 -8 -6 -8 -6 Veh -8 -6 -8 -6 -8 -6 -8 -6 Veh -8 -6 -8 -6 -8 -6 -8 -6
E2 P4 MPA DEX E2 P4 MPA DEX E2 P4 MPA DEX E2 P4 MPA DEX

10-8 M 10-6 M
Figure 5. MPA enhances the replication of HIV-1 in in vitro cell culture. A and B, CD8-depleted PBMCs (A) or purified CD4 T cells depleted of
CD14 cells (B) were incubated in the presence of hormonal regulators at the indicated concentration for 5 days before activation with either anti
CD2/CD3/CD28-coated microbeads (MACSi; bead to cell ratio of 1:2 for PBMCs and 1:1 for CD4 T cells) or 2 g/ml PHA-L for 24 hours. CCR5-
tropic (Bal) or CXCR4-tropic (NL4.3) virus encoding Renilla luciferase was added at a multiplicity of infection of 0.5, and the cells were cultured for
an additional 7 days before analysis. Luciferase activity is expressed as relative light units (RLU). The results of a representative experiment of 4
similar experiments are shown. Error bars depict SD. Statistically significant difference compared with vehicle control are indicated: * P .05;
** P .01; *** P .001 (analyzed using unpaired t test).

acterization of the effect of MPA on innate and adaptive in HIV-1 acquisition in women using high-dose DMPA
immune mechanisms as a function of concentration indi- contraception. Our observations are consistent with pre-
cated that MPA suppresses both arms of the immune sys- vious reports demonstrating that MPA suppresses the pro-
tem at a significantly lower concentration than unmodi- duction of IL-1, IL-12, IL-10, IL-13, and granulocyte
fied P4. At a concentration of 107M (100nM) and higher, colony-stimulating factor by Bacillus CalmetteGurin
MPA inhibited the production of IFN, IL-2, IL-4, IL-12, antigen-stimulated human PBMCs (44) and that admin-
IL-13, TNF, soluble CD40 ligand, and other cytokines istration of DMPA to mice reduces IFN production by
and chemokines by T cells activated via the TCR and the pDCs (51).
production of TNF by monocytes activated by LPS via The critical question is whether the concentration at
the CD14/MD-2/TLR4 complex (Figures 1 and 2). MPA which MPA exerts its immunosuppressive properties in
suppressed the proliferation of T cells after antigen pre- vitro is relevant to the physiological concentration in
sentation and, importantly, reduced the ability of pDCs to women using DMPA. Pharmacokinetic studies show that
respond to stimulation via TLR7/8 and TLR9 by produc- after administration of 150 mg DMPA by I.M. injection,
tion of IFN and TNF (Figure 3). The suppressive effect the serum concentration of MPA reaches up to 6.5
of MPA on IFN production is further supported by the 108M (25 ng/ml) within days after injection and de-
data demonstrating lower systemic and genital secretion creases to about 0.3 to 2 108M (1-9 ng/ml) in the
levels of IFN in women using DMPA (Figure 3C). pDCs following weeks (37, 64-67). The immunosuppressive ef-
are a key immune population functioning as a sentinel fect of MPA in vitro is generally observed at concentra-
recognizing early viral and bacterial infections and induc- tions equal and higher than the peak concentration de-
ing antiviral responses via a release of factors such as tected in plasma of DMPA-using women (107M or 38
class-I IFNs (52, 53). IFN-producing pDCs accumulate ng/ml); however, alteration of cytokine production was
beneath the genital epithelium at 1 day after SIV infection observed at concentrations as low as 108M in some in-
in rhesus macaques (63). Active suppression of central dividuals. Importantly, long-term exposure of immune
immune mechanisms, including the function of pDCs, an- cells and tissues to MPA in vivo is likely to exert a signif-
tigen presentation, and T cell effector mechanisms, may icant effect at lower concentrations (51). Furthermore, the
tip the balance between the proliferation of a founder viral effect of MPA may be enhanced locally in tissues such as
population and immune control at an early stage of infec- the genital mucosa due to the differences in the expression
tion toward the benefit of the virus. This may be the un- of GR and PR and the presence of cell types with increased
derlying mechanism contributing to the observed increase sensitivity to GR- and PR-mediated signaling (37). A po-

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Endocrinology, March 2013, 154(3):12821295 endo.endojournals.org 1291

tential limitation of the studies using VMMCs is that the eration at 1:8 DC to T cell ratio at 106M. Differences
cells were purified from the mucosa of postmenopausal between our study and previously published studies may
women. Cell type frequencies and properties of mucosa- be caused by variances in experimental conditions and
populating cells may differ between pre- and postmeno- methods of T cell activation. Concurrent stimulation of
pausal women. Because the data obtained in vitro cannot various steroid receptors may result in a synergistic or
be directly translated to the situation in vivo due to the antagonistic effect (58). In our experiments, the presence
variances in the dosage, pharmacokinetics, effect of serum of a combination of E2 and P4 at midproliferative
hormone-binding proteins, and length of exposure of im- (1010M and 109M) or midsecretory levels (109M and
mune cells, it is critical to study the effect of MPA and 107M) did not result in a change in the production of
other progestins on the immune system in vivo. In this IFN by activated PBMCs or modify the suppressive effect
respect, the observation of reduced levels of IFN in cir- of MPA (data not shown).
culation and CVL of women using DMPA (Figure 3C) Our data show that estrogen or progesterone at con-
represents important evidence of an effect of DMPA on centrations of up to 106M had no effect on TLR-stimu-
immune system in vivo. lated production of IFN and TNF by pDCs (Figure 3).
It is likely that MPA exerts its immunosuppressive This is consistent with a previous study demonstrating
properties via the engagement of the GR (36 40, 42). that P4 inhibits IFN production by CpG-stimulated pu-
Activated GR was shown to regulate the expression of a rified human pDCs at a concentration of 20 g/ml (6.4
number of cytokines and proinflammatory factors via a 105M), whereas no inhibition was observed at 0.2 g/ml
direct interaction with nuclear factor B (NFB) and ac- (6.4 107M) (51). Interestingly, pDCs isolated from
tivator protein-1 (AP-1), modification of the basal tran- mice treated with DMPA produced lower levels of IFN
scriptional machinery, chromatin remodeling, and repres- upon CpG stimulation and displayed lower levels of serum
sion of the induction of NFB inhibitor IB resulting in IFN after vesicular stomatitis virus infection (51). These
transrepression (68 72). The GR-ligand complex de- data are in concordance with the observation of decreased
creases the activity of the Th1 transcription factor T-box systemic and cervicovaginal levels of IFN in women us-
expressed in T cells (T-bet) and Th2 transcription factor ing DMPA described here. It has been reported that IFN
GATA-binding protein-3 (GATA-3) (73, 74). Interest- production upon stimulation with synthetic HIV-1 de-
ingly, HIV-1-infected patients display glucocorticoid hy- rived TLR7/8 ligand is higher in pDCs isolated from
persensitivity associated with reduced T cell function (75). women than men (49, 50). Estrogen signaling was not
HIV-1 accessory protein Vpr binds to the steroid receptor responsible for this effect (49), and a positive correlation
coactivator motif LXXLL on the GR, leading to enhanced was observed between plasma P4 levels (up to 5 108M)
GR activation and suppression of production of IL-12 and and IFN production by pDCs in women (50). We did not
other cytokines (72, 76). Thus, the sensitivity of immune observe an effect of P4 on IFN production by pDCs at
cells to the effect of MPA may be significantly enhanced in these concentrations.
the context of HIV-1 infection. In contrast to MPA, P4 Previously, it was reported that the use of oral hor-
does not efficiently activate the GR signaling cascade, and monal contraceptives is associated with increased expres-
its effect on immune processes is less pronounced. How- sion of HIV-1 coreceptor CCR5 on intraepithelial endo-
ever, it cannot be excluded that the effect of MPA is par- cervical CD4 T lymphocytes (54) and that the increased
tially mediated via the PR pathway because MPA has level of P4 in pregnancy directly correlates with CCR5
higher affinity to PR than P4 (37). Previous studies have expression on T cells in peripheral blood and genital tissue
demonstrated that P4 affects T cell-mediated immune (55). We show that T cells activated in the presence of
mechanisms by inhibition of cytokine production and cy- MPA display higher levels of surface HIV-1 coreceptors
tolytic activity (4, 77 85) and promotion of the expres- than either untreated or P4-treated cells (Figure 4). Our
sion of Th2-type cytokines (86 93). Estrogen was shown data are consistent with the observation that CXCR4 sur-
to exert a stimulatory or inhibitory effect on the expression face levels on T cells in cultured PBMCs are up-regulated
of a number of cytokines depending on the concentration under nonstimulating conditions within several hours of
and experimental system (4, 94 97). Here we show that culture and significantly contract after T cell activation
P4 at concentration 107M reduces the levels of IL-4, using anti-CD3 antibodies or PHA, leading to a decreased
IL-12 p70, IP-10 and MDC production after nonspecific rate of replication of CXCR4-tropic virus (98). Down-
stimulation of T cells. Treatment with estrogen at 106M modulation of surface CXCR4 after CD3 engagement is
resulted in reduced levels of IL-5, IL-12 p70, and TNF; no caused by endocytosis through clathrin-coated pits involv-
effect was discernible at 107M. P4 but not estrogen ing protein kinase C (PKC) signaling (56, 57). Down-mod-
caused a slight reduction of antigen-specific T cell prolif- ulation of CCR5 can occur via both clathrin-dependent

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1292 Huijbregts et al Hormonal Contraception and HIV-1 Endocrinology, March 2013, 154(3):12821295

and independent pathways (57, 99). The precise molecular cult to interpret (34, 35); however, the identification of
mechanisms of the effect of MPA on the endocytosis and biological mechanisms underlying the observed associa-
recycling of CXCR4 and CCR5 are unclear. The observed tion constitutes a strong argument against the use of high-
retention of surface coreceptor levels on activated T cells dose injectable DMPA in women at high risk of HIV-1
in the presence of MPA may explain the previous obser- infection. Importantly, immediate withdrawal of DMPA
vation of increased CCR5 expression on CD4 T cells in from family planning programs without offering other ef-
women using hormonal contraception (54). fective forms of contraception is not warranted because it
MPA at 106M resulted in a significant increase in the could result in a sharp increase in unwanted births and
rate of proliferation of CCR5- and CXCR4-tropic viruses maternal and infant mortality (110). At the current time,
in CD8 T cell-depleted PBMCs and purified CD4 T cells the risk of DMPA discontinuation without a switch to a
(Figure 5 and Supplemental Figure 5). The observed in- safe and effective method of contraception far outweighs
crease in HIV-1 proliferation may be caused by enhanced the risk associated with continuous DMPA use because in
retention of HIV-1 coreceptors on the surface of CD4 T some regions, up to 9 additional maternal deaths will oc-
cells as suggested by our data and reported previously cur for every case of HIV-1 averted (41). Women using
(98), by inhibition of production of antiviral factors, or by DMPA or other forms of hormonal contraception should
a direct effect on HIV-1 transcription. In this respect, we be strongly advised to use condoms, male or female, as
show that MPA reduced the levels of MIP-1 and - recommended by recent World Health Organization
(CCL3 and CCL4), major HIV-suppressive factors (100, guidelines (1). DMPA should be gradually replaced with
101), in activated PBMC cultures (Figure 2). It has been alternative modern methods of contraception such as con-
reported that MIP-1 and other CCR5 ligands induce en- traceptives with a lower impact on the immune system
docytosis of CCR5 and decrease infectability with CCR5- (41).
tropic HIV-1 (102104). The enhancement of HIV-1 pro-
liferation may be also mediated by direct binding of
activated GR-MPA complex to glucocorticoid response Acknowledgments
elements located within the HIV-1 long-terminal repeat
(105, 106). The data reported here are consistent with University of Alabama Center for AIDS Research Virology Core
led by Dr. J. Kappes was instrumental in the preparation of len-
previous reports demonstrating that HIV-1 replication
tiviral vectors. We thank Dr. J. Mestecky for critical reading of
can be stimulated by various glucocorticoid agonists (59-
this manuscript.
62). Importantly, activation of the GR releases unstimu-
lated PBMCs from an early block in HIV-1 replication via Address all correspondence and requests for reprints to:
a mechanism requiring Vpr (107). The presence of a GR Zdenek Hel, PhD, Department of Pathology, University of Al-
ligand results in a translocation of GR-Vpr complex into abama at Birmingham, 1825 University Boulevard, SHEL 603,
the nucleus and efficient proviral integration. This mech- Birmingham, Alabama 35294-2182. E-mail: zhel@uab.edu.
anism may be particularly important in the early stage of This work was supported by National Institutes of Health
infection. Grant PO1 AI083027.
Disclosure Summary: The authors declare no competing
In summary, we show that MPA may constrain immune
interests.
responses to HIV-1 by suppression of innate and adaptive
immune responses, by retention of HIV-1 coreceptors on
T cells after activation, and by increasing the rate of HIV-1
replication. These mechanisms may promote HIV-1 pro- References
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