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Sci Immunol. Author manuscript; available in PMC 2017 April 01.
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Published in final edited form as:

Sci Immunol. 2016 October ; 1(4): .
GROUP B STREPTOCOCCUS CIRCUMVENTS NEUTROPHILS

AND NEUTROPHIL EXTRACELLULAR TRAPS DURING
AMNIOTIC CAVITY INVASION AND PRETERM LABOR
Erica Boldenow1, Claire Gendrin1,*, Lisa Ngo1,*, Craig Bierle1,*, Jay Vornhagen1,2,*, Michelle
Coleman1, Sean Merillat1, Blair Armistead1,2, Christopher Whidbey1,2, Varchita Alishetti1,
Veronica Santana-Ufret1, Jason Ogle3, Michael Gough3, Sengkeo Srinouanprachanh4,
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James W MacDonald4, Theo K Bammler4, Aasthaa Bansal5, H. Denny Liggitt6, Lakshmi

Rajagopal1,2,#, and Kristina M Adams Waldorf7,#
1Department of Pediatric Infectious Diseases, University of Washington and Seattle Children’s
Research Institute, Seattle, Washington, United States of America
2Department of Global Health, University of Washington, Seattle, Washington, United States of
America
3Washington National Primate Center, University of Washington, Seattle, Washington, United
States of America
4Department of Environmental and Occupational Health Sciences, University of Washington,
Seattle, Washington, United States of America
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5Department of Pharmacy, University of Washington, Seattle, Washington, United States of

America
6Department of Comparative Medicine, University of Washington, Seattle, Washington, United
States of America
7Department of Obstetrics & Gynecology, University of Washington, Seattle, Washington, United
States of America
Abstract
Preterm birth is a leading cause of neonatal morbidity and mortality. Although microbial invasion
of the amniotic cavity (MIAC) is associated with the majority of early preterm births, the temporal
events that occur during MIAC and preterm labor are not known. Group B Streptococci (GBS) are
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β-hemolytic, gram-positive bacteria, which commonly colonize the vagina but have been
#
Address correspondence to Kristina Adams Waldorf, adamsk@u.washington.edu and Lakshmi Rajagopal,
lakshmi.rajagopal@seattlechildrens.org.
*Equal Contribution
AUTHOR CONTRIBUTIONS: E. B., C.G., J.V., L.R and K.A.W designed the research. E. B., C.G., L.N., C. B., J. V., M.C., S.M.,
B.A., C.W., V.A., V. S-U., J.O., M.G., S.S., L.R and K.A.W performed the experiments. E. B., C.G., L.N., C. B., J. V., M.C., S.M.,
B.A., C.W., V.A., S.S., J. W.M., T.K.B., A.B., H.D.L., L.R., and K.A.W analyzed the results. E.B., C.G., J. V., T.K.B., H.D.L., L.R.,
and K.A.W wrote the paper.
COMPETING INTERESTS: The authors declare no competing financial interests.
DATA AND MATERIALS AVAILABILITY: The microarray data for this study have been deposited in the GEO database and is
available through the GEO accession number GSE80248.
Boldenow et al. Page 2
recovered from the amniotic fluid in preterm birth cases. To understand temporal events that occur
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during MIAC, we utilized a unique chronically catheterized nonhuman primate model that closely
emulates human pregnancy. This model allows monitoring of uterine contractions, timing of
MIAC and immune responses during pregnancy-associated infections. Here, we show that adverse
outcomes such as preterm labor, MIAC, and fetal sepsis were observed more frequently during
infection with hemolytic GBS when compared to nonhemolytic GBS. Although MIAC was
associated with systematic progression in chorioamnionitis beginning with chorionic vasculitis and
progressing to neutrophilic infiltration, the ability of the GBS hemolytic pigment toxin to induce
neutrophil cell death and subvert killing by neutrophil extracellular traps (NETs) in placental
membranes in vivo facilitated MIAC and fetal injury. Furthermore, compared to maternal
neutrophils, fetal neutrophils exhibit decreased neutrophil elastase activity and impaired
phagocytic functions to GBS. Collectively, our studies demonstrate how a unique bacterial
hemolytic lipid toxin enables GBS to circumvent neutrophils and NETs in placental membranes to
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induce fetal injury and preterm labor.
INTRODUCTION
Preterm birth is a leading cause of neonatal morbidity and a direct cause of one-third of
neonatal deaths (1, 2). Intraamniotic infection and inflammation are major risk factors for
fetal injury, early preterm births, stillbirths, and early onset fulminant neonatal infections (3,
4). The infected amniotic fluid often contains organisms typically colonizing the lower
genital tract including Group B Streptococcus (GBS; Streptococcus agalactiae) (5, 6).
GBS are β-hemolytic, gram-positive bacteria that typically exist as recto-vaginal colonizers
in healthy adult women. However, during pregnancy ascending GBS infection can lead to
fetal injury, stillbirth or preterm birth. Despite the success of intrapartum antibiotic
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prophylaxis to prevent maternal to infant transmission during labor and delivery, GBS
remains a leading cause of neonatal morbidity and mortality (7, 8). Effective therapies to
prevent GBS fetal sepsis, preterm birth or stillbirth are lacking. A recent report indicated that
maternal colonization of GBS can be associated with increased rates of infants being
transferred to the neonatal intensive care unit (9). Furthermore, maternal sepsis due to GBS
can predispose infants to adverse outcomes that include preterm birth or stillbirth (10). A
better understanding of host immune responses in the placenta that normally protect the
fetus from ascending infection of lower genital tract organisms like GBS, is pivotal to
development of preventive therapies.
Although a comprehensive understanding of GBS virulence factors that enable the pathogen
to breach placental membranes and induce preterm birth or stillbirth are lacking, we recently
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showed that increased expression of the hemolytic pigment enables GBS to penetrate human
placental membranes ex vivo (11). We have also shown that hyperhemolytic/hyperpigmented
GBS strains, some with mutations in the transcriptional repressor of the hemolytic pigment
known as CovR/CovS (or CsrR/CsrS), can be isolated from the amniotic fluid and placental
(chorioamniotic) membranes of women in preterm labor (11). Further, we and others have
demonstrated that expression of the hemolytic pigment induces fetal death in pregnant
mouse models of GBS infection (12, 13).

Despite these advances, there are limitations to the above model systems. For example, the
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use of human placental membranes ex vivo does not permit investigation of host immune
cells, which may be recruited to prevent microbial invasion of the amniotic fluid and fetus
during pregnancy. Also, as antibiotics are routinely administered during Cesarean sections
(14–16), the transfer of these antibiotics to the human placenta can impose limitations on
bacterial studies performed with placental membranes ex vivo. Although animal models of
pregnancy address the role of host immune defenses during an active infection, lower
mammalian models differ significantly from human pregnancy in key respects including
dissimilarities in reproductive anatomy, placentation, mechanism of labor onset and
sensitivity to pathogens. In contrast, the pregnant nonhuman primate (NHP) emulates human
pregnancy and is considered the closest animal model for studies related to human
pregnancy (17–20). Similarities of NHP to humans include reproductive anatomy, number of
fetuses (singleton), long gestational period (160–170 days), type and structure of placenta
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(hemomonochorial), initiation of labor (hormonal control of parturition), sensitivity to

pathogens and timeline of fetal lung and brain development (19, 20). In our chronically
catheterized pregnant NHP model (21) we are able to inoculate bacteria at the
choriodecidual space, which lies between the uterine muscle and the placental membranes,
where bacteria first encounter the maternal-fetal interface during ascending infection from
the lower genital tract (4, 21).
To elucidate temporal events that occur during MIAC and preterm labor, we used the
chronically catheterized pregnant NHP model (21). Previous studies using this model
revealed that choriodecidual inoculation of a wild type GBS strain (serotype III, strain
COH1) induced cytokine production that was associated with fetal lung injury without
MIAC or overt chorioamnionitis or preterm labor (21). Interestingly, this human isolate of
GBS is mildly hemolytic/pigmented in contrast to certain other GBS strains (22). As GBS
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strains with increased expression of the hemolytic pigment were recovered from the
amniotic fluid (AF) of women in preterm labor (11), we utilized the chronically catheterized
pregnant NHP model to understand how hyperpigmented GBS (lacking the gene covR,
GBSΔcovR) evade host immune responses in vivo during MIAC. Although CovR/S is a
transcriptional repressor of the cyl genes important for hemolytic pigment expression, this
two-component system also controls the expression of more than 100 genes in GBS (23–25).
Therefore, to evaluate the role of the hemolytic pigment on MIAC and preterm labor in the
NHP model, we also included an isogenic, nonpigmented GBS covR mutant that lacked the
gene cylE important for hemolytic pigment expression (11, 22), as a control
(GBSΔcovRΔcylE). Here, we show that hyperpigmented GBS rapidly invaded the AF and
induced preterm labor in pregnant NHP due, in part, to the ability of the hemolytic pigment
to induce neutrophil cell death and evade killing by neutrophil extracellular traps (NETs).
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RESULTS
Hyperhemolytic GBS induces adverse pregnancy outcomes
To understand how the hemolytic pigment may promote GBS invasion of the amniotic fluid
and fetus, we utilized our unique chronically catheterized NHP model. Ten animals received
choriodecidual inoculations of 1–3 × 108 colony forming units (CFU) of either

hyperpigmented GBSΔcovR (n=5) or control nonpigmented GBSΔcovRΔcylE (n=5); these

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results were compared with saline controls (n=5) that were previously described (21).
The primary and secondary study outcomes from this study are shown in Table 1. Our
primary outcome was a composite of preterm labor and/or MIAC, because either event
results in a poor pregnancy outcome. We observed that inoculation of the hyperpigmented
ΔcovR was associated with an adverse pregnancy outcome in 5/5 (100%) animals when
compared to 2/5 (40%) animals inoculated with the nonhemolytic GBSΔcovRΔcylE or 0/5
(0%) saline controls (Table 1). Preterm labor occurred in 4/5 animals inoculated with
GBSΔcovR (excluding GBSΔcovR 3) compared to 1/5 with GBSΔcovRΔcylE and 0/5 saline
controls (Tables 1 and S1). In the other animal inoculated with hyperpigmented GBSΔcovR
(GBSΔcovR 3), the amniotic fluid (AF) became dark and cloudy due to MIAC at 12 hours
after inoculation, the decision was made to proceed with Cesarean section earlier than the
defined study endpoint (preterm labor) to avoid stillbirth due to fetal sepsis. At the time of
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Cesarean section at 48 hours post inoculation, this animal (GBSΔcovR 3), had a sustained
pattern of increased uterine contractions, but had not yet made cervical change to meet
criteria for development of preterm labor. Overall, MIAC and fetal sepsis were observed in
3/5 animals inoculated with GBSΔcovR versus 1/5 animals inoculated with
GBSΔcovRΔcylE and 0/5 saline controls (Tables 1 & S1, Figs. 1, S1 and S2). In all cases of
MIAC, the fetus became septic and GBS could be recovered from multiple organs. Overall,
the bacterial burden in GBS infected fetal organs ranged from 102–106 CFU/g tissue with
consistently more bacteria recovered from the fetal lung when compared to the other fetal
organs (Fig. S3).
An increase in uterine activity was often seen within a few hours following inoculation of
GBSΔcovR with MIAC and preterm labor occurred rapidly in most of these cases. Three
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animals from the GBSΔcovR group (GBSΔcovR 1, 2, 3; see Tables 1 & S1; Fig. 1B, S1A &
S1B) developed sustained uterine contractions within hours after inoculation; bacteria were
recovered as early as within 15 minutes (0.25 hours) in one animal, 45 minutes (0.75 hours)
in another and within 12 hours in the third case. Due to the increase in uterine contractions
and cervical dilation (GBSΔcovR 1, 2) or dark and cloudy AF (due to bacteria) that imposed
concerns for stillbirth (GBSΔcovR 3), a Cesarean section was performed within 6, 24 and 48
hours after inoculation, respectively (Table S1). In all these cases, GBS was recovered from
fetal organs (Table S1, & Fig. S3). In the remaining two animals infected with GBSΔcovR,
rapid uterine contractions and cervical dilation indicative of preterm labor were seen without
MIAC resulting in Cesarean section at 24 and 72 hours post inoculation, respectively
(GBSΔcovR 4 and 5; Table S1, Fig. S1C & D).
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In the 5 animals infected with the nonpigmented isogenic control GBSΔcovRΔcylE (see
Tables 1 & S1; Fig. 1C & S2), adverse outcomes were detected in two animals
(GBSΔcovRΔcylE 1, 3). Preterm labor developed in one animal without MIAC
(GBSΔcovRΔcylE 3; see Tables 1 & S1; Fig. S2D). In a second animal (GBSΔcovRΔcylE
1), MIAC without preterm labor was detected at the time of Cesarean section on day 3 (Fig.
S2C). Unfortunately, AF could not be recovered from the amniotic catheter until the
experimental end point on day 3 and thus we could not determine the time course of MIAC;
GBS were recovered from the fetal organs of this animal (Fig. S3). The other animals did

not exhibit signs of preterm labor or MIAC. Taken together, our results indicate that
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choriodecidual inoculation of hyperpigmented GBSΔcovR induced adverse outcomes such

as preterm labor, MIAC with concerns for stillbirth and fetal sepsis more frequently than in
animals inoculated with nonpigmented GBSΔcovRΔcylE or in saline controls (P =0.03,
GBSΔcovR vs. GBSΔcovRΔcylE; P =0.0009, GBSΔcovR vs. saline).
Increased amniotic fluid and fetal cytokines are observed in pregnant NHP infected with
hyperpigmented GBS
We then examined cytokine responses in the AF and fetal tissues. Levels of AF interleukin-1
beta (IL-1β), tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and interleukin 8
(IL-8) were all significantly higher in animals inoculated with hyperpigmented GBSΔcovR
versus saline controls (Table 1, all P <0.05). In animals inoculated with nonpigmented
GBSΔcovRΔcylE, AF IL-1β and IL-8 were significantly higher compared to saline controls
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(Table 1, P <0.05), but levels of TNF-α and IL-6 were not significantly different. The effect
of hemolytic pigment expression was assessed by comparing mean peak levels of AF
cytokines between animals infected with GBSΔcovR compared to GBSΔcovRΔcylE.
Notably, the AF mean peak levels of IL- 6 and IL-8 were significantly higher in
hyperpigmented GBSΔcovR inoculated animals versus those inoculated with nonpigmented
GBSΔcovRΔcylE (Table 1, P <0.05). At delivery, fetal plasma IL-1β, TNF-α, IL-6 and IL-8
levels were all significantly higher in the GBSΔcovR compared to saline controls (Table 1;
all P <0.05); fetal IL-8 showed a trend towards higher levels in animals inoculated with
GBSΔcovR compared to nonpigmented GBSΔcovRΔcylE groups (P =0.06). IL-1β and TNF-
α were also higher in animals infected with GBSΔcovRΔcylE compared to saline controls.
Regardless of MIAC, levels of TNF-α were significantly increased in fetal organs of animals
infected with GBSΔcovR compared to GBSΔcovRΔcylE (Fig. S3), suggesting the onset of
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severe fetal systemic inflammation (26).
Microbial invasion of the amniotic cavity correlated with neutrophil recruitment

We next compared histological sections of the chorioamniotic membranes at the inoculation
site from the hyperpigmented GBSΔcovR, nonpigmented GBSΔcovRΔcylE and saline
controls. Histological lesions associated with placental infection of GBSΔcovR appeared to
rapidly progress and were dominated by widespread accumulation of neutrophils and
necrosis as the time interval from inoculation to delivery increased. Within 6 hours of
GBSΔcovR inoculation neutrophils aggregated within blood vessels in the chorion,
marginated to the endothelium and occasionally were seen surrounding small vessels within
the chorionic trophoblast layer. At this early timepoint, neutrophils also lined the interface
between chorion and trophoblast layers, but did not extend into the chorion or amnion (Fig.
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2A and D, corresponds to animal shown in Fig. S1B). By 24 hours after inoculation,

neutrophil accumulation was more pronounced with wide, dense layers of neutrophils in the
chorion without invasion of the amnion (Fig. 2B and E, corresponds to animal shown in Fig.
S1A). Small vessels in the chorion and trophoblast layers appeared thrombosed with focal
areas of necrosis. At 48 hours post inoculation, there was widespread severe inflammation
and necrosis, which was predominantly neutrophilic and involved all layers of the
chorioamnion including the amniotic epithelium. (Fig. 2C and F, corresponds to animal
shown in Fig. 1B).

With the exception of a single animal where MIAC was observed, there was minimal to no
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inflammation within the chorioamniotic membranes from animals inoculated with

nonpigmented GBSΔcovRΔcylE; neutrophils were occasionally seen within deeper regions
of the placenta (Fig. 2H and J; corresponds to animal shown in Fig. 1C). In the case of the
animal with microbial invasion of GBSΔcovRΔcylE, neutrophil infiltration of the
membranes was severe, but the extent of necrosis and neutrophil density was less than in the
most affected hypervirulent GBSΔcovR infected animal (Fig. 2C and F).
Taken together, these observations indicate that neutrophil recruitment into the
chorioamniotic membranes is the primary placental innate immune response to bacterial
infection. However, frequent MIAC as observed with GBSΔcovR suggests that despite
neutrophil recruitment, the placental innate immune response may be inadequate or rendered
ineffective to prevent bacterial trafficking into the amniotic cavity.
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Hyperpigmented GBS induce transcription of genes associated with neutrophil

recruitment in chorioamniotic membranes
To further investigate the innate immune response following infection by hyperpigmented
(ΔcovR) and nonpigmented GBS (ΔcovRΔcylE), we performed microarray analysis on RNA
isolated from the inoculation site of the chorioamniotic membranes in GBS infected animals
versus saline controls (n=5/group). Out of a total of 31,740 probesets for host gene
expression, 797 were differentially regulated in chorioamniotic membranes between animals
inoculated with GBSΔcovR compared to saline controls and 530 were differentially
regulated between animals inoculated with GBSΔcovR compared to those inoculated with
GBSΔcovRΔcylE (≥1.5 fold change; P <0.005; heatmap shown in Fig. S4). A subset of
differentially expressed probe sets is shown in Table S2 and the entire list is available
through GEO link GSE80248 ( http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?
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token=cpifasaozdulpkl&acc=GSE80248.) Approximately half of the differentially regulated

probe sets for host gene expression (263) between GBSΔcovR and GBSΔcovRΔcylE
infected animals were also significant in the animals infected with GBSΔcovR versus saline
suggesting that the pigment has an outsized effect on gene expression compared to other
GBS factors (Venn diagram shown in Fig. S5A). However, many genes that showed
increased expression in the GBSΔcovR infected animals show similar trends in the one
GBSΔcovRΔcylE infected animal that exhibited MIAC (Fig. S4). These data suggest that
some of the pigment-mediated effect on host gene expression may, in part, be due to its
ability to promote MIAC.
Overall, significantly upregulated genes in animals inoculated with GBSΔcovR compared to

saline controls or GBSΔcovR compared to GBSΔcovRΔcylE included genes encoding
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factors associated with inflammation, chemokines, cytokines and cell adhesion molecules
such as matrix metallopeptidase 8 (MMP8), interleukin-1 alpha (IL-1A), interleukin-1 beta
(IL-1B), vanin 2 (VNN2), chemokine receptor (CXCR1), and Selectin-L (SELL).
Interestingly, apart from factors associated with preterm labor such as matrix
metalloproteinases and pro-inflammatory cytokines and chemokines, GBSΔcovR infected
membranes showed increased expression of cell adhesion molecules associated with

neutrophil rolling, transmigration and arrest such as CD177, Selectin-L, and ICAM-1 (27,
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28) which is consistent with the neutrophil influx observed in these membranes (Fig. 2).
Downregulated genes included genes important for maintenance of cell-cell junctions and
cytoskeletal organization such as desmocollin 2 (DSC2), desmoplakin (DSP), integrin alpha
6 (ITGA-6), neuroepithelial cell-transforming gene 1 (NET1), occludin (OCLN) and ADAM
metallopeptidase (ADAMTS6). These data indicate that the chorioamniotic membranes
responded to infection by hyperpathogenic GBS by increasing expression of factors that
directly promote neutrophil recruitment and also diminishing expression of factors that
maintain cytoskeletal organization and the adhesive properties of cell layers such as
desmosomes and tight junctions. Decreased expression of desmosome and tight junction
proteins may facilitate neutrophil transmigration (29). A panel of selected genes was
validated by real-time quantitative reverse transcriptase polymerase chain reaction (qRT-
PCR). Levels of gene expression obtained with amplified RNA samples were compared to
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β-actin as a control for each sample. There was a significant correlation between microarray
and qRT-PCR for differential gene expression between GBSΔcovR versus saline control
animals (Fig. S5B, all P <0.05) and for animals inoculated with the two GBS strains for
IL-1β, IL-1α, IL18RAP, CCDC6, NET1, SORBS2 and PIK3C2G (Fig. S5C, all P <0.05).
Other genes tested by qRT-PCR showed similar trends of positive or negative gene
expression when compared with the microarray analysis (Fig. S5C).
The hemolytic GBS pigment and hyperpigmented GBS induce neutrophil cell death
Given that neutrophils were the primary immune cells observed in the chorioamnion of
pregnant NHP infected with GBSΔcovR (Fig. 2), we asked if the frequent MIAC observed in
GBSΔcovR animals could, in part, be attributed to the ability of the hemolytic GBS pigment
to induce neutrophil cell death. While some studies have indicated that hemolytic GBS
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strains can induce either apoptosis or pyroptosis in macrophages (30, 31), how the GBS
hemolytic pigment modulates neutrophil function is not completely understood. Since our
discovery that the GBS hemolysin is the ornithine rhamnolipid pigment and not a protein
toxin (11), we have shown that the purified hemolytic pigment induces osmotic lysis of red
blood cells, is cytotoxic to human amniotic epithelial cells (hAECs) and promotes NLRP3
dependent pyroptosis in macrophages (11, 12). To test the possibility that the purified GBS
hemolytic lipid toxin may induce neutrophil cytotoxicity, primary human neutrophils were
isolated from fresh adult human blood. Using flow cytometry, we confirmed that ~92.7% of
the cells that were isolated were positive for the neutrophil markers CD15 and CD16 (Fig.
S6). The isolated neutrophils were then exposed to various concentrations of purified GBS
pigment (0.625–5μM) for four hours and cytotoxicity was estimated by measuring the
release of lactate dehydrogenase (LDH) as described (32). As controls, we included an
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equivalent amount of ΔcylE extract (i.e. pigment extraction procedure was performed using
the nonpigmented GBSΔcylE strain) or Buffer DTS (DTS: DMSO+0.1%TFA+20% starch;
see materials and methods and (11, 12)). The data shown in Fig. 3A indicates that
cytotoxicity or percent cell death was significantly higher in neutrophils treated with the
hemolytic GBS pigment when compared to control ΔcylE extract or DTS buffer. To confirm
that hemolytic pigment mediated neutrophil cell death was also observed with live bacteria,
neutrophils were treated with GBS strains namely WT COH1 or isogenic mutants

(hyperpigmented ΔcovR or nonpigmented strains i.e. ΔcovRΔcylE or ΔcylE) at various

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multiplicity of infection (MOI: 1, 10, 100) for four hours and cytotoxicity was measured by
LDH release as described (12, 32). Similar to observations with purified pigment,
hyperpigmented GBSΔcovR induced neutrophil cell death in a dose dependent manner (Fig.
3B), whereas no significant neutrophil cell death was observed with WT GBS or the
nonpigmented GBS strains ΔcovRΔcylE and ΔcylE at any MOI tested. These data indicate
that GBS hemolytic pigment induces neutrophil cytotoxicity in a dose dependent manner.
The hemolytic GBS pigment and hyperpigmented GBS induce morphological changes in
neutrophils
We next performed scanning electron microscopy (SEM) to determine if GBS pigment or
hyperpigmented GBS induce morphological changes to neutrophils. To this end, adult
neutrophils were treated with 0.5 μM pigment or an equivalent amount of control ΔcylE
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extract for 10 min. Untreated neutrophils were included as controls. The results shown in
Fig. 3C (see top panel) indicate that the morphology of untreated or resting neutrophils (see
Media in Fig. 3C) is rounded as shown previously (33). In contrast, neutrophils exposed to
the GBS hemolytic pigment exhibit severe morphological changes when compared to
untreated neutrophils or those treated with the control ΔcylE extract (Fig. 3C). Consistent
with these observations, neutrophils exposed to the nonpigmented GBSΔcovRΔcylE strain
also had a greater number of rounded or partially rounded cells when compared to
neutrophils exposed to the hyperpigmented GBSΔcovR strain (see bottom panel in Fig. 3C).
Collectively, these data indicate that the hemolytic pigment of GBS accelerates
morphological changes in neutrophils ultimately leading to neutrophil cell death.
GBS pigment induced neutrophil cell death is independent of apoptosis or pyroptosis

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As neutrophils typically undergo cell death via apoptosis (34), we first examined the
possibility that the GBS hemolytic pigment may accelerate neutrophil apoptosis. Therefore,
we compared the ability of GBS pigment/lipid toxin to induce membrane permeabilization
and apoptosis in neutrophils. To this end, neutrophils were either treated with GBS pigment
(0.5μM) or controls (ΔcylE extract or buffer DTS). Then, we measured uptake of the
membrane impermeable dye propidium iodide (PI) at various times post treatment. A
significant increase in the number of fluorescent (PI positive) cells was observed in
neutrophils at 30 and 60 min post treatment with GBS pigment indicating loss of membrane
integrity, which was not seen in controls (ΔcylE or buffer DTS, see Fig. S7 A–C). To
determine if the neutrophil membrane damage induced by pigment was preceded by
classical events in apoptosis [extracellular exposure of phosphatidylserine (PS)], we
examined binding of fluorescent Annexin V (AV). We observed that uptake of fluorescent PI
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(Fig. S7 A, B, C) preceded AV staining (Fig. S7 D, E, F) in pigment treated neutrophils.

These data suggest that pigment induced neutrophil cell death was independent of apoptosis.
We next examined if the purified hemolytic pigment or hyperpigmented GBS induced

neutrophils to undergo an inflammatory form of cell death known as pyroptosis, similar to
our observations with macrophages (12). As release of IL-1β is a characteristic feature of
pyroptosis (35), we measured cytokine levels using luminex bead assays on supernatants
obtained from neutrophils treated with GBS strains (WT, ΔcovR, ΔcovRΔcylE, ΔcylE) or

pigment and controls for 4 hours (see materials and methods). Interestingly, levels of
cytokines IL1β, IL-6, TNFα, GRO- α, IFNγ or IL-10 were all below the limit of detection.
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Notably, only WT and nonpigmented/nonhemolytic GBS strains (ΔcovRΔcylE, ΔcylE)

triggered the release of IL-8 from neutrophils. IL-8 release was not observed with
hyperpigmented GBS (ΔcovR, Fig. S8A) likely due to pigment mediated neutrophil cell
death. Also, significant activation of caspase 3/7 was not observed in pigment treated
neutrophils when compared to the positive control staurosporine (Fig. S8B). Collectively,
these data suggest that GBS pigment induction of neutrophil cell death is not due to
apoptosis or pyroptosis but may involve other pathways (i.e. lysis or necrosis), which likely
contribute to the adverse outcomes observed during GBS pregnancy associated infection.
GBS pigment and hyperhemolytic GBS induce reactive oxygen species production in
neutrophils
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Neutrophil activation is associated with generation of reactive oxygen species (ROS) (36).
To determine if exposure to the hemolytic GBS pigment induced the generation of ROS,
neutrophils were pre-treated with dihydrorhodamine123 (DHR) and exposed to GBS
pigment (0.5 μM) or an equivalent amount of control ΔcylE extract or buffer DTS. The
conversion of DHR to fluorescent monohydrorhodamine (MHR) indicates generation of
ROS and was measured by flow cytometry. Treatment of neutrophils with GBS pigment
stimulated the generation of ROS, which was not observed with control ΔcylE extract or
buffer DTS (Fig. S9 A, B).
To confirm that GBS strains also induced ROS production, neutrophils were pre-treated with
dihydrorhodamine123 (DHR) and exposed to GBS strains (WT, hyperhemolytic ΔcovR or
nonhemolytic ΔcovRΔcylE, ΔcylE) at an MOI of 100. Notably, hyperhemolytic GBS
accelerated the production of ROS in neutrophils within 15 minutes (Fig. S9C, D).
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Subsequently, we observed that most neutrophils treated with pigment or hyperpigmented

GBS succumbed to cell death and therefore generation of ROS was not sustained.
The hemolytic pigment enables GBS to resist neutrophil killing

We next compared the ability of various GBS strains that exhibit differences in hemolytic
pigment expression for their ability to resist neutrophil killing. To this end, we compared
survival of the GBS strains (WT, hyperpigmented ΔcovR, nonpigmented ΔcovRΔcylE,
ΔcylE and capsule deficient ΔcpsK) in the presence of purified human neutrophils. These
experiments were performed in the absence of serum as described previously (37), in order
to understand the role of the hemolytic pigment, independent of complement opsonization.
We observed that hyperpigmented GBSΔcovR exhibit increased survival in the presence of
neutrophils when compared to nonhemolytic or acapsular GBS strains (Fig. S9E). These
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data suggest that despite inducing generation of ROS, increased expression of the hemolytic
toxin enables GBS to subvert neutrophil killing mechanisms likely by promoting neutrophil
cell death.

The purified hemolytic pigment/lipid toxin induces but is resistant to neutrophil

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extracellular traps
Extrusion of neutrophil contents has been associated with formation of neutrophil
extracellular traps (NETs, (38, 39)). Formation of NETs is dependent on neutrophil
components that include NADPH oxidase, generation of ROS, myeloperoxidase, neutrophil
elastase and histone deamination (39–41). While one function of NETs is to trap and
promote extracellular killing of bacterial and fungal pathogens (33, 42, 43), certain bacterial
toxins such as the Staphylococcus aureus leukotoxin GH have been shown to induce NETs
(44). As GBS-infected neutrophils in vitro exhibited DNA extrusion (45), we were interested
to determine if the purified hemolytic GBS pigment is sufficient for induction of NETs. To
this end, neutrophils in NET assay buffer were treated with either GBS pigment (5μM),
equivalent amount of control (ΔcylE extract) or with the GBS (hyperhemolytic ΔcovR, or
nonhemolytic ΔcovRΔcylE) at an MOI of ~ 10 for 4 hours at 37°C. Phorbol myristate
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acetate (PMA, 20 nM) was included as a positive control. SEM was then performed and the
results shown in Fig. 4A. The top panel in Fig. 4A shows that the hemolytic GBS pigment
induced the formation of NETs similar to PMA, whereas the control ΔcylE extract did not
induce significant NET formation. Similarly, the hyperhemolytic GBSΔcovR strain robustly
induced NET formation whereas the ΔcovRΔcylE strain showed slightly attenuated NET
formation (Fig. 4A, lower panel). As neutrophil elastase activity is associated with NET
formation (46), we measured NET associated neutrophil elastase. Briefly, neutrophils in
NET assay buffer were treated with either pigment or the GBS strains as described above.
After removal of soluble neutrophil elastase and treatment with S7 nuclease, NET associated
neutrophil elastase activity was measured as described in Materials and Methods. The results
shown in Fig. 4B indicates that, consistent with the SEM images, increased NET associated
neutrophil elastase activity was observed with pigment and hyperhemolytic GBSΔcovR
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compared to controls or nonpigmented GBS ΔcovRΔcylE strain, respectively.
We then examined the ability of the GBS strains to resist killing by NETs. To this end,
neutrophils were induced to produce NETs and the ability of the GBS strains (WT COH1,
hyperhemolytic ΔcovR, nonhemolytic ΔcovRΔcylE,) to resist killing by NETs was
evaluated. As sialic acid deficiency on the GBS polysaccharide capsule was previously
described to increase sensitivity to NET killing (47), we included the sialic acid deficient
GBSΔcpsK strain (48) as a control in the NET killing assay. The results shown in Fig. 4C
indicates that the hyperhemolytic GBSΔcovR strain was resistant to killing by NETs unlike
the nonhemolytic ΔcovRΔcylE strain or the control capsule deficient ΔcpsK. Taken together,
our observations suggest that the increased virulence of the hyperhemolytic GBSΔcovR
strain may primarily be due to its ability to induce neutrophil cell death and may be
augmented by resistance to NETs.
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To confirm that NET formation occurs during GBS infection in vivo, we stained the
chorioamniotic membranes of nonhuman primates (NHP) infected with either GBSΔcovR or
GBSΔcovRΔcylE for neutrophil-derived elastase and DNA (DAPI) as described (33, 49).
Membranes from saline controls were also included. The results shown in Fig. 5
demonstrate increased NETs (i.e. co-localization of extracellular DNA with neutrophil
elastase) in chorioamniotic membranes of NHP infected with GBSΔcovR (animal from Fig.

1B) when compared to membranes infected with GBSΔcovRΔcylE (animal from Fig. 1C) or
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the saline control. Overall, increased NETs were observed in chorioamniotic membranes of
ΔcovR infected samples when compared to GBSΔcovRΔcylE (Fig. S10). Collectively, these
data indicate that NETs are formed in the chorioamniotic membranes during ascending GBS
infection and that expression of the hemolytic pigment enables GBS to circumvent
extracellular killing by NETs.
Decreased ROS production and neutrophil elastase activity in fetal neutrophils exposed to
GBS
We also tested the hypothesis that fetal neutrophils may be more susceptible to the hemolytic
GBS pigment when compared to maternal neutrophils. To test this hypothesis, we obtained
maternal and cord blood from non-laboring pregnant women undergoing elective Cesarean
section at term. Neutrophils were isolated from maternal and cord blood pairs and flow
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cytometry confirmed that the isolated cells were enriched for the neutrophil markers CD15
and CD16 (Fig. S11). We first compared the sensitivity of maternal and fetal neutrophils to
the GBS pigment by measuring LDH release (Fig. 6A) or PI uptake (Fig. S12). While both
maternal and fetal neutrophils were sensitive to GBS pigment compared to controls (ΔcylE
extract and DTS buffer), fetal neutrophils were similar in their sensitivity to the GBS
pigment when compared to maternal neutrophils for LDH release (Fig. 6A) and PI uptake
(Fig. S12). We next tested the release of ROS from maternal and fetal neutrophils after
exposure to GBS pigment or controls. Here, we observed that conversion of DHR to
fluorescent MHR indicative of ROS generation was significantly attenuated in fetal
neutrophils when compared to maternal neutrophils (compare panels ii and iii to v and vi in
Fig. 6B). We then compared the survival of various GBS strains (WT, hyperhemolytic
ΔcovR and nonhemolytic ΔcovRΔcylE) in the presence of maternal and fetal neutrophils.
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Notably, while survival of the nonhemolytic GBSΔcovRΔcylE was significantly attenuated

when compared to the hyperhemolytic ΔcovR in maternal neutrophils, this trend was not
observed with fetal neutrophils (Fig. 7A). We then compared the release of NET-associated
neutrophil elastase between maternal and fetal neutrophils exposed to hemolytic pigment or
GBS strains. The results shown in Fig. 7B indicates that fetal neutrophils showed
significantly lower NET associated neutrophil elastase activity when compared to maternal
neutrophils exposed to GBS pigment or hyperpigmented GBS. These data indicate that
decreased ROS generation and NET formation by fetal neutrophils may contribute to
increased susceptibility of neonates to many GBS strains.
Collectively, the results presented in this study indicate that GBS utilizes a unique
rhamnolipid pigment toxin to circumvent neutrophils and neutrophil extracellular traps in
the chorioamnion to facilitate microbial trafficking into the amniotic cavity and fetus during
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pregnancy.
DISCUSSION
GBS infections during pregnancy remain a significant public heath concern (8). Although
current methods of prevention of GBS transmission from mother to fetus during labor and
delivery has made remarkable progress in decreasing early onset disease, intrapartum

antibiotic prophylaxis does not address or decrease ascending GBS infection leading to
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preterm births or stillbirths. Recent reports indicate the increasing prevalence of GBS in
pregnant women and preterm delivery (50) and GBS infection associated stillbirths are
currently estimated at 12.1% (51). The lack of a mechanistic understanding of events that
enable the pathogen to circumvent the maternal and fetal immune system during pregnancy
imposes limitations in developing alternate methods of prevention and treatment.
In this study, we used a highly relevant, chronically catheterized pregnant nonhuman primate
model that closely emulates human pregnancy. The tethered chronic catheter preparation
was used for all in vivo experiments and is ideal for studying maternal-fetal immunologic
responses. This model allows us to investigate uterine responses and immune mechanisms
during the course of a bacterial infection beginning from the time of inoculation until
preterm labor, which would be impossible in humans. Using this model, we demonstrate
how increased expression of the hemolytic pigment enables GBS to penetrate the placental
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chorioamniotic membranes and infect the amniotic fluid and fetal organs. Interestingly, we
observed that inoculation of hyperpigmented GBS into the choriodecidual space can result in
bacterial invasion of the amniotic cavity and fetus as early as within 15 minutes to within a
few hours post infection. GBS invasion of the amniotic activity induced increased expression
of neutrophil recruiting cytokines and chemokines in the chorioamniotic membranes and
amniotic fluid. Consistent with these findings, we observed significant recruitment of
neutrophils in the chorioamnion of NHP infected with hyperpigmented GBS. These results
indicate that neutrophils comprise the initial and primary host defense mechanism utilized
by the chorioamnion to combat invasive pathogens such as GBS. However, despite the
recruitment of neutrophils, the hyperhemolytic GBS strain was able to traffic across the
chorioamniotic membranes in 3 of the 5 animals and bacteria were recovered from multiple
fetal organs. These observations suggest that although neutrophils are recruited to the site of
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infection, increased expression of the hemolytic pigment may impair their phagocytic
function. Our findings in the pregnant NHP model are consistent with previous reports on
the role of hemolysin in exacerbating GBS infections such as meningitis, (52), experimental
sepsis (53, 54), lung injury (55, 56), urinary tract infections (57) and fetal demise (12, 13) in
murine, rat or rabbit models. Although expression of the GBS hemolytic pigment is typically
associated with generation of a proinflammatory response and neutrophil recruitment, this
appears ineffective in curtailing bacterial burden as observed by others ((13, 52, 57) and this
work).
We then examined how increased expression of the hemolytic pigment may enable GBS
subvert neutrophils. Our studies revealed that the hemolytic pigment and hyperpigmented
GBS induced neutrophil cell death within four hours, in a dose dependent manner. The
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neutrophil cell death observed with the GBS pigment is independent of apoptosis or even
pyroptosis. While we previously noted that macrophages lacking the NLRP3 inflammasome
were able to recover from the membrane permeability induced by the GBS hemolytic
pigment (12), the short lived nature of neutrophils may prevent remodeling of neutrophil
membranes thereby increasing their susceptibility to the GBS lipid toxin. These data indicate
that cell death due to GBS pigment may be due to direct lysis or necrosis. Indeed, SEM of
neutrophils exposed to the GBS toxin revealed severe morphological changes and
interestingly, the toxin induced NET formation both in vitro and in vivo. To our knowledge,

NET formation in placental membranes due to microbial infections has not been shown.
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Prior to our study, NET formation in placental membranes was only reported in association
with a non-infectious condition i.e. preeclampsia (49). Here, we demonstrate that in vivo
bacterial infections induce NET formation in placental membranes. Recently, studies using
mouse models have shown that live neutrophils form NETs in vivo to limit systemic
bacterial infection (58). It is likely that in NHP infected with hyperpigmented GBS, both live
and dying neutrophils contribute to NET formation in vivo. Whether NETs themselves
contribute to placental dysfunction as suggested (59) is not known.
Typically NET formation is associated with the ability of neutrophils to ensnare bacteria for
further antimicrobial action. However, we observed that GBS strains that overexpress the
hemolytic pigment were resistant to killing by NETs when compared to the nonhemolytic
strain. This may in part be due to the ability of the unsaturated polyene chain in the
hemolytic pigment toxin to quench reactive oxygen species (ROS). Previous work by Liu et.
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al. (30) demonstrated the antioxidant nature of the GBS pigment. They observed that
hyperpigmented GBS strains are more resistant to ROS such as hydrogen peroxide,
superoxide and singlet oxygen in vitro and in macrophages (30). Although expression of an
extracellular nuclease (nuclease A) in GBS was shown to degrade NETs (60), previous work
by others and us showed that expression of nuclease A is not under CovR/S regulation in
GBS (23, 61). This suggests that differences in resistance to NET killing between
GBSΔcovR and GBSΔcovRΔcylE is likely not due to altered endogenous DNAse activity.
Our observations suggests that increased expression of the hemolytic toxin enables GBS to
induce neutrophil cell death and likely resist killing by NETs, which may promote MIAC
during ascending infection. It is also noteworthy that a few reports have indicated that NETs
may only entrap bacteria to prevent dissemination and wall off infection, without actually
inducing bacterial cell death (62, 63). Addition of DNase at the end of the NET killing assay
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is thought to relieve the clumping effect and provide accurate results for discrimination
between ensnaring of bacteria by NETs versus bacterial killing by NETs (62), While we
repeated these experiments with addition of DNase at the end of the experiment to confirm
increased sensitivity of the GBSΔcovRΔcylE to NETS, it remains plausible that GBS
entrapment by NETs rather than NET associated killing regulates MIAC in vivo.
Interestingly, we observed that two of five animals inoculated with the hypervirulent GBS
strain did not develop MIAC despite exhibiting preterm labor. We predict that this may in
part be due to early neutrophil recruitment wherein the number of neutrophils recruited may
have surpassed the bacterial load to effectively curtail GBS invasion of the amniotic fluid
and fetus. Recently, using a mouse vaginal colonization model, we showed that mast cell
degranulation can promote neutrophil recruitment and enable eradication of hypervirulent
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GBS from the vaginal tract (32). While the mast cell response of humans and NHP to
vaginal microorganisms remains unknown, we posit that an early and effective neutrophilic
inflammatory response is essential for prevention of ascending GBS infection. It is likely
that in the absence of neutrophil cell death, the expression of neutrophil recruiting cytokines
and chemokines by chorioamniotic membranes and the release of IL-8 from activated
neutrophils themselves may support the recruitment of additional neutrophils that assist in
GBS phagocytosis. Consistent with this hypothesis, Mohammadi et al. recently reported that
murine bone marrow derived neutrophils released TNF and IL-1β when exposed to GBS for

24 hours (64). Taken together, these observations suggest that when neutrophils escape
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cytotoxic killing by the GBS hemolytic pigment, cytokine responses may promote additional
neutrophil recruitment.
Our results also revealed the attenuated ability of human fetal neutrophils to combat GBS.
When compared to maternal neutrophils, fetal neutrophils exhibited decreased ROS
generation and NET associated neutrophil elastase activity when exposed to GBS pigment or
hyperpigmented GBS strains. These data are consistent with previous observations of
decreased or delayed NET formation by neonatal neutrophils upon treatment with
inflammatory stimuli such as PMA and LPS (65, 66). Furthermore, fetal neutrophils were
not as efficient as maternal neutrophils in curtailing nonhemolytic GBS strains. While
previous studies reported that pregnancy can been associated with diminished oxidative burst
function in maternal neutrophils (67), our data suggests that compared to fetal neutrophils,
maternal neutrophils play a critical role in restraining lower genital tract pathogens such as
Author Manuscript
GBS from accessing fetal compartments and tissues.
Although in this study, we describe the role of the hemolytic pigment in MIAC and preterm
labor in the NHP model, many virulence factors are likely to play critical roles in the
outcome of GBS infections. It is also important to note that differences between clinical
strains of GBS are not always limited to differences in hemolytic pigment expression but can
include differences in expression of many other virulence factors, which were not examined
in this study. Further studies that explore the role of other pathogenic determinants in GBS
infection-associated preterm birth in pregnant animal models will provide new insight into
the repertoire of virulence factors utilized by this pathogen to cause fetal injury, preterm
birth or stillbirth.
Author Manuscript
In summary, we show that GBS utilizes the hemolytic pigment to invade the amniotic fluid
and fetus during pregnancy by circumventing neutrophils and NETs in placental membranes.
Understanding how pathogens circumvent host immune responses at the maternal/fetal
interface is a critical first step for determining strategies to prevent microbial trafficking
across the chorioamniotic membranes during pregnancy.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
We are grateful to the human subjects who participated in our study. We thank Amy Gest, Jan Hamanishi, Gina
Heidel, Danny Power, Joyce Karlinsey and Connie Hughes for their assistance. We acknowledge Drs. Craig Rubens
Author Manuscript
and Michael Gravett for study design related to performance of the original nonhuman primate experiments.
FUNDING: This work was supported by funding from the National Institutes of Health, Grant # R01AI100989 to
L.R and K. A. W, R56AI070749, R01AI112619 and R21AI109222 to L. R, and P30HD002274. The NIH training
grants T32 HD007233 (PI: Lisa Frenkel) and T32 AI07509 (PI: Lee Ann Campbell) supported E.B and J.V,
respectively.
The content is solely the responsibility of the authors and does not necessarily represent the official views of the
National Institutes of Health or other funders. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.

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Fig. 1. Choriodecidual inoculation of hyperpigmented GBSΔcovR induced rapid preterm labor,

microbial invasion of the amniotic cavity (MIAC) and elevated amniotic fluid (AF) cytokines
Chronically catheterized pregnant Macaca nemestrina at 118–125 days gestation (term=172
days) received choriodecidual inoculations of saline (n=5), hyperpigmented GBS
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COH1ΔcovR (n=5) or nonpigmented GBS COH1ΔcovRΔcylE (n=5). Shown are uterine

contractions (vertical grey lines), cytokines (IL-8, IL-1β, TNF-α), prostaglandin (PGE2) and
GBS CFU from AF of a representative animal that received either saline (A), GBSΔcovR
(B) or GBSΔcovRΔcylE (C).
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Fig. 2. Neutrophil infiltration is associated with MIAC in the chorioamnion of pregnant NHP
infected with hyperpigmented GBS
H & E sections of the chorioamniotic membranes from NHPs infected with hyperpigmented
GBSΔcovR or nonpigmented GBSΔcovRΔcylE. Panel A and magnified image in panel D
shows chorioamniotic membranes from GBSΔcovR infected animal with preterm labor and
necropsy at 6 hours post inoculation (HPI). Panel B and magnified image in panel E shows
chorioamniotic membranes from GBSΔcovR infected animal with preterm labor and
necropsy at 24 HPI. Panel C and magnified image in panel F shows chorioamniotic
membranes from GBSΔcovR infected animal with signs of stillbirth and necropsy at 48 HPI.
Panel G and magnified image in panel I shows chorioamniotic membranes from saline
control. Panel H and magnified image in panel J shows chorioamniotic membranes from
GBSΔcovRΔcylE inoculated control animal with necropsy at 72 HPI. Scale bar =100μm.
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Fig. 3. The hemolytic pigment/lipid toxin is cytotoxic to neutrophils

(A) Neutrophils were incubated with various concentrations of GBS pigment, control ΔcylE
extract or buffer DTS. Cytotoxicity was measured by LDH release. Data shown are the
average of three independent experiments performed in triplicate, error bars ± SEM.
Significance was determined using Tukey’s multiple comparison test following ANOVA;
n=3, ****P = <0.0001, ***P = <0.001. (B) Neutrophils were treated with GBS (WT COH1,
hyperpigmented ΔcovR, nonpigmented ΔcylE, or ΔcovRΔcylE). Cytotoxicity was measured
by LDH release and data shown are the average of three independent experiments performed
in triplicate, error bars ± SEM. Significance was determined using Sidak’s multiple
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comparison test following ANOVA; n=3, ****P <0.0001. (C) Top Panel: Scanning electron
micrographs (SEM) of neutrophils treated with either GBS pigment (0.5μm) or controls
(media, ΔcylE extract) for 10 minutes. Bottom panel: SEM of neutrophils treated with
hyperpigmented GBSΔcovR or nonpigmented ΔcovRΔcylE at MOI 100 for 10 minutes. A
representative image from two independent experiments is shown. A minimum of 30 cells
was examined in a blinded fashion.

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Fig. 4. Increased expression of the hemolytic pigment enables GBS to resist killing by neutrophil
extracellular traps
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(A) Top Panel: SEM of neutrophil extracellular traps (NETs) in neutrophils treated with
GBS pigment (5 μM), negative control (ΔcylE extract) or positive control PMA (20 nM).
Bottom panel: SEM of NETS due to hyperpigmented GBSΔcovR or nonpigmented
ΔcovRΔcylE. (B) NET associated neutrophil elastase activity was measured after induction
of NETs. Data shown are the average of three independent experiments performed in
duplicate, error bars ± SEM. Significance was determined using Students t-test, ** P <0.01.
(C) Neutrophils were incubated with cytochalasin D and B (10 μM) to block phagocytosis
and NETs were induced by stimulation with PMA. Subsequent NET killing of the GBS

strains (WT COH1, isogenic hyperhemolytic ΔcovR, nonhemolytic ΔcovRΔcylE & ΔcylE
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and capsule deficient ΔcpsK) was compared. To inhibit NET-mediated bacterial killing,
control wells were treated with DNase I. Data shown are the average of three independent
experiments performed in duplicate, error bars ± SEM. Significance was determined using
Bonferroni’s multiple comparison test following ANOVA, * P <0.05.
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Fig. 5. Neutrophil extracellular traps (NETs) are formed in the chorioamnion during in vivo GBS
infection
Immunofluorescence staining for neutrophil elastase and extracellular DNA was performed
on chorioamniotic membranes from NHP inoculated with either hyperpigmented
GBSΔcovR, nonpigmented GBSΔcovRΔcylE or saline. Data shown is representative of 5
animals from each group that were examined for NETs. Neutrophil elastase staining is show
in gray scale mode for ease of visualization.
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Fig. 6. Decreased ROS generation in fetal neutrophils compared to maternal neutrophils

(A) Neutrophils isolated from maternal and fetal (cord) blood were treated with various
concentrations of GBS pigment or controls (ΔcylE extract or buffer DTS). Cytotoxicity was
measured by LDH release. Data shown are the average of three independent experiments
performed in triplicate, error bars ± SEM. Significance was determined using Tukey’s
multiple comparison test following ANOVA; NS = not significant, **** P = <0.0001. (B)
Maternal or fetal neutrophils were pretreated with 84μM dihydrorhodamine 123.
Subsequently, the neutrophils were treated with either 0.5 μM pigment or controls and
oxidation to fluorescent mono-hydrorhodamine 123 was monitored. Panels i, ii, iii show
fetal neutrophils at 0, 30 and 60 min after treatment with pigment and controls, respectively.
Panels iv, v, vi show maternal neutrophils at 0, 30 and 60 min after treatment with pigment
and controls, respectively. Gates shown reflect the % cell number with various treatments.
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Data shown are representative of three independent experiments.

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Fig. 7. Fetal neutrophils are more sensitive to nonpigmented GBS strains and exhibit decreased
neutrophil elastase activity when compared to maternal neutrophils
(A) Maternal and fetal neutrophils were incubated with either WT GBS COH1, or isogenic
mutants (hyperpigmented ΔcovR, nonpigmented ΔcovRΔcylE) at an MOI of 1. The total
surviving bacteria (intracellular and extracellular) were enumerated 1 hour after incubation.
The survival index was calculated as the ratio of CFU recovered in the presence of
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neutrophils to CFU recovered in the absence of neutrophils. Data shown are the average of
four independent experiments with neutrophils isolated from different donors and each
experiment was performed in duplicate, error bars ± SEM. Significance was determined
using Sidak’s multiple comparison test following ANOVA; n=3, ** P <0.01. (B) NET
associated neutrophil elastase activity was measured after induction of NETs in maternal and
fetal neutrophils treated with GBS strains (WT ΔcovR, ΔcylE, ΔcovRΔcylE). Data shown
are the average of three independent experiments with neutrophils isolated from different

donors, error bars ± SEM. Significance was determined using Students t test, * P <0.05. ** P
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<0.01.
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Table 1
Summary of pregnancy outcomes, cytokines and prostaglandins in pregnant NHP
Saline (N=5) GBSΔcovR (N=5) GBSΔcovRΔcylE (N=5) P value Saline vs. GBSΔcovR P value GBSΔcovR vs. P value Saline vs.
GBSΔcovRΔcylE GBSΔcovRΔcylE
Boldenow et al.
PRIMARY AND COMPOSITE OUTCOMES
Adverse Outcomes# 0 (0%) 5 (100%) 2 (40%) 0.0009 0.03 0.1
Preterm Labor@ 0 (0%) 4 (80%) 1 (20%) 0.002 0.01 NS
MIAC and Fetal Sepsis 0 (0%) 3 (60%) 1 (20%) 0.03 0.16 NS
PEAK AMNIOTIC FLUID CYTOKINES AND PROSTAGLANDINS
IL-1β 0.005 (0.003) 5.1 (3.1) 0.5 (0.4) 0.009 NS 0.02
TNF-α 0.019 (0.01) 2.0 (1.2) 0.07 (0.04) 0.029 0.15 NS
IL-6 10.71(2.2) 22.7 (1.5) 6.2 (4.3) 0.017 0.049 NS
IL-8 1.45 (0.3) 15.7 (4.2) 5.3 (2.3) <0.001 0.032 0.03
PGE2 0.72 (0.4) 4.5 (4.1) 0.7 (0.3) NS NS NS
PGF2α 0.78 (0.2) 98.5(98.0) 0.3 (0.1) NS NS 0.05
FETAL CYTOKINES*
IL-1β 0.6 (0) 42.31 (23.2) 13.63 (5.9) 0.008 NS 0.001

TNF-α 1.21 (0.31) 7.7 (0.8) 8.30 (0.4) <0.001 NS <0.001
IL-6 1.73 (0.37) 675.34 (413.0) 88.59 (73.9) 0.019 0.16 NS
IL-8 309.54 (83.32) 4721.72 (1745.0) 1828.41(1039.3) 0.002 0.06 NS
MIAC, Microbial invasion of the amniotic cavity.

The primary outcomes are shown as number (%). Amniotic fluid cytokines and prostaglandins are shown as mean peak (standard error of the mean) in ng/mL. Fetal plasma cytokines are levels taken at the
time of delivery (GBS) or peak (saline controls) and shown as pg/mL.
#
Adverse outcomes represent preterm labor or increase in uterine contraction and MIAC.
@
Preterm labor was defined as a progressive increase in uterine contraction accompanied by cervical change.
*
In 2 saline controls, fetal blood could not be obtained, but there was no detectable AF inflammation and fetal lung histology was normal. In this category, saline controls represent n=3. Barnards or Chi
square test was used to compare adverse outcomes, preterm labor and MIAC. Amniotic fluid cytokines (IL-1β, IL-6, IL-8, TNF-α,) and prostaglandins (PGE2, PGF2α) were compared using analysis of
covariance (ANOVA) to allow for adjustment of each animal’s baseline value. Statistical analyses were conducted using Intercooled STATA 8.2 for Windows 2000 (StatCorp, College Station, TX) or
SciStatCalc and P <0.05 are indicated in bold.
NS: P > 0.2.

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GROUP B STREPTOCOCCUS CIRCUMVENTS NEUTROPHILS

James W MacDonald4, Theo K Bammler4, Aasthaa Bansal5, H. Denny Liggitt6, Lakshmi

5Department of Pharmacy, University of Washington, Seattle, Washington, United States of

induce fetal injury and preterm labor.

Sci Immunol. Author manuscript; available in PMC 2017 April 01.

(hemomonochorial), initiation of labor (hormonal control of parturition), sensitivity to

Sci Immunol. Author manuscript; available in PMC 2017 April 01.

hyperpigmented GBSΔcovR (n=5) or control nonpigmented GBSΔcovRΔcylE (n=5); these

Sci Immunol. Author manuscript; available in PMC 2017 April 01.

choriodecidual inoculation of hyperpigmented GBSΔcovR induced adverse outcomes such

severe fetal systemic inflammation (26).

Microbial invasion of the amniotic cavity correlated with neutrophil recruitment

2A and D, corresponds to animal shown in Fig. S1B). By 24 hours after inoculation,

Sci Immunol. Author manuscript; available in PMC 2017 April 01.

inflammation within the chorioamniotic membranes from animals inoculated with

Hyperpigmented GBS induce transcription of genes associated with neutrophil

token=cpifasaozdulpkl&acc=GSE80248.) Approximately half of the differentially regulated

Overall, significantly upregulated genes in animals inoculated with GBSΔcovR compared to

Sci Immunol. Author manuscript; available in PMC 2017 April 01.

Sci Immunol. Author manuscript; available in PMC 2017 April 01.

(hyperpigmented ΔcovR or nonpigmented strains i.e. ΔcovRΔcylE or ΔcylE) at various

GBS pigment induced neutrophil cell death is independent of apoptosis or pyroptosis

(Fig. S7 A, B, C) preceded AV staining (Fig. S7 D, E, F) in pigment treated neutrophils.

We next examined if the purified hemolytic pigment or hyperpigmented GBS induced

Sci Immunol. Author manuscript; available in PMC 2017 April 01.

Notably, only WT and nonpigmented/nonhemolytic GBS strains (ΔcovRΔcylE, ΔcylE)

Subsequently, we observed that most neutrophils treated with pigment or hyperpigmented

The hemolytic pigment enables GBS to resist neutrophil killing

Sci Immunol. Author manuscript; available in PMC 2017 April 01.

The purified hemolytic pigment/lipid toxin induces but is resistant to neutrophil

compared to controls or nonpigmented GBS ΔcovRΔcylE strain, respectively.

Sci Immunol. Author manuscript; available in PMC 2017 April 01.

Notably, while survival of the nonhemolytic GBSΔcovRΔcylE was significantly attenuated

Sci Immunol. Author manuscript; available in PMC 2017 April 01.

Sci Immunol. Author manuscript; available in PMC 2017 April 01.

Sci Immunol. Author manuscript; available in PMC 2017 April 01.

GBS from accessing fetal compartments and tissues.

Sci Immunol. Author manuscript; available in PMC 2017 April 01.

human placenta. J Exp Med. 2013; 210:1265–1281. [PubMed: 23712433]

administration. Obstet Gynecol. 2010; 116:791–792. [PubMed: 20733474]

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171:1660–1667. [PubMed: 7802084]

eukaryotic-type and two-component signalling mechanisms in Streptococcus agalactiae. Mol

group B streptococcal beta-hemolysin/cytolysin and carotenoid pigment function to subvert host

Sci Immunol. Author manuscript; available in PMC 2017 April 01.

176:231–241. [PubMed: 17210947]

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6427. [PubMed: 19221037]

amniotic epithelium: a pathway to membrane weakening. PLoS Pathog. 2014; 10:e1003920.

Sci Immunol. Author manuscript; available in PMC 2017 April 01.

Sci Immunol. Author manuscript; available in PMC 2017 April 01.

Fig. 1. Choriodecidual inoculation of hyperpigmented GBSΔcovR induced rapid preterm labor,

COH1ΔcovR (n=5) or nonpigmented GBS COH1ΔcovRΔcylE (n=5). Shown are uterine

Sci Immunol. Author manuscript; available in PMC 2017 April 01.

Sci Immunol. Author manuscript; available in PMC 2017 April 01.

Fig. 3. The hemolytic pigment/lipid toxin is cytotoxic to neutrophils

Sci Immunol. Author manuscript; available in PMC 2017 April 01.

Sci Immunol. Author manuscript; available in PMC 2017 April 01.

Sci Immunol. Author manuscript; available in PMC 2017 April 01.

Sci Immunol. Author manuscript; available in PMC 2017 April 01.

Fig. 6. Decreased ROS generation in fetal neutrophils compared to maternal neutrophils