CERVICAL VIRAL INFECTION CAUSES PREMATURE CERVICAL RIPENING IN A
MOUSE MODEL OF PREGNANCY
By
Devin McGee

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Animal Science—Master of Science
2017

ABSTRACT
CERVICAL VIRAL INFECTION CAUSES PREMATURE CERVICAL RIPENING IN A
MOUSE MODEL OF PREGNANCY
By
Devin McGee
Preterm birth (PTB), or birth before 37 weeks of gestation, occurs in about 1 in every 10
pregnancies in the United States and is the leading cause of neonatal morbidity and
mortality in the developed world. Furthermore, surviving preterm infants are at increased
risk for brain, heart, breathing, metabolic, and immune function problems, with an
estimated cost of $26 billion each year. Despite the health and financial implications of
PTB, 50% of cases still have no attributed cause. Recently, cervical viral infection has
been identified as a risk factor for PTB. The implication of viruses, specifically herpes
simplex virus-2 (HSV-2), in pregnancy outcomes has not been well defined. Our approach
was to utilize both in vivo and in vitro models to study cervical viral infection. Using a
mouse model of pregnancy, we determined that intra-vaginal HSV-2 infection resulted in
changes in both cervical structure and function.

Viral infection caused stromal

disorganization and increased risk for PTB associated with ascending bacterial
infection. Using a cervical epithelial cell line, we discovered HSV-2 mediated changes in
cell function by activating Src kinase, which affected hormone receptor stability and other
down-stream effectors. We conclude that viral infection of the cervix leads to alterations
in key pathways that are important to parturition and ultimately increase risk for PTB.

Copyright by
DEVIN MCGEE
2017

iii

Dedicated to my Mom for instilling a sense of wonder and appreciation for how all things
work and to my Dad for challenging me to be well-informed in what I discuss, and to
think critically.

iv

ACKNOWLEDGEMENTS

I would like to thank my mentor Dr. Karen Racicot for all the time, effort, and patience
she gave me as she helped me grow both as scientist, and as a hard-working student. I
want to thank Dr. Arianna Smith for keeping me sane by making me laugh every day
and for putting up with my shenanigans. A special thanks to Sharra Poncil is necessary
for teaching me many of the techniques I utilized in the lab. I also want to acknowledge
and thank my committee members Dr. Fazleabas, Dr. Petroff, and Dr. Knott for their
time and for their support and advice. Dr. Knott also helped me by allowing me lab
space and equipment for a semester in East Lansing which I am very grateful for as it
keep me off the snowy roads! A thank you is necessary to each of Dr. Rooney and
Chris Svoboda from Michigan State’s Geology Department for their generous time and
space to allow me to borrow a special microscope to image a critical part of my work. I
thank the members of the Teixeira lab in Grand Rapids for the advice and unselfish
sharing of space. And finally, I want to thank my friends, family, and relatives for their
words of encouragement and support as I pursued my degree.

v

TABLE OF CONTENTS

LIST OF FIGURES………………………………………………………………….………..vii
KEY TO ABBREVIATIONS……………………………………………………………..…..viii
CHAPTER 1: REVIEW OF THE LITERATURE…………………………………..………..1
Significance of Preterm Birth…………………………………………………………2
Risk Factors for Preterm Birth………………………………………………………..3
The Function of the Cervix During Pregnancy.....................................................3
Cervical Softening……………………………………………………………………..4
Cervical Ripening……………………………………………………………………...5
Hormonal Regulation of Cervical Ripening…………………………………………6
Immune Regulation of Cervical Ripening……………………………………..…....8
Cervical Viral Infections and PTB………………………………………………..…10
Herpes Simplex-2 Virus……………………………………………………………..11
CHAPTER 2: CERVICAL VIRAL INFECTION CAUSES PREMATURE CERVICAL
RIPENING IN A MOUSE MODEL OF PREGNANCY…………………………………....13
Abstract………………………………………………………………………………………..14
Introduction……………………………………………………………………………………15
Materials and methods……………………………………………………......................…18
Pregnant mice and infections…………………………………………………….…18
Histology………………………………………………………………......................18
Cell culture and treatments………………………………………………………….19
Virus production and quantification……………………………………………...…20
RNA, cDNA synthesis and qPCR…………………………………………………..20
Hyaluronic Acid ELISA…………………………….............................…………....21
Western blot analysis…………………………………………….......……………...22
Statistical analysis……………………………………………………………………22
Results and figures………………………………………………………….......................23
Cervical viral infection affects stromal tissue organization………………...…....23
Viral infection increases ER-α protein in cervical epithelial cells.......................26
Hyaluronic acid is increased by HSV-2 in the pregnant cervix and ECT1
cells………………………………………………………………………………...….28
Herpes simplex virus-2 activation of Src kinase stabilizes ER-α and regulates
HA………………………………………………………………………………...…...30
Hyaluronic acid mediates HSV-2 infection………....……………………………..33
CHAPTER 3: CONCLUSIONS AND DISCUSSION ……………………………….…....35
REFERENCES……………………………………………………………………….………42

vi

LIST OF FIGURES

Figure 1: Cervical viral infection affects stromal tissue organization……...…………….24
Figure 2: Viral infection increases ER-α protein in cervical epithelial
cells………………………………………………………………………………………….....27
Figure 3: Hyaluronic acid is increased by HSV-2 in the pregnant cervix and
ECT1…………………………………………………………………………………………...29
Figure 4: Herpes simplex virus-2 regulates ER-α and HA via Src kinase
activation……………………………………………………………………………………….31
Figure 5: Hyaluronic acid mediates HSV-2 infection……………………………………...34
Figure 6: Proposed model of cervical viral infection during pregnancy…………………40

vii

KEY TO ABBREVIATIONS

PTB

Preterm Birth

Src

Src Kinase

4-MU

4-Methylumbelliferone

IVAG

intravaginal

sPTB

Spontaneous preterm birth

Src

Src kinase

ERα

Estrogen Receptor Alpha

i.p.

intraperitoneal

IVAG

intravaginal

SKI

Src Kinase inhibitor

PP1

Src Kinase inhibitor

C.I.

Cervical insufficiency

E.O.

External orifice

TFF1

Trefoil factor 1

SPINK5

Serine protease inhibitor Kazal type 5

GAG

Glycosaminoglycans

MMP

Matrix metalloproteinases

PG

Protaglandins

PGE2

Prostaglandin E2

HA

Hyaluronic acid or Hyaluronan

HAS

Hyaluronic acid synthase

viii

MW

Molecular weight

KO

Knockout

17βHSD

17β-hydroxysteroid dehydrogenase type 2

20αHSD

20α-hydroxy-steroid dehydrogenase

5α-SRD

Steroid 5α-reductase

HPV

Human Papilloma Virus

HSV-2

Herpes Simplex Virus type 2

HSV-1

Herpes Simplex Virus type 1

MHV

Murid Herpes Virus

LPS

Lipopolysaccharide

IFN

Interferon

NK

Natural Killer Cells

pDC

plasmacytoid dendritic cells

PR

Pregesterone receptor

PgR

Progesterone receptor gene

P4

Progesterone

ECM

Extracellular matrix

E2

17 beta-estradiol

DME

Differentiated mucosal epithelial cells

NDE

Non-differentiated mucosal epithelial cells

SFK

Src Family Kinases

Gd

Gestational day

MHV

Murid Herpesvirus

ix

CMV

Cytomegalovirus

HHV

Human Herpes virus

EBV

Epstein-Barr virus

Tyr

Tyrosine residue

DMSO

Dimethyl sulfoxide

ANOVA

Analysis of Variance

n.s.

Not significant

x

CHAPTER 1:
REVIEW OF THE LITERATURE

1

Significance of Preterm Birth
Preterm birth (PTB), or birth before 37 weeks of gestation, accounts for over 10%
of all pregnancies worldwide and over 11% in the United States[1-4] and rates continue
to rise, despite continued attempts at intervention [5] [6, 7]. Preterm delivery is the
leading cause of neonatal morbidity and mortality resulting in nearly 1 million neonatal
deaths per year [4, 8, 9]. These deaths stem from preterm infants’ increased risk for
neurologic and developmental disabilities [10], respiratory distress [11], dysregulated
metabolism function [12], bone disease [13], heart defects [14], and immune function
impairment [15]. Many of these problems arise because preterm infants have
underdeveloped organs or organ systems [11]. Even when preterm infants survive past
5 years of age, they typically have poor long-term outcomes that include higher risk for
vision and hearing impairment, chronic lung diseases, cardiovascular diseases, brain
development and behavior problems such as increased hyperactivity, anxiety, and
depression [16].
The cost associated with caring for a preterm infant is, understandably, quite
high. The Institute of Medicine’s Committee on Understanding Premature Birth and
Assuring Healthy Outcomes found that the minimum cost burden of PTB on the US
economy is predicted to be 26.2 billion dollars per year with 5.7 billion of that total
coming from the disabilities associated with PTB. These estimates only consider four
major debilitating conditions: cerebral palsy, mental retardation, vision impairment, and
hearing loss; therefore, the estimates could be missing a significant amount of cost.
Furthermore, the average cost per infant born preterm is about $51,600 [16], which can
be an incredible financial burden to a new family, especially to an uninsured family.
2

Unfortunately, research to understand and prevent PTB is underfunded [17] and there is
still much to learn about the causes and mechanism(s) of PTB [17].

Risk Factors for Preterm Birth
Bacterial infections and cervical insufficiency have been identified as important risk
factors for PTB [18-20]. Up to 40% of preterm births are associated with intra-uterine
infection [21] and, intra-vaginal, intra-uterine and/or systemic injection with bacteria or
their conserved microbial motifs can induce PTB in animal models [22-28]. The majority
of intra-uterine infections ascend from the vagina [29], and this is associated with
cervical insufficiency [30]. Cervical insufficiency (CI) is characterized as mechanical
weakness of the cervical tissue that affects the capacity of the cervix to support the
fetus, and protect it from bacteria in the lower reproductive tract [31]. Unfortunately,
diagnosing cervical insufficiency is challenging because of the limited criteria for use by
clinicians [31]. While factors such as “short cervix” can be used as indicators of CI, by
this time PTB is typically inevitable [32, 33].

The Function of the Cervix During Pregnancy
Throughout gestation the cervix functions as a structural and immune barrier for
the developing fetus. During pregnancy, the cervical stroma is characterized by a
dense extracellular matrix of proteins, including highly cross-linked fibrillar collagen,
which provides mechanical strength to the tissue [34]. In humans and rodents, the
cervical lumen is lined by epithelial cells that generate a cervical mucus plug, which
3

provides physical and immune protection against the vaginal flora [35-37]. At the end of
gestation, the cervix must undergo a significant transformation into a pliable tissue to
allow for the fetus to pass through the vaginal canal. The changes in the cervical
structure, or “cervical remodeling”, is a tightly regulated process that can be grouped
into two different stages: cervical softening and cervical ripening [38].

Cervical Softening
In mid-gestation the cervix begins undergoing structural changes, appropriately
named “cervical softening” by Dr. Word and colleagues, resulting in a progressively
more compliant tissue but without loss of its mechanical competence [39]. In women
softening begins at the end of the first trimester, and in mice is initiated by day 12 [39,
40]. Cervical softening is associated with decreased collagen cross-linking in the
cervical stroma [41]. Collagen undergoes continuous turn-over, and during this time
there is a down-regulation of genes, such as Plod2 [41], that mediate the formation of
the cross-linked collagen [42]. There are also changes in cervical epithelial cell function
that could affect the characteristics of the cervical mucus barrier [38, 39]. For example,
trefoil factor 1 (TFF1) and serine protease inhibitor Kazal type 5 (SPINK5) proteins
incrementally increase in the epithelia during softening [39]. Trefoil factor 1 is an
estrogen-regulated peptide, secreted by mucosal cells, that has a role in mucosal
protection and epithelial repair [43]. The protease inhibitor encoded by SPINK5 also
functions to maintain the epithelial barrier [44]. Increased expression of factors that
protect the epithelial barrier is understandably important in preparation for vaginal

4

delivery. However, it is not known how changes in these factors or others affect the
mucosal protection against bacteria.

Cervical Ripening
Over the last 2-3 days of gestation in the mouse, or in the last weeks of pregnancy
in women, the cervix enters the phase of “cervical ripening” resulting in complete
structural reorganization of the cervical stroma in preparation for labor. The stromal
compartment becomes increasingly hydrated and the collagen fibers become larger in
diameter and increasingly disorganized, a process initiated during cervical softening [41].
The changes in collagen structure are associated with increased collagen solubility,
decreased cross-linking and down-regulation of collagen accessory proteins such as
thrombospondin-2 and tenascin C [41].

Collagen degradation can also result from

activation of matrix metalloproteinases in fibroblasts and activated immune cells [45].
Cervical ripening is also associated with changes in ECM components such as
proteoglycans [46, 47]. Proteoglycan metabolism increases before the onset of labor in
the term cervix and in preterm labor and elastin is low in women with cervical
insufficiency [46, 47]. Interestingly, versican, dercorin, and heparan sulphate are
proteoglycans that are highly expressed in the cervix, but don’t change over the course
of pregnancy [39, 48]. Perhaps what is more important than their regulation or
expression is their interaction with other components in the cervix. These factors could
still regulate cervical structure and function via their interactions with collagen or
immune cell recruitment [38, 49]. For example, decorin can cause an increase in
collagen fibrils and veriscan can also affect ECM disorganization in tissue remodeling
5

[50]. Heparan sulphate can interact with cytokines, such as IL-8 [49, 51], and can affect
their half-life and functional potency within the tissue [52-54].
Hyaluronan, also known as Hyaluronic acid (HA), is a non-sulfated
glycosaminoglycan that is a component of the ECM of most tissues [55]. It has been
shown to affect tissue hydration [56], collagen interactions [57], and the immune
response [58]. Hyaluronan is produced by epithelial, stromal, and immune cells in the
cervix [55], then excreted into the ECM in multiple forms, ranging from high molecular
weight (MW) HA, thought to influence tissue structure, and low molecular weight HA,
potentially mediating tissue repair or the immune response [59]. It is present in the
cervix in low amounts throughout pregnancy, but drastically increases during cervical
ripening when the hyaluronan synthase enzyme gene, Has2, is upregulated [55, 60].
The HA that increases during ripening is high molecular weight, and has been
implicated in weakening fibronectin-collagen interactions, thus destabilizing the ECM
and making collagen vulnerable to collagenases [61]. Interestingly, when HA function
was studied in the pregnant cervices of HA knockout mice, it was determined to have a
role in epithelial and mucosal barrier function against pathogenic E. coli [62]. Indeed,
loss of HA resulting in increased ascending bacterial infection and higher rates of
infection-associated PTB[62].

Hormonal Regulation of Cervical Ripening
Cervical ripening is associated with the functional loss of progesterone (P4)
signaling within the cervix. Cervical ripening can be induced in women and rodents by

6

anti-progestin treatment, and vaginal P4 can delay ripening and labor in women with
anatomically short cervix [63]. Interestingly, the loss of cervical P4 does not appear to be
a result of declining P4 in the circulation. In women, circulating P4 does not significantly
decrease before labor. In rodents, circulating P4 does decline before labor, but cervical
ripening is initiated prior to the decline [64]. Therefore, it is postulated that loss of P4
function in the cervix is the result of tissue-specific changes in P4 concentration and/or
function [65]. The loss of P4 function in the cervix preceding cervical ripening is not well
defined but elegant work has demonstrated that there are changes in local P4 metabolism.
Enzymes that metabolize progesterone and estradiol are expressed by cervical
cells and are regulated over gestation. Enzymes that convert P4 to an inactive form
increase during ripening while enzymes that synthesize estradiol (E2) increase at this time
[66].

For example in early pregnancy, epithelial cells express 17β-hydroxysteroid

dehydrogenase-2 (17β-HSD-2), which catalyzes the conversion of estradiol to inactive
estrone. It also oxidizes its partner enzyme, 20α-HSD, which maintains progesterone
concentrations. During cervical ripening, 17β-HSD is downregulated in epithelial cells,
resulting in decreased local progesterone and increased estradiol [66-68].

The

expression of the enzyme steroid 5α-reductase (5α-SRD) also converts progesterone to
an inactive form and is upregulated in the cervical epithelia of mice during ripening.
Indeed, 70% of mice lacking 5α-SRD had delayed parturition because local progesterone
remained high, resulting in the inability to undergo cervical ripening [69].

Interestingly,

there is little known about the role of estrogen in cervical ripening, although several
studies suggest potential functions. For example, it was previously determined that
estrogen administration induced cervical collagenase activity in women and rodents,

7

which could potentially affect collagen remodeling [70-73]. In vitro models have also
demonstrated that estrogen induces collagen degradation activity, which can be blocked
by progestin treatment [73, 74]. The key enzyme responsible for up-regulation of cervical
HA during ripening, HAS2, is also regulated by estradiol [75], suggesting a role in HAmediated immune protection.

Immune Regulation of Cervical Ripening
Immune cell recruitment, pro-inflammatory cytokines, prostaglandins and protease
activation are also implicated in the regulation of cervical ripening. It is proposed that
activated immune cells induce ripening by expressing proteases that degrade collagen
and cytokines that affect tissue hydration [76]. These cells are also potential sources of
prostaglandins, which regulate progesterone and induce labor [77-79]. There is some
debate about the role of inflammation in the regulation of ripening. Some propose that
infection induces cervical ripening via an inflammatory mechanism [80], and this is distinct
from the mechanism associated with the functional loss of local progesterone.
Graham Liggins (1981) initially theorized cervical ripening was an inflammatory
reaction mediated by immune cells [81]. It was postulated that neutrophils infiltrated
during cervical ripening and mediated collagen degradation by secreting MMPs [82-85].
Recent studies, however, suggest neutrophils might have a more important role in
cervical repair, postpartum [86-88]. These studies found that women and mice did not
have increased neutrophils in the cervix at ripening, but they were increased postpartum [87, 88]. Also, depletion of neutrophils did not affect ripening or parturition in

8

mice, and there was no increase in myeloperoxidase, a marker of neutrophil function,
until the period of post-partum repair [86, 88].
Monocyte recruitment and macrophage activation have also been characterized
in the cervix during cervical ripening, although there is debate about the origin of these
cells and how they function. Some have reported monocyte recruitment to the cervix
during cervical ripening in mice, but without increased differentiated macrophages [89].
These data have led some to suggest that, like neutrophils, the role of macrophages is
in post-partum cervical repair [38, 88]. Others have reported increased macrophage
numbers and changes in functional markers at the time of cervical ripening, in direct
conflict with the prior reports [90, 91]. In addition, macrophage depletion was able to
prevent LPS-induced preterm birth in a mouse model [92]. These discrepancies are
most likely associated with different methodologies, as evidenced by an elegant report
from Steve Yellon’s laboratory. They determined that if the cervices of mice were
perfused prior to tissue collection and dispersion there was no increase in monocyte
numbers. This observation suggested increased monocyte numbers previously
reported were likely a result of the increased cervical vasculature, not a specific
recruitment to the tissue. They also performed highly validated, 8-marker flow
cytometric analysis of macrophage populations within the cervix, including markers for
cell viability and activation. They discovered robust changes in functional populations of
macrophages associated with ripening, supporting a potential role for these cells prior to
post-partum repair.

9

Cervical Viral Infections and PTB
An increasing number of clinical studies suggest that cervical viral infection is a
risk factor for PTB. Meta-analysis of Human Papilloma Virus (HPV) infections and
preterm birth showed that HPV infections could more than double risk for preterm
delivery [93]. This is important because, while HPV vaccines are used to combat
cervical dysplasia and cancer, the HPV vaccine may provide an additional obstetrical
benefit, and screens for HPV could be used as a predictor for PTB [93]. Several recent
studies have also linked HSV-2 infections with increased risk for PTB, including a study
with nearly 700,000 women that showed untreated genital HSV-2 infection nearly
doubled the risk for PTD [94]. Another study in 2017 confirmed treatment of genital
herpes with acyclovir decreased the chances of preterm birth in a cohort of with HSV-2
positive women in Uganda [95]. Early evidence of HSV-2’s relationship to preterm birth
was found in a 1996 study that associated asymptomatic genital shedding of the virus
with increased incidence of preterm delivery [96]. Despite these associations there
have been few studies into the mechanistic role that HSV-2 may have in incidences of
preterm deliveries.

A mouse model of viral infection during pregnancy also suggests that cervical viral
infection increases risk of PTB in the presence of bacteria. This mouse model was
developed using a gammaherpes virus, MHV68, a latent DNA virus from the
herpesviridae family, the same family of nearly all of the viruses that infect the woman’s
reproductive tract (HSV, CMV, EBV, HHV6, HHV7). When pregnant mice received an
intraperitoneal (i.p.) injection of MHV68 on embryonic day (E)8.5 of pregnancy it
10

sensitized the response to a secondary i.p. injection of LPS on E15.5, resulting in PTB
[97]. The same dose of LPS on that day did not induce PTB in mice without viral infection
[97]. This suggested that while viral infection, alone, was not enough to induce labor, it
acted as a “risk factor” to those with a secondary infection. Importantly, the pregnant
cervix was the primary reproductive tract target for MHV68, and infection affected cervical
function by decreasing cervical protection against ascending bacterial infections. This
suggests the mouse can be a reasonable model for herpes viral-infection associated PTB,
which is generally associated with cervical HSV-2 infection in women.

Herpes Simplex-2 Virus
The Herpesviridae family is a large group of double-stranded DNA viruses that
are ubiquitous in the human population [98-101]. Herpes Simplex Virus (HSV) -1 is
predicted to be present in 67% of the world’s population [98], and HSV-2 in over 11%
[99]. HSV-2 generally manifests as a genital infection, and has received special
attention during pregnancy for the its potential for vertical transmission from mother to
neonates [99, 100]. HSV-1 remains important since there has been a rising number of
genital herpes cases documented as being caused by HSV-1 [102].
HSV-2 poses a unique problem for its host because, after an infection episode, it
has the ability to establish latency within neurons and later reactivate in a lytic cycle
[103]. HSV-2 primarily and efficiently infects mucosal epithelial cells, which is the
classical site of primary infection and HSV is also present in these cells during its lytic
cycle which is essential for transmission [104]. HSV-2 targets sensory neurons for long
term residency, important for its latent life [104]. There are a few well documented
11

stimuli that induce reactivation of viruses including UV light exposure, emotional stress,
fever, tissue damage, and immune suppression [103]. HSV-2 also has documented
cases of lytic cycles, which result in viral shedding, occurring without the presence of
clinically noticeable symptoms, and this is postulated to be why HSV-2 is so effective in
its virulence [105-108].
Herpes simplex virus -2 is an enveloped virus that utilizes host cell surface
receptors for attachment and subsequent breach of cells [109]. Herpes simplex virus
has been shown to bind to glycosaminoglycans and interacts with plasma membrane
integrin αvβ3 for entry into genital tract epithelial cells [109, 110]. Interestingly, HSV-2
causes phosphorylation of the integrin’s downstream focal adhesion kinase even in the
absence of viral entry [109]. Specifically, herpes viruses have been shown to regulate
Src family kinase activity as a means of producing efficient viral yields [111, 112]. It is
not known how these cellular changes might affect the cervical epithelial cells in the
context of pregnancy.

12

CHAPTER 2:
CERVICAL VIRAL INFECTION CAUSES PREMATURE CERVICAL RIPENING IN A
MOUSE MODEL OF PREGNANCY

13

Abstract
Preterm birth occurs in about 10% of all pregnancies worldwide, and about 12% of
pregnancies in the United States. Preterm infants have an increased risk for brain, heart,
breathing, metabolism, and immune function problems, which is, in-part, why it is the
leading cause of neonatal morbidity and mortality in the developed world. 50% of all
spontaneous PTB cases have no cause attributed to them. Viruses, specifically HSV-2,
have been implicated in potentially contributing to preterm delivery (PTD) and require a
deeper examination. In order to do this, we utilized both an in vivo mouse model and in
vitro cell line model of virus infections to study both outcomes and mechanisms by which
the virus acts on cervix tissue and cells. We observed that intravaginal infections with
HSV-2 of our pregnant E15.5 mice show cervices with characteristics of cervical
insufficiency compared to controls. Viral infection of both mice and ECT1 cells with HSV2 results in a stabilization of the transcription factor estrogen receptor alpha (ERa) which
is mediated by the focal adhesion kinase, Src.

We also found an upregulation of

progesterone receptor (PR) in our virally treated mice cervices.

ELISA analysis of

hyaluronic acid (HA) showed that it is upregulated in response to viral infection in mice
cervical flushes and human cervical epithelial cells. HA was linked as an important factor
for high viral copy numbers, and to the focal adhesion kinase, Src, by its downregulation
when pSrc is inhibited. Our results show that intravaginally infected mice by E15.5 show
characteristics of cervical insufficiency (i.e. dilute collagen, tissue spacing) and HSV-2
infected cervical cells show increases in factors important for parturition suggesting that
viral infections of the cervix could contribute to compromised cervix integrity, and
subsequent preterm labor.

14

Introduction
Preterm birth (PTB), or birth before 37 weeks’ gestation, affects approximately
12% of pregnancies in the United States [9, 113-118] and is the leading cause of neonatal
mortality worldwide. Unfortunately, there has been little progress in predicting, preventing
and treating PTB due to the complexity of the condition. Indeed, PTB is more accurately
defined as a syndrome, resulting from an accumulation of risk factors that, in isolation,
may not precipitate labor [114]. While some risk factors have been identified, including
infection/inflammation, low progesterone (P4), cervical insufficiency, and race/ethnicity,
we have had limited success using them to predict PTB. Furthermore, nearly half of PTB
have no known risk factors and are unexplained. Therefore, the best strategy for
predicting or preventing PTB will be to identify the common biological pathways that can
be influenced by multiple risk factors.
Appropriate regulation of cervical structure and function is essential to pregnancy
maintenance. Throughout most of gestation, the cervical stroma contains a dense
extracellular matrix (ECM) rich in highly organized fibrillar collagen.
structural support for the developing fetus [119].

This provides

The cervical epithelial cells also

synthesize a highly effective mucus barrier that protects the uterus from ascending
bacteria.

This barrier consists of mucins, proteoglycans, anti-microbial peptides,

cytokines, proteases and associated protease inhibitors [120]. At the end of gestation
there is profound remodeling of the stromal ECM resulting in a structurally compliant
tissue in preparation for vaginal birth. At this time the collagen fibers become larger in
diameter and increasingly disorganized as a result of increased collagen solubility,
decreased cross-linking, increased stromal hydration and/or enzymatic degradation [39].

15

There are also significant changes in the mucus barrier and epithelial cell function.
Collectively, this process is termed “cervical ripening”, and premature cervical ripening
precedes preterm labor.
The initiation and regulation of ripening is not well-understood, but there is
evidence it is influenced by a tissue-specific decline in progesterone (P4) concentration
and/or function[65]. Cervical ripening can be induced in women and rodents by antiprogestin treatment, and vaginal P4 can delay ripening and labor in women. Changes in
P4 concentration could be mediated by an increase in local P4 metabolism or changes in
the expression of steroid hormone receptors genes for P4 and E2 (PgR/ESR1).
Interestingly, very little is known about the role of E2 in cervical ripening.
Ripening is also associated with increased synthesis of hyaluronan (HA). The
function of HA in the pregnant cervix is not fully understood, but it has been identified as
an important mediator of epithelial cell differentiation, epithelial barrier permeability and
ascending bacterial infections [121]. The concentration and size of HA may also affect
stromal remodeling. High molecular weight (HMW) HA increases in the stroma during
ripening, and may contribute to ECM disorganization and loosening because of its large
size (>500 kDa) and high level of glycosylation [60].
One of the most common causes of PTB is ascending bacterial infection, which
typically indicates cervical function is compromised. Normally, cervical epithelial cells
synthesize a highly effective mucus barrier that protects the uterus from ascending
bacteria.

This barrier consists of mucins, proteoglycans, anti-microbial peptides,

cytokines, proteases and associated protease inhibitors. During cervical ripening there is
increased epithelial cell proliferation [122], increased synthesis of hyaluronan (HA) [55],

16

activation of proteases [123], and immune cell infiltration resulting in the enzymatic
degradation of the mucus barrier[121]. When ripening is premature, these changes can
lead to ascending infection, which can trigger in utero inflammation and PTB. Therefore,
anything that can induce cervical ripening, or in any way compromise the mucus barrier,
can increase risk for PTB in response to secondary bacterial infection.
Very little is known about how viral infection affects cervical function during
pregnancy. Since HSV-2 was shown to increase risk of sPTB, we hypothesized that intravaginal herpesvirus infection would affect the function and structure of the cervix, resulting
in reduced cervical competency. We discovered that herpesvirus infection increased ER
in cervical epithelia and upregulated cervical HA synthesis, in vitro and in vivo.
Furthermore, these changes were associated with dramatic remodeling of collagen within
the stromal ECM, in vivo, suggesting that viral infection affected the structural integrity of
the cervix.
Previously, a study including nearly 700,000 women reported that untreated genital
herpes simplex virus-2 (HSV-2) infection nearly doubled the risk of sPTB[124].
Furthermore, in a mouse model of pregnancy, herpesvirus infection significantly
increased the risk for PTB in the presence of low levels of bacterial endotoxin, another
risk factor. In this model, the cervix was the primary viral target in the reproductive tract
[125]. Infection decreased expression of numerous innate immune genes in the cervix
and reduced cervical protection from artificial ascending bacterial infection. Based on
these data, we hypothesize herpesviruses can increase risk for sPTB by affecting cervical
function.

17

Materials and methods
Pregnant mice and infections
Animals were maintained at Michigan State University’s Animal Facility and all
procedures were approved by Michigan State University’s Institutional Animal Care and
Use Committee (IACUC #11-14-198-00). C57BL/6 mice were purchased from Jackson
Laboratory (Bar Harbor, ME); adult female mice (8–12 wks of age) were mated and
confirmed pregnant when vaginal plug was detected. Mice received HSV-2 (106), MHV68
(106), or vehicle, intra-vaginally, on E10.5 after sedation with isoflurane. Redness and
swelling was observed with mice treated with HSV-2, indicating infection. Mice were
humanely sacrificed on E15.5 using CO2 followed by cervical dislocation. The cervix was
flushed with sterile 1XPBS (3x with 30µl) and fixed in 4% PFA overnight. Animal numbers:
HSV-2, n=4; MHV68, n=4; control, n=8.

Histology
Cervices were harvested from mice and immediately placed into 4% paraformaldehyde
rocking at room temperature overnight. Tissues were then washed in 70% ethanol,
placed into Thermo Scientific Excelsior ES tissue processor for routine processing, and
embedded in paraffin wax. For immunohistochemical (IHC) analysis of ER protein, 6µm
sections underwent antigen retrieval by boiling in sodium citrate buffer and treatment with
endogenous biotin inhibitor (X059030-2, Dako, Santa Clara, CA). Tissue sections were
incubated with primary antibody (1:100, ER-α, MC20, Santa Cruz Biotechnology, CA) in

18

antibody diluent (S080981-2, Dako, Santa Clara, CA) overnight at 4C. The following day,
tissue was incubated with secondary antibody (ab6720, 1:200, goat anti-rabbit IgGbiotinylated, Abcam, Cambridge, ME) for 1h, treated with an inhibitor of endogenous
peroxidase (S200380-2, Dako, Santa Clara, CA), and developed with diaminobenzidine
(DAB) substrate-chromogen (K346711-2, Dako, Santa Clara, CA). Negative controls
underwent the same protocol but without primary antibody. For collagen staining, 10µm
tissue sections were stained with picrosirius red (Polysciences Inc, Warrington, PA).
Using a 40x objective and polarized light, a photomicrograph was taken of 10 sections,
at least 20µm apart, per animal. The optical density (OD) of three non-overlapping
sections of each photo was analyzed. Specifically, after photomicrographs were
converted to gray scale, they were inverted and OD was calculated using a calibrated
threshold and the Rodbard standard curve (NIH Image J software). In this analysis, areas
of dark collagen staining have low OD values, and areas with light staining have high OD
values. To account for cell density, the OD was divided by cell nuclei/area. Animal
numbers per group: HSV-2, n=4; MHV68, n=4; control, n=8.

Cell culture and treatments
Immortalized human ectocervical cells (ECT1, CRL-2614, ATCC, Grand Island, NY) were
cultured in keratinocyte serum free medium (17005-042, Gibco, Grand Island, NY) with
bovine pituitary extract and hEGF supplementation as recommended by ATCC under 5%
CO2 at 37°C. Cells were confirmed to be mycoplasma free (13100-01, Southern
Biotechnology, Birmingham, Al). Inhibitors included Src inhibitor cocktail, SKI-1/PP1
(ab120839, ab120859, Abcam, Cambridge, ME) resuspended in DMSO (5µM) and an

19

inhibitor of HA synthesis, 4-methylumbelliferone (4-MU, M1381, Sigma Aldrich, St Louis,
MO) resuspended in methanol (600nM). Cells were treated with inhibitors for 1h prior to
HSV-2 infection (106). Supernatants and cells were collected 24 or 48h after HSV-2
infection, as indicated. To infect ECT-1 with HSV-2, cells were inoculated with HSV-2
(106) in 500 µl for 1h prior to addition of 2.5 mL media (protocol for 35mm plate). Results
from cell culture experiments are representative of 4 independent experiments.

Virus production and quantification
MHV68 was passaged on M2-10B4 cells (CRL-1972, ATCC, Grand Island, NY) in DMEM
plus 10% FBS. HSV-2 was passaged in Vero Cells (CL-81, ATCC, Grand Island, NY) in
DMEM plus 10% FBS. After lysis supernatants were harvested, filtered (0.45 µm pore)
and titered by 10-fold serial dilutions on confluent monolayers. To detect viral titers in
mice, DNA was extracted from the cervix using DNeasy blood and tissue kit (69504,
Qiagen, Valencia, CA). 100ng total DNA was then assayed using primers specific for
MHV68 (forward primer: 5’-CCA-CTG-AGG-GAC-GTA-TGT-GT-3’, reverse primer: 5’TGG-GAT-CAC-CCA-AGA-AAC-CAC-3’) or HSV-2 (forward primer: 5’-GCT-CGA-GTGCGA-AAA-AAC-GTT-3’, reverse primer: 5’-TGC-GGT-TGA-TAA-ACG-CGC-AGT-3’),
and compared to a standard curve. Results reported as copies/100ng DNA.

RNA, cDNA synthesis and qPCR
RNA was extracted from ECT-1 cells using RNeasy RNA extraction kit (74104, Qiagen,
Valencia, CA). RNA concentration and purity was analyzed using spectrophotometric
analyses of 260/280 ratios with exclusions for samples that were below 1.7. For real-time
20

quantitative analysis of mRNA, 1 µg of RNA was reverse transcribed for each sample
using iScript cDNA synthesis kit (170-8891, Bio-Rad, Hercules, CA). The cDNA was
diluted 1:20 in nuclease-free water and 5µl was mixed with SsoAdvanced Universal SYBR
green superscript (172-5270, Bio-Rad, Hercules, CA) and gene specific primers for ERα (forward primer: 5’-GGC-CCC-AGC-TCC-TCC-TCA-T-3’, reverse primer: 5’-ACG-TTCTTG-CAC-TTC-ATG-CTG-TA-3’) and GAPDH (forward primer: 5’-AGG-GCT-GCT-TTTAAC-TCT-GGT-3’, reverse primer: 5’-CCC-CAC-TTG-ATT-TTG-GAG-GGA-3’). Samples
were evaluated with the Applied Biosystems Q-RT PCR machine.

Values were

normalized to GAPDH and calculated using delta delta Ct method; delta delta Ct= delta
ct treated- delta Ct control; results expressed as fold differences are 2ˆ-(average delta
delta Ct) for negative delta delta Ct values or – (2ˆ[average delta delta Ct]) for positive
delta delta Ct values.

Hyaluronic Acid ELISA
Hyaluronic acid concentration was assessed using ELISA (DHYAL0, R&D systems,
Minneapolis, MN).

ECT1-conditioned medium was diluted 1:40, cervical flushes were

diluted 1:160, and samples were assayed according to manufacturer's protocol.
Wavelength correction was used by subtracting 540 nm readings from all readings at 450
nm.

The subtraction was used to correct for optical imperfections in the plate as

recommended by manufacturer.

21

Western blot analysis
Cells were lysed in M-PER Mammalian Protein Extraction Reagent buffer (78503, Pierce,
Rockford, IL), with HALT protease inhibitor. Total protein concentrations were quantified
using BCA assay (23227, Pierce, Rockford, IL). Twenty-five micrograms of total proteins
were dissolved in 1X sample buffer, boiled for 5 minutes and separated on a 5-20% SDSPAGE gel in 1X Tris-Glycine SDS running buffer (Novex, Carlsbad, CA) at a constant
voltage of 125V for 2h. The proteins were transferred to PVDF membranes (0.45 μm,
Novex, Carlsbad, CA) in an XCell II Blot module apparatus (Novex, Carlsbad, CA) at a
constant 25 V for 2hrs. Non-fat milk (NFM) (5%) was used to block non-specific signals
and immunoblotting was performed with a 1:1000 dilution of primary antibodies against
total Src and phospho-Src-Tyr527 (2105, 32G6, Cell Signaling, Danvers, MA), ER-α
(MC20, Santa Cruz Biotechnology, CA), or 1:10,000 dilution of beta-actin (ab16039,
Abcam, Cambridge, ME) in 2% NFM at 4°C overnight. Membranes were washed in 1X
PBST and a 1:10,000 dilution of goat anti-rabbit or goat anti-mouse IgG-horseradish
peroxidase (HRP) conjugate (Cell Signaling, Danvers, Mass) was used as appropriate.
Membranes were developed using HRP substrate (Amersham ECL Prime Detection
Reagent; General Electric, Buckinghamshire, UK) and immunoreactive proteins were
visualized using the Bio-Rad ChemiDoc XRS+.

Statistical analysis
Differences were determined using analysis of variance (ANOVA) (multiple comparisons,
Tukey’s test), or independent t-test functions of Graph pad inSTAT statistical software (La

22

Jolla, CA). A p-value of ≤0.05 was considered significant, different letters denote
significant differences. Data is presented as mean ± standard error of the mean (SEM).
Results and figures
Cervical viral infection affects stromal tissue organization
We first determined if viral infection affected the cellular and/or extra-cellular
structure of the cervical stroma during pregnancy. Mice were infected with MHV68, HSV2
or vehicle, intra-vaginally, at gestational day (gd)10.5, and cervices were collected at
gd15.5. Tissue was fixed, stained for collagen, and examined using bright light, and
polarized light, microscopy. Under bright light, it was readily apparent that infected
cervices had a looser arrangement of collagen fibers compared to controls, as
demonstrated by representative sections from cervices of mice treated with HSV2,
MHV68 or vehicle (Figure 1a). To quantify collagen objectively, the sections were first
viewed under polarized light and converted to gray scale (Figure 1b). Images were then
inverted and OD was calculated using a calibrated threshold and the Rodbard standard
curve (NIH Image J). In this analysis, areas of dark collagen staining have low OD values,
and areas with light staining have high OD values, and the OD is normalized to cell
number by dividing by number of nuclei per μm2. The infected cervix tissue had higher
OD compared to the controls, indicating the collagen was less dense in infected tissue
(Figure 1c), (t-test, p=.0007). The cervical stroma was also less cellular in infected
animals, which was quantified by analyzing the number of nuclei per μm2 (Figure 1d), (ttest, p=.02).

23

Figure 1: Cervical viral infection affects stromal tissue organization
Histological sections from cervices of animals infected with MHV68, HSV-2 or vehicle
were stained with picrosirius red to characterize collagen organization.

(A) A

representative section from pregnant mice infected with HSV2, MHV68 and
representative sections from two control mice (VEH) were imaged under bright light
microscopy (4x) and shown in gray scale. (B) Representative images from control (VEH)
and infected (VIRUS) animals under polarized light and gray scale conversion (40x). (C)
Collagen content and structure was quantified by calculating the optical density (OD)
using 3 non-overlapping photos from 10 sections per animal, normalized to cell
nuclei/μm2. Specifically, after photomicrographs were converted to gray scale (as shown

24

Figure 1 (cont’d): in (B)), they were inverted and OD was calculated using a calibrated
threshold and the Rodbard standard curve. In this analysis, areas of dark collagen
staining have low OD values, and areas with light staining have high OD values (t-test,
p=.0007). (D) Cell density analysis, represented as the number of nuclei per μm2 (t-test,
p=.02). Animal treatments: HSV-2, n=4; MHV68, n=3; vehicle, n=8.

25

Viral infection increases ER-α protein in cervical epithelial cells
Normally, at term, cervical remodeling is mediated by changes in steroid hormone
concentrations within the tissue, characterized by a shift from progesterone to estrogen
dominance [126]. Since viral infection induced similar changes in the cervical stroma, we
examined how infection affected the known mediators of the local hormonal shift. Initially,
the human cervical epithelial cell line, ECT-1, was used to screen the effect of HSV-2
infection on known regulators of cervical endocrinology. While HSV-2 did not affect
progesterone receptor (PR), or 17β -hydroxysteroid dehydrogenase (17 β -HSD)(data not
shown), the infection did increase ER-α (Figure 2a). Furthermore, there was no change
in ESR1 mRNA (Figure 2b), (analysis of variance (ANOVA), n.s.), suggesting HSV-2
stabilized the protein, as opposed to increasing transcription.
We also examined ER-α expression in cervices from infected and control mice at
gd15.5 using immunohistochemistry. In infected mice, there were irregular pockets of
cervical epithelium expressing ER-α, which can be identified by the brown positive
staining (Figure 2c). The cellular staining pattern suggested the receptor was localized
in the cytoplasm, as well as in or around the nucleus of the non-differentiated epithelial
cells (NDE), which were small, lacked mucus and resided beneath the differentiated
mucosal epithelial cells (DME) (Figure 2c). The DME contained large mucus vacuoles
and had little to no positive ER-α staining (Figure 2c). This was in stark contrast to control
tissues that had very little, if any, epithelial cells positive for ER-α (Figure 2c).

26

Figure 2: Viral infection increases ER-α protein in cervical epithelial cells
ECT-1 cells were treated with vehicle (VEH) or HSV-2 for 24, 48h and 72h. (A)
Representative Western blot analysis of ER-α, and actin loading control. (B) Quantitative
PCR was used to analyze ERA mRNA expression in ECT-1 with and without HSV-2
(ANOVA, n.s.). (C) Representative images from control (VEH) and infected (HSV-2)
animals analyzed for ER-α protein using immunohistochemistry. NDE, non-differentiated
epithelial cells; DME, differentiated mucosal epithelial cells. Figures represent results of
4 independent in vitro experiments. Animal treatments: HSV-2, n=4; MHV68, n=3;
vehicle, n=8.

27

Hyaluronic acid is increased by HSV-2 in the pregnant cervix and ECT1 cells
We then considered how the virus, and the associated increase in ER-α protein in
the epithelia, might be related to the changes in the tissue stroma. Hyaluronic acid (HA)
is an important component of the cervical mucus barrier and the stromal ECM. It is also
regulated by estradiol and increases at the end of gestation. Therefore, we analyzed
cervical flushes from pregnant mice with and without viral infection and discovered HA
was significantly increased in the infected mice (Figure 3a, t-test, p=.012). Similarly, HSV2 increased HA in ECT-1 conditioned medium at 48h post-infection (Figure 3b), (24h NT
vs HSV2: t-test, n.s.; 48h NT vs HSV2: t-test, p=.017).

28

Figure 3: Hyaluronic acid is increased by HSV-2 in the pregnant cervix and ECT1
(A) Hyaluronic acid was quantified at gd15.5 in cervical flushes from infected and control
animals using ELISA (t-test, p=.012).

(B) Hyaluronic acid was quantified in ECT-1

conditioned medium 24 or 48h after treatment with vehicle or HSV-2 using ELISA (24h
Veh vs Virus: t-test, n.s.; 48h Veh vs Virus: t-test, p=.017). Animal treatments: HSV-2,
n=4; MHV68, n=3; vehicle, n=8.

HSV-2 and MHV68 treatments analyzed together

compared to vehicle. In vitro results represent 3-4 independent experiments.

29

Herpes simplex virus-2 activation of Src kinase stabilizes ER-α and regulates HA
We next determined how HSV-2 might stabilize ER-α protein and/or induce
changes in HA expression in cervical epithelial cells. The tyrosine kinase Src was
previously shown to be activated by toll-like receptor signaling following viral infection
[125], and Chu et al. showed it to interact with ER-α, therefore we first tested its role in
HSV-associated ER-α stabilization. First we treated ECT-1 with HSV-2 and measured
phosphorylation of Src-Tyr527 at multiple time points from 30m up to 8h post-infection.
This phosphorylation site is an inhibitor of function and acts as the primary regulator of
Src, therefore loss of phosphorylation at Tyr527 indicates Src is in its active form. We
found that Src Tyr527 phosphorylation was not changed at early timepoints following
infection (30m-2h, data not shown), but viral infection reduced phosphorylation, or
activated Src, at 6h and 8h post-infection (Figure 4a). To determine if Src could also affect
ER-α, ECT-1 cells were treated with Src inhibitor, SKI/PP1, which resulted in decreased
ER-α protein (Figure 4b). To test if HSV-associated changes in ER-α were mediated by
Src kinase, we inhibited Src (SKI-PP1) prior to HSV-2 infection and again measured ERα protein. When ECT-1 cells were treated with the Src inhibitor, HSV-2 infection no longer
caused an increase in ER-α protein (Figure 4c) suggesting HSV-2 mediates ER-α protein
stability by activating Src kinase in cervical epithelial cells. Finally, we tested how
activation of Src affected HSV-associated changes in HA. When we infected ECT-1 with
HSV-2 in the presence of Src inhibitors (SKI-PP1), the virus-associated increase in HA
was diminished (Figure 4d), (ANOVA; Tukey’s multiple comparison; a<b, p=.012; a>c,
p=.026; b>c, p=.0003). Therefore, Src kinase could be an important mediator of the
cervical response to HSV-2.

30

Figure 4: Herpes simplex virus-2 regulates ER-α and HA via Src kinase activation
(A) Src kinase activation was characterized in ECT-1 cells 4, 6, and 8h post-infection with
HSV-2 using Western blot analysis with an antibody specific for Src Tyr527
phosphorylation or total Src. (B) ECT-1 cells were treated with vehicle (DMSO) or Src
inhibitor (SKI/PP1) and ER-α protein was quantified using Western blot analysis. (C)
ECT-1 cells were treated with HSV-2, HSV-2 and DMSO, or HSV-2 and Src kinase
inhibitor, and ER-α protein was quantified using Western blot analysis. (D) ECT-1 were
treated with vehicle (DMSO), vehicle and HSV-2, or Src inhibitor (SKI-PP1) and HSV-2

31

Figure 4 (cont’d): for 24h and HA was quantified in the conditioned medium using ELISA
(ANOVA with Tukey’s multiple comparison; a<b, p=.012; a>c, p=.026; b>c, p=.0003).
Figures represent results of 4 independent in vitro experiments.

32

Hyaluronic acid mediates HSV-2 infection
Interestingly, HA has also been shown to facilitate viral infection of host cells [127].
Therefore, since HSV-2 upregulated HA, we tested its role in mediating HSV-2 infection
of cervical epithelial cells. ECT-1 cells were treated with 4-methylumbelliferone (4-MU),
an inhibitor of HA synthesis, which resulted in down-regulation of HA (Figure 5a), (t-test,
p=.003). Cells were then infected with HSV-2, and viral titers were analyzed 24h postinfection by measuring gene copy number. Inhibition of HA resulted in a drastic decrease
in HSV-2 infection (Figure 5b), (t-test, p=.019). Inhibition of Src kinase (SKI-PP1) also
resulted in a similar decrease in HSV-2 infection (Figure 5c), (t-test, p=.006). Therefore,
the HSV-associated increases in HA could result in increased susceptibility to epithelial
cell infection, in addition to the potential changes in cervix tissue structure.

33

Figure 5: Hyaluronic acid mediates HSV-2 infection
(A) ECT-1 cells were treated with vehicle (VEH, methanol) or 4-MU, an inhibitor of HA
synthesis, and HA was quantified in conditioned medium (t-test, p=.003). (B) ECT-1 cells
were treated with vehicle (VEH) or 4-MU preceding HSV-2 infection, and viral copy
number was analyzed using qPCR (t-test, p=.019). ECT-1 were treated with vehicle
(VEH) or Src inhibitor (SKI-PP1) preceding HSV-2 infection and HSV-2 copy number was
analyzed using qPCR (t-test; p=.006). Figures represent results of 4 independent in vitro
experiments.

34

CHAPTER 3:
CONCLUSIONS AND DISCUSSION

35

For the first time, to our knowledge, we report that intra-vaginal herpesvirus
infection causes cervical remodeling in pregnant mice. Herpesvirus infection of the
cervical epithelium was associated with increased expression of ER-α, increased cell
proliferation, and upregulation of HA synthesis and/or secretion. These changes were
associated with significant disorganization and/or loss of collagen within the cervical
stroma.

Using ECT-1 cells, we discovered that HSV-2 activated Src kinase, which

mediated the increase in ER and HA. Infection with HSV-2, activation of Src, and
increased HA also increased the susceptibility of cervical epithelial cells to HSV-2
infection. These results demonstrate viral infection can significantly change the cervix
during pregnancy. We propose viral infection can induce cervical insufficiency, which
increases risk of ascending infection, intra-amniotic inflammation and preterm labor[128].
Cervical ripening in response to hormones has been best characterized in rodent
models. Typically, and in the absence of bacterial infection, cervical ripening is initiated
by the switch from high P4 to high E2 at the end of pregnancy. Previously this raised
concerns about the model, since rodents experience a sharp decline in circulating P4 at
the end of pregnancy, while P4 is maintained in women. These concerns were in some
way overcome by the discovery that, in both species, cervical ripening was regulated by
changes in local hormone metabolism, which resulted in the loss of P4 within the tissue.
In rodents, upregulation of SRD5α1 decreases P4 concentration, while in humans
downregulation of 17β-HSD reduces P4 and increased E2 synthesis in the cervix.
Therefore, since we considered the rodent model reasonable, we used a mouse model
to study herpesvirus infection of the cervix during pregnancy.

36

We were intrigued to discover viral infection increased ER-α in cervical epithelial
cells. Availability of ER has been shown to be directly associated with E2 function in
multiple cell types, therefore viral infection likely increases E2 sensitivity in the epithelium.
We also observed dramatic reorganization of the cervical stroma, including reduced cell
density and loosening of the collagen fiber network. These changes can be triggered by
the hormone environment, or by an inflammatory cascade mediated by immune cells
[129]. We speculate that viral infection could affect the cervix both ways, by increasing
local E2-sensitivity and activating immune cells. Future investigation into the mechanism
of virus-induced cervical changes will be important for development of clinical
interventions.
Hyaluronic acid synthesis is also associated with hormone changes [130], and
infection [62], induced cervical ripening and is a result of increased expression of HA
synthase-2 or -3 (HAS2/3). Interestingly, HAS2 is positively regulated by estradiol in the
cervix[131], which is relevant to our study because HA was increased in infected cervices,
which was associated with the increased ER-α in cervical epithelia. We could not use the
ECT-1 to determine the role of ER in HSV-induced HA synthesis because cervical
epithelial cells do not properly respond to E2 without the stroma, but we did determine HA
synthesis was dependent on Src kinase activation following viral infection. Therefore, Src
kinase could be an important mediator of the virus-associated changes in cervical
function. Although the role of ER in our model is still unknown, we hypothesize it mediates
some of the cervical changes downstream of viral infection and Src activation. This is
being tested in ongoing in vivo studies of infection with an ER antagonist.

37

Finally, we tried to identify factors that might affect the susceptibility of the cervix
to HSV-2 infection. Herpes simplex virus-2 can readily infect primary human cervical
epithelial cells, and infection can be reduced using the antiviral drug, Acyclovir, or the
ligand for toll-like receptor-3, Poly (I:C) [132]. Infection can also be affected by sexhormone treatment. In primary human epithelial and stromal cell co-cultures, treatment
with E2 resulted in increased infection while P4 decreased HSV-2 infectivity [132]. In a
mouse model, there was no susceptibility to genital infection during estrus (high E2), while
animals in diestrus or treated with P4 had high susceptibility to HSV-2 [133, 134]. It is still
unclear how genital tract infection is affected by sex hormones and susceptibility to HSV2 during pregnancy has not been established.

Interestingly, HA and other

glycosaminoglycans, such as heparin sulfate, are established mediators of herpesvirus
viral entry into host cells. We determined that HA did affect HSV-2 infection of cervical
epithelial cells. Indeed, loss of HA by direct inhibition with 4-MU or inhibition of Src kinase
drastically reduced HSV-2 infection of ECT-1. Collectively, the data suggests that HSV2 infection upregulates HA, which in turn, further increases viral susceptibility.
In conclusion, we propose that virus-associated activation of Src kinase, and the
associated stabilization of ER, and HA synthesis in the epithelium, affects the structure
of the cervical stroma and further increases the susceptibility to infection (Figure 6).
Ongoing studies are investigating the causal link between the virus-associated changes
in the epithelia and the stromal remodeling. Also, while not addressed in this study, local
immune cells are also affected by cervical viral infection, and it is likely that these cells
also contribute to the remodeling of the ECM.

Studies like this are necessary to

understand how viral infection could increase the risk for sPTB in women. Based on these

38

results, we could predict that HSV-2 infection might weaken the cervix or even induce
premature cervical ripening in some women. It is our hope that understanding the
mechanism of virus-induced cervical remodeling will help us provide targets for clinical
intervention.

39

Figure 6: Proposed model of cervical viral infection during pregnancy
Herpesvirus infection of the pregnant cervix bind surface receptors, such as integrins and
HA, resulting in viral entry. Infection causes activation of Src kinase, which stabilizes ER,
increasing ER concentration and potentially estrogen sensitivity. These changes result
in increased HA expression on the surface of epithelial cells, which in turn, increases viral
entry into the cells. Increased soluble HA affects the structure of the cervical stroma.
These changes are associated with decreased collagen and cellular density of the
stroma.

40

As we continue this investigation we hope to address several remaining questions.
One of the primary questions is the potential role that immune cells are playing in
remodeling after the initial infection. If immune cells are recruited to the cervix, what is
their function there? We want to study MMPs and their potential role in the changing
collagen. Understanding if MMPs are being created immune cells, fibroblasts, or both
could create potential for development of therapeutic treatments for at-risk women. Better
understanding the role of estrogen and its receptors can be achieved with utilizing an
estrogen receptor antagonist, and would allow us to both identify critical factors controlled
by estrogen and turn to the potential of rescuing a phenotype of term-birth. Finally, to
address the idea of PTB as a syndrome, we could attempt to test viral infections with
other causes of preterm birth in mice.
.

41

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