Viral Infection of the Pregnant Cervix
Predisposes to Ascending Bacterial Infection
This information is current as
of June 17, 2017.
Karen Racicot, Ingrid Cardenas, Vera Wünsche, Paulomi
Aldo, Seth Guller, Robert E. Means, Roberto Romero and
Gil Mor
J Immunol 2013; 191:934-941; Prepublished online 10 June
2013;
doi: 10.4049/jimmunol.1300661
http://www.jimmunol.org/content/191/2/934
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References
The Journal of Immunology
Viral Infection of the Pregnant Cervix Predisposes to
Ascending Bacterial Infection
Karen Racicot,* Ingrid Cardenas,† Vera Wünsche,* Paulomi Aldo,* Seth Guller,*
Robert E. Means,‡ Roberto Romero,x and Gil Mor*
P
reterm birth is the major cause of neonatal mortality and
morbidity worldwide, yet the underlying etiologies remain
poorly understood (1, 2). Preterm labor is a syndrome
diagnosed in the presence of increased uterine contractility, cervical ripening, and/or membrane rupture or activation, which may
occur in response to multiple pathological processes (1, 2). Neonates born with a fetal inflammatory response are more likely to
develop short- and long-term complications above those expected
for the gestational age at birth (3–5).
Numerous studies suggest that intrauterine infection is an important mechanism leading to preterm labor and may account for
40% of preterm births (6–8). However, this number may be higher
because many infections are likely to be subclinical and the pathogenesis is not detected due to the lack of sensitivity of conventional culture techniques (9, 10). Furthermore, our understanding of
the normal flora of the female reproductive tract (FRT) is insufficient and we have limited knowledge of the mechanisms controlling
pathogenic bacteria and their relationship with intrauterine infection
and preterm labor (11, 12).
All women have microorganisms in the lower genital tract
(vulva, vagina, and cervix); however, most studies indicate that
amniotic fluid is normally sterile and does not contain microbial
products such as endotoxin. During pregnancy, intrauterine infec*Department of Obstetrics and Gynecology, Yale University School of Medicine,
New Haven, CT 06520; †Department of Obstetrics and Gynecology, Tufts University,
Boston, MA 02111; ‡Department of Pathology, Yale University School of Medicine,
New Haven, CT 06520; and xPerinatology Research Branch, Eunice Kennedy Shriver
National Institute of Child Health and Human Development, National Institutes of
Health, Department of Health and Human Services, Detroit, MI 48201
Received for publication March 11, 2013. Accepted for publication May 8, 2013.
This work was supported in part by National Institutes of Health Grants NICDH
P01HD054713 and 3N01 HD23342 and the Intramural Research Program of the
Eunice Kennedy Shriver National Institute of Child Health and Human Development,
National Institutes of Health, Department of Health and Human Services.
Address correspondence and reprint requests to Prof. Gil Mor, Department of Obstetrics, Gynecology, and Reproductive Sciences, Reproductive Immunology Unit,
Yale University School of Medicine, 333 Cedar Street, LSOG 305A, New Haven, CT
06520. E-mail address: [email protected]
Abbreviations used in this article: AMP, antimicrobial peptide; Ct, cycle threshold;
FRT, female reproductive tract; HPV, human papillomavirus; MHV68, murine gammaherpesvirus 68; ORF, open reading frame; qPCR, quantitative PCR; RFP, red
fluorescent protein.
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300661
tions begin in the decidua, extend to the amnion and chorion, and
finally reach the amniotic cavity and the fetus (3). Bacteria gain
access to gestational tissues through one of three major routes:
ascending into the uterus from the lower tract, descending into the
uterus from the peritoneal cavity, or via the maternal circulation
(6, 13). Bacterial infections that ascend from the lower FRT are the
most common route of uterine infection, and it is not known why
some women suffer such infections that threaten pregnancies and
fetal survival (14). In a healthy pregnancy, the uterus and growing
fetus are protected from ascending infection by the cervix (12, 15,
16). The cervix has a unique role in the FRT in that it actively
controls and limits microbial access by the production of mucus,
inflammatory cytokines, and antimicrobial peptides (AMP) (16,
17). In nonpregnant women, the cervical mucus is a viscous fluid in
the endocervical canal; however, after conception the endocervical
canal develops a structure called the cervical mucus plug, which
is an anatomical and immunological barrier against ascending infection (18). Indeed, analysis of the composition and antimicrobial
activity of the cervical mucus plug revealed the presence of AMPs
with potent antimicrobial activity (19). Also expressed in the cervical epithelia are the pattern recognition receptors such as TLRs
capable of sensing the presence of microorganisms and eliciting an
innate immune response characterized by the production of cytokines and AMPs (20–23). Collectively, the cervix plays a key role
in the protection against ascending intra-amniotic infection. If the
mucus plug is expelled or cervical length is short, the risk of ascending uterine infection increases.
Herpesviruses are the most common cause of viral-related perinatal neurologic injury in the United States (24). However, HSV-1,
HSV-2, and CMV (25) are among the eight known human herpesviruses reported to induce adverse pregnancy and neonatal outcomes
and have been found in the amnion, placenta, and even the lower
reproductive tract of pregnant women (26). Murine gammaherpesvirus
68 (MHV68; Murid herpesvirus 4 [NC_001826.2]) is a gammaherpesvirus of rodents that shares significant genomic colinearity
with two human pathogens, EBV and Kaposi’s sarcoma-associated
herpesvirus (27).
Viral survival depends on their capacity to disable host defenses,
especially the innate immune system, establish latency, and secure
mechanism for reactivation. One way in which viruses could un-
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Preterm birth is the major cause of neonatal mortality and morbidity, and bacterial infections that ascend from the lower female
reproductive tract are the most common route of uterine infection leading to preterm birth. The uterus and growing fetus are
protected from ascending infection by the cervix, which controls and limits microbial access by the production of mucus, cytokines,
and antimicrobial peptides. If this barrier is compromised, bacteria may enter the uterine cavity, leading to preterm birth. Using
a mouse model, we demonstrate, to our knowledge for the first time, that viral infection of the cervix during pregnancy reduces the
capacity of the female reproductive tract to prevent bacterial infection of the uterus. This is due to differences in susceptibility of the
cervix to infection by virus during pregnancy and the associated changes in TLR and antimicrobial peptide expression and function.
We suggest that preterm labor is a polymicrobial disease, which requires a multifactorial approach for its prevention and
treatment. The Journal of Immunology, 2013, 191: 934–941.
The Journal of Immunology
Materials and Methods
Animals and treatments
C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor,
ME); adult mice (8–12 wk of age) with vaginal plugs were infected i.p. at
embryonic day 8.5 postconception with either 1 3 106 PFU MHV68 in 200
ml or DMEM (vehicle). To develop the ascending bacteria model, Escherichia coli (BL21) expressing red fluorescent protein (RFP) induced by
isopropyl b-D-thiogalactoside and arabinose (pZS2, Addgene plasmid
26598; Dr. M. Elovitz, University of Pennsylvania, Philadelphia, PA) were
collected after reaching an OD = 0.6, and resuspended gently in 40 ml PBS.
This was delivered in the vagina of mice sedated with isoflurane using a
200-ml gel tip, and imaging was performed after 24 h using the Carestream
In Vivo Imaging System FX PRO (Bruker). Lymphoid aggregates were
dissected from the stratum basalis after gently removing the implantation
site. For experiments determining gene expression and MHV68 titers in
cervix without hormone treatments, mice were sacrificed 7 d postinfection,
and organs were removed and stored at 280˚C. In experiments comparing
nonpregnant and pregnant mice, nonpregnant mice in diestrus were used.
This was determined by morphology of the reproductive tract at time of
sacrifice. For experiments determining the effect of systemic hormones,
mice were ovariectomized, and after 21 d treated with progesterone (500
mg) and estrogen (500 ng) or vehicle, s.c. for 3 d. Both treatment groups then
received injections of MHV68 and continued to get hormone or vehicle
every 2 d for 7 d. Cervix and spleen were then collected, and MHV68 infection was determined by quantitative PCR (qPCR). All animals were
maintained in the Yale University School of Medicine Animal Facility under
specific pathogen-free conditions, and all procedures reported in this manuscript were approved by the Yale University Institutional Animal Care and
Use Committee. All in vivo experiments used between three and six mice in
at least two separate independent experiments.
Mycoplasma
Ureaplasma urealyticum was purchased (27618) and reconstituted in
American Type Culture Collection media 2616, special modified formulation, as indicated by American Type Culture Collection. Serial dilutions
of bacteria were made and incubated at 37˚C until medium changed color,
indicating growth. At time of color change, individual tubes were stored at
4˚C until remaining dilutions exhibited growth. After 12 h, bacteria were
gently pelleted and resuspended in growth medium. A small aliquot was
then used to determine CFUs (1 3 104/ml), whereas the remaining bacteria
were injected intravaginally into mice at pregnancy E16.5 with or without
MHV68 infection (MHV68 at day 8.5).
Cell culture
Immortalized human ectocervical cells (ECT1; American Type Culture
Collection, CRL-2614) were cultured in keratinocyte serum-free medium
(17005-042; Life Technologies, Grand Island, NY) with bovine pituitary
extract and human epidermal growth factor supplementation, as recommended by American Type Culture Collection, under 5% CO2 at 37˚C. In
blocking studies, 500 mg fibronectin (Life Technologies) was added to
ECT1 cells for 30 min and removed. Cells were then infected with MHV68
for 30 min, washed, and maintained in medium for 24 h. To block integrin
a3, the same protocol was used, but with blocking Abs for a3 (P1B5;
Millipore); all in vitro experiments were repeated three times.
MHV68 production and quantification
MHV68 expressing GFP was passaged in NIH 3T3 cells with DMEM plus
10% FBS. After lysis, supernatants were harvested, filtered (0.45 mM pore),
and titered by 2-fold serial dilutions. To detect viral titers in mice, tissues
were homogenized, and ∼25 mg tissue from the reproductive tract or 10 mg
spleen was cut into small pieces and added to lysis buffer supplemented with
proteinase K. Samples were incubated at 56˚C with shaking for 4–6 h, as
recommended for the DNeasy blood and tissue kit (Qiagen, Valencia, CA).
Cells from culture were lysed with the same buffer and vortexed at room
temperature. All samples were then processed according to the DNeasy
protocol. DNA concentration and purity were assessed using spectrophotometric analyses of 260/280 and 260/230 ratios. A quantity amounting to
100 ng total DNA was then assayed using primers directed against MHV68
open reading frame (ORF)53 and compared with a standard curve created
using serial dilutions of purified virus. Results are reported as copies per 100
ng DNA.
RNA synthesis, cDNA synthesis, and qPCR
RNA was extracted using the TRIzol method (Invitrogen, Carlsbad, CA).
RNA concentration and purity were assessed using spectrophotometric
analyses of 260/280 ratios, and only samples with values of 1.7 or higher
were used for PCR analysis. For quantitative analysis of mRNA, 1 mg RNA
was reverse transcribed for each sample using oligo(d)T priming and Verso
cDNA kit, which includes a DNase enzyme (Invitrogen). Syber Green
master mix (KAPA Biosystems) and gene-specific primers were added to
the reverse-transcriptase reactions that were diluted 1:10 with nucleasefree water and run on the CFX96, C1000 system qPCR machine (BioRad). No reverse-transcriptase controls were used to confirm that values
did not represent amplification of genomic DNA, and no template controls
were used to confirm lack of contamination by any reagents. Values represented were normalized to b-actin and were calculated using the d d
cycle threshold (Ct) method, as follows: d d Ct = d ct treated 2 d Ct
control; results expressed as fold differences are 2^2(average d d Ct) for
negative d d Ct values or 2(2^[average d d Ct]) for positive d d Ct values.
Cytokines
Cytokine concentrations were determined using cytokine multiplex assays
from Bio-Rad. Briefly, wells were either loaded with 50 ml prepared
standard or 50 ml cell-free supernatant and incubated on an orbital shaker
at 500 rpm for 2 h in the dark at room temperature. Wells were washed
three times with Bio-Rad wash reagent, and samples were then incubated
with 25 ml biotinylated detection Ab for 30 min, washed, and incubated
with 50 ml streptavidin-PE for 10 min. After final wash, samples were
resuspended in assay buffer and measured using LUMINEX 200 (LUMINEX,
Austin, TX). Cytokines included in this assay are as follows: IL-1b, IL-10,
GM-CSF, IFN-g, TNF-a, IL-1a, IL-6, IL-12p40, IL-12p70, G-CSF, KC, MIP1a, RANTES, MCP-1, and MIP-1b.
Western blot analysis
Tissues were homogenized in phospho-cell lysis buffer (Cell Signaling,
Danver, MA), and total protein concentrations were quantified using
bicinchoninic acid assay (Pierce, Rockford, IL). Twenty micrograms of total
proteins were dissolved in 13 sample buffer, boiled for 5 min, and separated on a 12% SDS-PAGE gel with 6% stacking gel in 13 electrode
buffer at a constant current of 70 mA for ∼2 h. The proteins were transferred to nitrocellulose membranes (Protran, 0.2 mM; Schleicher &
Schuell, Keene, NH) in a Mini-Protean II Cell apparatus (Bio-Rad Laboratories, Hercules, CA) at a constant 70 V for 90 min with an ice pack.
Nonfat milk (5%) was used as a blocker (Fisher Scientific, Pittsburgh, PA),
and immunoblotting was performed with a 1:500 dilution of primary Abs
in 2% nonfat milk at 4˚C overnight. Abs were Cell Signaling CS4749S (b1
integrin) and Millipore AB1920 (a3 integrin). Membranes were washed,
and a 1:10,000 dilution of goat anti-rabbit or goat anti-mouse IgG–HRP
conjugate (Pierce) was added, as appropriate. Membranes were washed
and incubated with Western Lighting Plus (PerkinElmer, Waltham, MA) to
detect immunoreactive proteins.
Statistics
Differences between means (three groups or more) were determined using
ANOVA, and differences between two groups were analyzed using independent t test functions of GraphPad inSTAT statistical software (La Jolla,
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dermine host immunity is through the manipulation of innate immune receptors such as TLRs. We have previously reported the use
of MHV68 as a murine model to determine whether a subclinical
viral infection sensitized the mother to other microorganisms and
induced preterm birth (15). Our published data suggested that
bacteria or virus alone was not enough to evoke preterm labor, but
the combination was a threat. HSV and CMV have latency periods
and can be reactivated via TLR signaling (28); therefore, it can be
postulated that they could affect pregnant women the way MHV68
affects pregnant mice in our model.
We have now tested the hypothesis that a viral infection reduces
the ability of the pregnant cervix to protect against ascending
bacterial infection. In this study, we show a dramatic difference in
the capacity of the cervix to prevent ascending intrauterine bacterial infection in nonpregnant and pregnant mice, and furthermore,
we demonstrate that viral infection compromises the nature of the
innate immune response of the pregnant cervix predisposing to
ascending intrauterine infection. Our results may explain the differential sensitivity observed in pregnant women to ascending bacterial infections.
935
936
CA). A p value #0.05 was considered significant, and data are presented as
mean 6 SEM.
Results
Development of a murine model of ascending bacterial
infection
Movement of pathogenic bacteria through the mouse female
reproductive tract
Because we found that the pregnant FRT is capable of preventing an
ascending E. coli bacterial infection, we investigated whether this
FIGURE 1. Murine model of ascending bacterial infection. Nonpregnant (A) and pregnant (B) mice were visualized 24 h after receiving
intravaginal injections of E. coli labeled with RFP. Legend indicates that
red color in nonpregnant animal has ascending bacterial infection in utero.
NT = no bacterial injection; Bacteria = intravaginal bacterial injection;
n = 6. (C) Time course of murine model of ascending bacterial infection.
Nonpregnant mice were visualized 12, 24, 48, and 72 h after receiving
intravaginal injections of E. coli labeled with GFP. Bacteria are visualized
in the vagina and cervix at 12 h, found throughout the reproductive tract at
24 and 48 h, and are diminishing 72 h postinfection.
protection is operative with the bacteria most frequently found
in the amniotic fluid of pregnant women with preterm labor, U.
urealyticum (29–31). U. urealyticum was inoculated intravaginally
on E15.5, and, after 24 h, decidua and lymphoid aggregates were
harvested to determine whether U. urealyticum was present in
these tissues. Similar as observed with E. coli, the pregnant FRT
was able to prevent the migration of U. urealyticum toward the
endometrial cavity (Fig. 2B).
Because we observed that the normal FRT, during pregnancy,
is highly protective of bacterial infection, we evaluated potential
factors that would alter this protection and consequently would
lead to an ascending bacterial infection as observed in pregnancy
complications. Having previously established a mouse model of
viral infection during pregnancy using a MHV68, which causes
mice to be more susceptible to bacterial products (bacterial endotoxin or LPS) (14), we tested whether a systemic viral infection
in pregnant animals could alter the resistance to microbial invasion of the uterine cavity. MHV68 was injected i.p. on E8.5 of
pregnancy, and U. urealyticum was inoculated intravaginally on
E15.5 (Fig. 2A). Twenty-four hours after bacterial inoculation, we
harvested decidua and lymphoid aggregates to determine whether
U. urealyticum bacteria was present in these tissues as determined
using a specific PCR assay for U. urealyticum. U. urealyticum
signal was significantly higher in the decidua and lymphoid
aggregates in MHV68-infected mice compared with mice that had
not been infected with MHV68 virus (Fig. 2B) or a control group
of pregnant animals not exposed to bacteria or virus (data not
shown). These data suggest that a viral infection during pregnancy
alters the capacity of the FRT to control ascending bacterial infection.
Expression of antimicrobial peptides and TLR by the uterine
cervix after viral infection with MHV68
The cervix has a unique role functioning as an interface between the
upper and lower FRT, providing protection to the uterine cavity
against ascending intrauterine infection from the lower genital
tract. During pregnancy, epithelial cells of the cervix are responsible for the formation of the mucus plug that provides a mechanical and biochemical barrier to ascending infection (19).
Because we observed that a viral infection resulted in dramatic
FIGURE 2. U. urealyticum ascends into the uterus in MHV68-infected
animals. Mice were infected with MHV68 or DMEM (control) at E8.5 of
pregnancy, received an intravaginal injection of pathogenic U. urealyticum
at E15.5, and were sacrificed (Sac) at E16.5 (A). NT = mice with no
MHV68; MHV68 = virus-treated animals. Both groups received U. urealyticum; decidua and lymphoid aggregates are represented as fold difference from the same tissue from mice with no viral infection. Mice with
MHV68 had higher concentrations of bacteria in the decidua and lymphoid
aggregates (Lymph Agg) compared with mice without viral infection (B).
n = 5, *p = 0.05.
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Our first objective was to determine whether a nonpathogenic form
of E. coli could ascend through the FRT in pregnant and nonpregnant mice. Consequently, we inoculated genetically engineered
bacteria, E. coli expressing RFP (RFP-E. coli), into the vagina of
healthy nonpregnant and pregnant mice and monitored their location using an imaging system. In nonpregnant animals, bacteria
were clearly detected in the cervix and endometrial cavity within
24 h (Fig. 1A). In contrast, bacteria were not detectable in the
cervix or uterine cavity of pregnant animals (Fig. 1B), indicating a
dramatic resistance to microbial invasion of the uterine cavity during
pregnancy. Bacteria were not observed at earlier time points in
pregnant animals, suggesting there was no access, and not that the
infection was there, but rapidly eliminated (data not shown).
To confirm the specificity of the signal and time course of ascending microbial invasion of the endometrial cavity, mice were
inoculated in the vagina with the same E. coli, but expressing GFP
as the reporter gene. Animals were euthanized after 12, 24, 48, and
72 h. Imaging of the genital tract was performed, and, as observed
with RFP-E. coli, GFP-E. coli ascended through the uterus of the
nonpregnant mice; furthermore, we observed an increase in fluorescent signal as a function of time (Fig. 1C). That was not the case
in the FRT of pregnant mice, in which there was no signal at any
time (data not shown). These observations indicate a dramatic
difference in the permissiveness of the nonpregnant and pregnant
uterus to ascending infection from the lower genital tract.
POLYMICROBIAL INFECTION AND PREGNANCY
The Journal of Immunology
In addition, AMP mRNA expression was lower in the pregnant
cervix of MHV68-infected mice than in that of pregnant animals
not exposed to the virus (Fig. 4D). These observations suggest that
the decreased TLR gene expression in the cervix of MHV68infected mice is associated with a lack of response to bacteria,
as demonstrated by the absence of proinflammatory cytokines and
decreased expression of AMPs. These changes in the MHV68infected pregnant mice may be functionally linked to the increased susceptibility to ascending bacterial infection.
Systemic administration of MHV68 results in viral infection of
the cervix of pregnant mice
The differences in the cervical response to viral and bacterial infections of pregnant and nonpregnant mice could be attributed to
variations in the systemic response to the virus or changes in cervical
viral infection. To test this postulate, we sought to determine whether
the virus was directly infecting the cervix. Pregnant and nonpregnant
mice received i.p. injections with MHV68, as described above;
cervix and spleen samples were collected 7 d after injection and
analyzed for MHV68 infection using qPCR for MHV68 ORF53.
MHV68 viral infection was observed at similar concentrations in the
spleen of pregnant and nonpregnant mice (Fig. 5A), but, surprisingly, MHV68 viral infection was observed in the cervix of pregnant mice, but was absent in the cervix of nonpregnant mice (Fig.
5B). These results indicate pregnancy renders the uterine cervix
susceptible to a systemic viral infection.
Sex hormones increase the susceptibility of the cervix to viral
infection
Estrogen and progesterone are major factors associated with morphologic and functional changes of the cervix, during pregnancy
(18). Because we found that virus was only infecting the cervix
of pregnant mice, we hypothesized high hormone concentrations
during pregnancy induced modifications in the epithelium of the
cervix that increase its susceptibility to viral infection. To test this
premise, we ovariectomized mice and they received hormone
treatment (estradiol [500 ng] and progesterone [500 mg]) or placebo
(vehicle) 21 d postsurgery for 3 d (Fig. 6A), mimicking the hormonal status during pregnancy. Both treatment groups then received injections of MHV68 i.p. and continued to get hormone
therapy or vehicle every 2 d for 7 d (Fig. 6A). Afterward, the cervix
was collected and it was determined whether sex hormones made
the cervix more susceptible to MHV68 infection. As suspected,
MHV68 was detected in the cervix of animals receiving hormone
therapy, but not in ovariectomized mice receiving only vehicle (Fig.
6B). These data suggest that hormonal changes associated with
pregnancy induced modifications of the cervix, leading to increased
susceptibility to viral infection, and this could in turn affect the role
of the uterine cervix in protecting against ascending bacterial infection.
Integrins are increased in the cervix of pregnant mice and have
a role in viral entry
FIGURE 3. MHV68 infection decreases TLR expression in the pregnant
cervix. NT = no MHV68; MHV68 = mice infected with virus. TLR mRNA
expression was higher or did not change in the cervix of nonpregnant
MHV68-infected animals relative to noninfected controls (A), but specific
TLR mRNA was significantly decreased in the cervix of MHV68-infected
pregnant animals (B). Significance as denoted by bars is *p , 0.05. n = 5.
Having determined that the high levels of sex hormones found during pregnancy might increase the susceptibility of the cervix to viral
infection, we hypothesized that this was due to increased expression
of a protein or proteins on cervical cells that may act as a receptor
for viral entry. Although the specific receptor for MHV68 entry is
unknown, integrins are common entry receptors for related viruses
(31, 32). To test the possibility that integrins are responsible for
MHV68 infection of cervical cells, we established an in vitro model
using human ectocervical cells clone ECT1 that are permissive to
MHV68 infection. Fibronectin is a high m.w. glycoprotein present
in the extracellular matrix that binds to integrins (33); therefore, we
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susceptibility to microbial invasion of the uterine cavity in pregnant animals, we explored whether this could be due to a change
in the capacity of the cervix to control microorganisms due to
alteration in the expression and function of pattern recognition
receptors (TLRs), responsible for sensing microorganisms. The
uterine cervix from nonpregnant and pregnant mice was analyzed
for changes in TLR mRNA expression in the presence or absence
of a systemic MHV68 infection. Systemic MHV68 viral infection,
in the nonpregnant mice, either did not change or increased the
expression of specific TLRs (Fig. 3A). In contrast, the cervix of
pregnant mice that had been exposed to MHV68 showed a substantial decrease in the expression of specific TLRs (Fig. 3B).
TLRs play a central role in maintaining the control of microorganisms in the cervix by sensing microbial products and eliciting
an innate immune response. To determine whether these changes
in TLR gene expression resulted in functional differences in the
capacity of the cervix to respond to bacterial infection, pregnant
mice were exposed to either MHV68 or vehicle at pregnancy E8.5,
followed by an intravaginal challenge of E. coli bacteria at E15.5
of pregnancy. Because TLR ligation induces changes in cytokine/
chemokine expression and AMP secretion, we characterized the
cervical cytokine/chemokine profile and AMP mRNA expression.
We observed a robust proinflammatory cytokine response to bacteria
in the cervix of pregnant mice not exposed to MHV68 infections
characterized by high tissue concentration of IL-1b, IL-6, KC,
MCP-1, MIP-1a/b, IL-12, and RANTES (Fig. 4A–C). This cytokine/
chemokine profile is stereotypic for a strong proinflammatory antibacterial response. A dramatically different profile was observed
in MHV68-infected pregnant animals. The presence of bacteria in
the lower genital tract in MHV68-infected mice was not able to
elicit a similar inflammatory response as the one observed in mice,
which received the same type of bacteria (Fig. 4A–C). Indeed, the
cervical protein concentration of IL-12, MIP-1a and b, RANTES,
and IL-6 of mice with MHV68 viral infection and bacteria was
similar to those of the control group (not exposed to either bacteria
or virus).
937
938
POLYMICROBIAL INFECTION AND PREGNANCY
postulated that fibronectin would bind to integrins present in cervical cells and block viral entry. To test this premise, we incubated
human cervical cells with fibronectin (500 mg/ml), followed by
MHV68 infection. We observed a significant decrease in MHV68
infection in cells pretreated with fibronectin compared with the
nonfibronectin-treated control, suggesting an integrin might be
FIGURE 5. MHV68 infects the cervix of pregnant mice. Both pregnant
and nonpregnant mice had similar systemic infections, as determined by
MHV68 ORF53 in the spleen (A), but pregnant mice also had high
MHV68 titers in the cervix, whereas nonpregnant mice had no cervical
infection (B). n = 4, *p , 0.001.
involved in viral entry into cervical cells (Fig. 7A). We then examined integrins in the cervix of nonpregnant and pregnant mice
to determine whether their expression was correlated with MHV68
infectivity. Our data showed integrin a3 and b1 expression was
positively correlated with MHV68 infection in the cervix of
infected pregnant (high expression) and nonpregnant (low expression) mice (Fig. 7B). This was not the case for integrin a4, which
was equally expressed in nonpregnant and pregnant cervices (Fig.
7B). Finally, to determine whether integrin a3, specifically, had
a role in the susceptibility of MHV68 infection in the cervix, we
evaluated whether specific blocking Abs for this integrin could
FIGURE 6. Sex hormones play a role in MHV68 infection of cervix
tissue. Mice were overiectomized (OVX) and, after 3 wk, given either P4 +
E2 or vehicle for 3 d; they were then injected with MHV68 and remained
on either hormone treatments or vehicle for 7 d (A). Seven days postinfection, MHV68 was detected in the cervices of mice treated with hormones, but absent in vehicle-treated controls (B). n = 4, *p , 0.05.
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FIGURE 4. MHV68 infection reduces cytokine response to bacteria in the cervix. Cervical cytokines were measured using luminex technology; “NT” are
cytokines from the cervix from animals that did not receive MHV68 or bacteria. “Bacteria” are cytokines from the cervix of animals with an intravaginal
bacteria injection, but no MHV68. “MHV + Bacteria” are cytokines from the cervix of animals that received an intravaginal injection of bacteria while
having a MHV68 infection. A number of cytokines and chemokines (A–C) were upregulated in the cervices of animals with artificial intravaginal bacterial
infection, whereas this response was significantly reduced in animals with MHV68 infection. b defensin 1 (BD1), b defensin 3 (BD3), b defensin 4 (BD4),
and b defensin 14 (BD14) mRNA expression was also reduced in cervices from MHV68-infected animals (D). Significance is denoted by bars and is *p ,
0.05. n = 5.
The Journal of Immunology
939
prevent MHV68 viral infection, in vitro. As shown in Fig. 7C, we
found that there was a significant decrease in the MHV68 titers in
human cervical cells pretreated with a3 blocking Ab (Fig. 7C), but
not with the control IgG. Collectively, these data suggest a possible
mechanism whereby pregnancy can increase the susceptibility of
the cervix to viral infection with MHV68 (Fig. 8).
Discussion
FIGURE 7. Integrins play a role in MHV68 entry into cervical cells.
Human ectocervical cells (ECT1) were pretreated with fibronectin to block
integrin–ligand interactions and then infected with MHV68; 24 h postinfection, MHV68 was found to be partially blocked from cells treated with
fibronectin compared with cells treated with MHV68, but no fibronectin
(NT) (three independent experiments) (A). Western blot analysis showed
integrin a3 and b1 were upregulated in the pregnant cervix of infected mice
(B), n = 3. Abs blocking integrin a3 (MHV68 + anti-integrin a3 Ab)
partially blocked MHV68 infection, whereas treatment with nonspecific
IgG (MHV68 + IgG) did not block MHV68 infection as compared with
cells only treated with MHV68 (MHV68 only) (representative of three
independent experiments with a minimum of three animals per group) (C).
Significance is denoted by bars and is *p , 0.05.
FIGURE 8. Model of polymicrobial disease during pregnancy. (A) Commensal bacteria are located in the lower reproductive tract, and, during
healthy pregnancy, the uterine cervix provides protection against bacteria
ascending into the upper reproductive tract. If the protection provided by the
uterine cervix is jeopardized, bacteria can ascend from the lower tract, to the
decidua and amnion, leading to inflammation and pregnancy complications,
such as preterm birth. We propose a model of polymicrobial disease during
pregnancy, as follows: in this model, pregnancy and the associated sex
hormones increase the susceptibility of the cervix to viral infection. Viral
infection then results in a decrease in the protection against ascending
bacteria. The decrease in protection can then lead to intrauterine inflammation in response to bacteria and preterm birth (B).
amniotic cavity elicits an intense inflammatory response, which
may evolve into a fetal inflammatory response syndrome. The
latter predisposes preterm neonates to short- and long-term consequences, such as neonatal sepsis, bronchopulmonary dysplasia,
and cerebral palsy. It has been estimated that one of every three
preterm neonates is born to a mother with intra-amniotic infection,
identified with either cultivation or molecular microbiologic techniques (36).
Two major events need to occur for bacteria to induce preterm
labor, as follows: 1) bacteria need to breach the cervix and reach the
decidua, amnion, and potentially the fetus; 2) bacterial numbers
must reach a threshold that will trigger an inflammatory response
that in turn induces preterm labor (37). A major question is how
bacteria can breach the cervical barrier and reach the maternal–
fetal interface. All women have microorganisms in the lower
genital tract, yet these microorganisms do not gain access to the
amniotic cavity during normal pregnancy. What are the mechanisms responsible for preventing ascending intrauterine infection
in most pregnant women? It is widely believed that the cervical
mucus plug plays an important role in host defense against microbial invasion of the amniotic cavity (19, 38). Indeed, the cervical
mucus plug has antimicrobial activity (38), contains antimicrobial
peptides, as well as Igs and cellular components of the innate
immune system (macrophages, neutrophils, etc.) (39). Therefore,
alterations of the composition or formation of the cervical plug
may affect the protection against ascending bacteria.
In this study, we first evaluated the regulation of bacteria in the
mouse FRT. We provide novel insights into the regulation of
bacterial movement through the reproductive tract and reveal
major differences depending on the reproductive stage. We observed, in our murine model, that bacteria delivered in the lower
FRT is able to ascend to the uterine cavity, confirming new findings in women, showing that the nonpregnant uterus has a normal
flora (2, 40). In contrast, pregnant mice were able to prevent ascending bacteria from the lower FRT. These findings could sug-
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In the current study, we demonstrate, to our knowledge for the first
time, that a viral infection of the cervix during pregnancy reduces
the capacity of the lower reproductive tract to prevent bacterial
infection of the pregnant uterus. Furthermore, we report that
pregnancy increases the sensitivity of the cervix to viral infection,
leading to changes in the expression and function of TLRs and
antimicrobial products. This sensitivity is partially caused by hormonal regulation of proteins in the cervix that cause it to be permissive to virus.
Approximately 30% of all neonatal death is directly caused by
preterm birth, and 50% of premature births are idiopathic (1, 34,
35). Because preterm birth represents a major health problem, it
is, therefore, of the greatest importance to determine its causes,
which then will allow us to develop novel ways for prevention and
treatment. A strong body of evidence suggests that a majority of
intrauterine bacterial infections during pregnancy result from
bacteria ascending from the lower reproductive tract (2, 6). The
amniotic cavity is normally sterile, and a major transition during
life is the emergence from a sterile to a nonsterile environment at
the time of birth. Microbial invasion of the amniotic cavity can
lead to preterm labor with intact membranes, preterm premature
rupture of membranes, cervical insufficiency, a short cervix, fetal
sepsis, and preterm delivery. The presence of organisms in the
940
the site of bacterial invasion. The epithelial cells further help to
protect against pathogens by the appropriate expression of TLRs,
secretion of cytokines, and antimicrobial peptides (53, 54). We
demonstrate that MHV68 infection of the cervix decreases TLR
expression and consequently dampens the antimicrobial and inflammatory response to bacteria. This effect could be part of the
viral mechanism of evading host immune recognition. In contrast,
TLR3, TLR9, and TLR2 are upregulated in the cervix of nonpregnant animals, potentially for heightened protection against viral
infection. These results show that systemic infection regulates specific TLRs in mucosal epithelia quite differently, as compared with
direct infection.
The pregnant state requires dramatic remodeling of the uterus.
Sex steroid hormones and, in particular, estrogen and progesterone
have a powerful effect in remodeling the uterine cervix and the
myometrium. Because only the pregnant cervix was infected by
MHV68, this suggested that the changes of the cervix are associated
with the hormonal profile characteristic of pregnancy. This was
confirmed with the observation that ovariectomized mice receiving
estrogen and progesterone were more susceptible to viral infection
of the cervix, suggesting that a systemic endocrine milieu can create
conditions favoring viral invasion and multiplication. We were able
to establish that integrin a3 plays a role in the permissiveness of the
cervix to MHV68 infection, although additional factors may also
be involved because the blocking studies did not completely diminish MHV68 infection.
Based on these findings, we propose the following model:
commensal bacteria are located in the lower reproductive tract,
and, during healthy pregnancy, the uterine cervix provides protection against bacteria ascending into the upper reproductive
tract (Fig. 8A). If the protection provided by the uterine cervix
is jeopardized, bacteria can ascend from the lower tract, to the
decidua and amnion, leading to inflammation and pregnancy
complications such as preterm birth (Fig. 8B). In summary, to our
knowledge, these experiments are the first to describe a viral
infection during pregnancy that alters the physiologic protection
of the cervix against intrauterine infection. These studies also
provide evidence that such changes are potentially mediated by
altered components of the innate immune response. Together our
results suggest that preterm labor is a polymicrobial disease,
which requires a multifactorial approach for its prevention and
treatment.
Acknowledgments
We acknowledge Dr. Michael Elovitz for the generous gift of the pZS2 plasmid used in this study and Melinda Wells for the model illustration.
Disclosures
The authors have no financial conflicts of interest.
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