Research ArticleINFECTIOUS DISEASE

Group B Streptococcus circumvents neutrophils and neutrophil extracellular traps during amniotic cavity invasion and preterm labor

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Science Immunology  14 Oct 2016:
Vol. 1, Issue 4, eaah4576
DOI: 10.1126/sciimmunol.aah4576

NETting group B strep

Group B Streptococcus (GBS) infection in pregnant women can lead to preterm birth and fetal injury. Boldenow et al. report that a hemolytic pigment toxin from GBS contributes to these effects by subverting neutrophils and neutrophil extracellular traps (NETs) in placental membranes. They found in a nonhuman primate model that adverse outcomes were more closely associated with hemolytic than with nonhemolytic GBS, and that GBS hemolytic pigment toxin induced cell death in neutrophils and prevented killing by NETs, allowing GBS to invade the amniotic fluid. This toxin therefore could serve as a target to prevent complications from GBS in pregnant women.

Abstract

Preterm birth is a leading cause of neonatal morbidity and mortality. Although microbial invasion of the amniotic cavity (MIAC) is associated with most early preterm births, the temporal events that occur during MIAC and preterm labor are not known. Group B streptococci (GBS) are β-hemolytic, Gram-positive bacteria, which commonly colonize the vagina but have been recovered from the amniotic fluid in preterm birth cases. To understand temporal events that occur during MIAC, we used a 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. We show that adverse outcomes such as preterm labor, MIAC, and fetal sepsis were observed more frequently during infection with hemolytic GBS when compared with 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 with maternal neutrophils, fetal neutrophils exhibit decreased neutrophil elastase activity and impaired phagocytic functions to GBS. Collectively, our studies demonstrate how a bacterial hemolytic lipid toxin enables GBS to circumvent neutrophils and NETs in placental membranes to 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). Intra-amniotic 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 (AF) often contains organisms that typically colonize 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 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 the 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 is lacking, we recently 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 of which have mutations in the transcriptional repressor of the hemolytic pigment known as CovR/CovS (or CsrR/CsrS), can be isolated from the AF and placental (chorioamniotic) membranes of women in preterm labor (11). Further, we and others have demonstrated that the 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 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 AF and fetus during pregnancy. Also, because antibiotics are routinely administered during cesarean sections (1416), 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 substantially 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 (1720). Similarities of NHPs to humans include reproductive anatomy, number of fetuses (singleton), long gestational period (160 to 170 days), type and structure of placenta (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 can 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 microbial invasion of the amniotic cavity (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 (WT) 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). This human isolate of GBS is mildly hemolytic/pigmented in contrast to certain other GBS strains (22). Because GBS strains with increased expression of the hemolytic pigment were recovered from the AF of women in preterm labor (11), we used 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 that are important for hemolytic pigment expression, this two-component system also controls the expression of more than 100 genes in GBS (2325). 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 NHPs due, in part, to the ability of the hemolytic pigment to induce neutrophil cell death and evade killing by neutrophil extracellular traps (NETs).

RESULTS

Hyperhemolytic GBS induces adverse pregnancy outcomes

To understand how the hemolytic pigment may promote GBS invasion of the AF and fetus, we used our unique chronically catheterized NHP model. Ten animals received choriodecidual inoculations of 1 × 108 to 3 × 108 colony-forming units (CFU) of either hyperpigmented GBSΔcovR (n = 5) or control nonpigmented GBSΔcovRΔcylE (n = 5); these 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 five of five (100%) animals when compared with two of five (40%) animals inoculated with the nonhemolytic GBSΔcovRΔcylE or zero of five (0%) saline controls (Table 1). Preterm labor occurred in four of five animals inoculated with GBSΔcovR (excluding GBSΔcovR 3) compared with one of five animals inoculated with GBSΔcovRΔcylE and zero of five saline controls (Table 1 and table S1). In the other animal inoculated with hyperpigmented GBSΔcovR (GBSΔcovR 3), the AF became dark and cloudy because of MIAC at 12 hours after inoculation; the decision was made to proceed with cesarean section earlier than the defined study end point (preterm labor) to avoid stillbirth due to fetal sepsis. At the time of cesarean section at 48 hours after inoculation, this animal (GBSΔcovR 3) had a sustained pattern of increased uterine contractions but had not yet made cervical change to meet the criteria for development of preterm labor. Overall, MIAC and fetal sepsis were observed in three of five animals inoculated with GBSΔcovR versus one of five animals inoculated with GBSΔcovRΔcylE and zero of five saline controls (Table 1, table S1, Fig. 1, and figs. 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 to 106 CFU per gram of tissue, with consistently more bacteria recovered from the fetal lung than from the other fetal organs (fig. S3).

Table 1 Summary of pregnancy outcomes, cytokines, and prostaglandins in pregnant NHPs.

The primary outcomes are shown as number (%). AF cytokines and prostaglandins are shown as mean peak (SEM) and are expressed as nanograms per milliliter. Fetal plasma cytokines are levels taken at the time of delivery (GBS) or peak (saline controls) and are expressed as picograms per milliliter. In this category, saline controls represent n = 3. Barnard’s or χ2 test was used to compare adverse outcomes, preterm labor, and MIAC. AF cytokines (IL-1β, IL-6, IL-8, and TNF-α) and prostaglandins (PGE2 and PGF) were compared using analysis of covariance (ANCOVA) to allow for adjustment of each animal’s baseline value. Statistical analyses were conducted using Intercooled STATA 8.2 for Windows 2000 (StataCorp) or SciStatCalc, and P values of <0.05 are indicated in bold. NS, P > 0.2.

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Fig. 1 Choriodecidual inoculation of hyperpigmented GBSΔcovR induced rapid preterm labor, MIAC, and elevated AF cytokines.

Chronically catheterized pregnant Macaca nemestrina at 118 to 125 days gestation (term, 172 days) received choriodecidual inoculations of saline (n = 5), hyperpigmented GBS COH1ΔcovR (n = 5), or nonpigmented GBS COH1ΔcovRΔcylE (n = 5). Shown are uterine contractions (vertical gray lines), cytokines (IL-8, IL-1β, and TNF-α), prostaglandin (PGE2), and GBS CFU from AF of a representative animal that received either saline (A), GBSΔcovR (B), or GBSΔcovRΔcylE (C).

An increase in uterine activity was often seen within a few hours after inoculation of GBSΔcovR with MIAC, and preterm labor occurred rapidly in most of these cases. Three animals from the GBSΔcovR group (GBSΔcovR 1, 2, and 3; see Table 1, table S1, Fig. 1B, and fig. S1, A and B) developed sustained uterine contractions within hours after inoculation; bacteria were recovered as early as within 15 min (0.25 hours) in one animal, 45 min (0.75 hours) in another, and within 12 hours in the third case. Because of the increase in uterine contractions and cervical dilation (GBSΔcovR 1 and 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 and 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 after inoculation, respectively (GBSΔcovR 4 and 5; table S1 and fig. S1, C and D).

In the five animals infected with the nonpigmented isogenic control GBSΔcovRΔcylE (see Table 1, table S1, Fig. 1C, and fig. S2), adverse outcomes were detected in two animals (GBSΔcovRΔcylE 1 and 3). Preterm labor developed in one animal without MIAC (GBSΔcovRΔcylE 3; see Table 1, table S1, and 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. Together, our results indicate that 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 versus GBSΔcovRΔcylE; P = 0.0009, GBSΔcovR versus saline).

Increased AF and fetal cytokines are observed in pregnant NHPs infected with hyperpigmented GBS

We then examined cytokine responses in the AF and fetal tissues. The levels of AF interleukin-1β (IL-1β), tumor necrosis factor–α (TNF-α), IL-6, and IL-8 were all significantly higher in animals inoculated with hyperpigmented GBSΔcovR than in saline controls (Table 1, all P < 0.05). AF IL-1β and IL-8 levels were significantly higher in animals inoculated with nonpigmented GBSΔcovRΔcylE than in saline controls (Table 1, P < 0.05), but the 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 and those infected with GBSΔcovRΔcylE. The AF mean peak levels of IL-6 and IL-8 were significantly higher in hyperpigmented GBSΔcovR–inoculated animals than in 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 GBSΔcovR-inoculated animals than in saline controls (Table 1, all P < 0.05); fetal IL-8 showed a trend toward higher levels in animals inoculated with GBSΔcovR compared with the nonpigmented GBSΔcovRΔcylE group (P = 0.06). IL-1β and TNF-α were also higher in animals infected with GBSΔcovRΔcylE than in saline controls. Regardless of MIAC, TNF-α levels were significantly increased in fetal organs of animals infected with GBSΔcovR than in those infected with GBSΔcovRΔcylE (fig. S3), suggesting the onset of severe fetal systemic inflammation (26).

MIAC correlated with neutrophil recruitment

We next compared histological sections of chorioamniotic membranes at the inoculation site from animals of the hyperpigmented GBSΔcovR group, the nonpigmented GBSΔcovRΔcylE group, 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 time point, neutrophils also lined the interface between chorion and trophoblast layers but did not extend into the chorion or amnion (Fig. 2, A and D, corresponding to the 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. 2, B and E, corresponding to the animal shown in fig. S1A). Small vessels in the chorion and trophoblast layers appeared thrombosed with focal areas of necrosis. At 48 hours after inoculation, there was widespread severe inflammation and necrosis, which was predominantly neutrophilic and involved all layers of the chorioamnion including the amniotic epithelium (Fig. 2, C and F, corresponding to the animal shown in Fig. 1B).

Fig. 2 Neutrophil infiltration is associated with MIAC in the chorioamnion of pregnant NHPs infected with hyperpigmented GBS.

Hematoxylin and eosin sections of the chorioamniotic membranes from NHPs infected with hyperpigmented GBSΔcovR or nonpigmented GBSΔcovRΔcylE. (A and magnified image D) Chorioamniotic membranes from a GBSΔcovR-infected animal with preterm labor and necropsy at 6 hours post-inoculation (HPI). (B and magnified image E) Chorioamniotic membranes from a GBSΔcovR-infected animal with preterm labor and necropsy at 24 HPI. (C and magnified image F) Chorioamniotic membranes from a GBSΔcovR-infected animal with signs of stillbirth and necropsy at 48 HPI. (G and magnified image I) Chorioamniotic membranes from a saline control. (H and magnified image J) Chorioamniotic membranes from a GBSΔcovRΔcylE-inoculated control animal with necropsy at 72 HPI. Scale bars, 100 μm.

With the exception of a single animal where MIAC was observed, there was minimal to no inflammation within the chorioamniotic membranes from animals inoculated with nonpigmented GBSΔcovRΔcylE; neutrophils were occasionally seen within deeper regions of the placenta (Fig. 2, H and J, corresponding to the 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 that in the most affected hypervirulent GBSΔcovR-infected animal (Fig. 2, C and F).

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.

Hyperpigmented GBS induce transcription of genes associated with neutrophil recruitment in chorioamniotic membranes

To further investigate the innate immune response after 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 per group). Out of a total of 31,740 probe sets for host gene expression, 797 were differentially regulated in chorioamniotic membranes between animals inoculated with GBSΔcovR and saline controls, and 530 were differentially regulated between animals inoculated with GBSΔcovR and those inoculated with GBSΔcovRΔcylE (fold change ≥ 1.5; P < 0.005; heat map shown in fig. S4). A subset of differentially expressed probe sets is shown in table S2, and the entire list is available through the Gene Expression Omnibus (GEO) link GSE80248 (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=cpifasaozdulpkl&acc=GSE80248). About 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 with 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 up-regulated genes in animals inoculated with GBSΔcovR compared with either saline controls or animals inoculated with GBSΔcovRΔcylE included those associated with inflammation and cell adhesion molecules, such as matrix metallopeptidase 8 (MMP8), interleukin-1α (IL-1A), interleukin-1β (IL-1B), vanin 2 (VNN2), chemokine receptor (CXCR1), and Selectin-L (SELL). Apart from factors associated with preterm labor such as matrix metalloproteinases and proinflammatory 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, 28), which is consistent with the neutrophil influx observed in these membranes (Fig. 2).

Down-regulated genes included genes important for maintenance of cell-cell junctions and cytoskeletal organization, such as desmocollin 2 (DSC2), desmoplakin (DSP), integrin α6 (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 not only by increasing the expression of factors that directly promote neutrophil recruitment but also by diminishing the 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 transcription polymerase chain reaction (qRT-PCR). The levels of gene expression obtained with amplified RNA samples were compared with β-actin as a control for each sample. There was a significant correlation between microarray and qRT-PCR for differential gene expression between animals inoculated with GBSΔcovR and 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 NHPs infected with GBSΔcovR (Fig. 2), we asked whether 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. Although some studies have indicated that hemolytic GBS 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, and promotes NLRP3-dependent pyroptosis in macrophages (11, 12). To test the possibility that the purified GBS hemolytic lipid toxin may induce neutrophil cytotoxicity, we isolated primary human neutrophils 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 to 5 μM) for 4 hours, and cytotoxicity was estimated by measuring the release of lactate dehydrogenase (LDH) as previously described (32). As controls, we included an equivalent amount of ΔcylE extract (i.e., pigment extraction procedure was performed using the nonpigmented GBSΔcylE strain) or buffer DTS [dimethyl sulfoxide + 0.1% trifluoroacetic acid + 20% starch; see Materials and Methods and (11, 12)]. The data shown in Fig. 3A indicate that cytotoxicity or percent cell death was significantly higher in neutrophils treated with the hemolytic GBS pigment than in those treated with control ΔcylE extract or DTS buffer. To confirm that hemolytic pigment–mediated neutrophil cell death was also observed with live bacteria, we treated neutrophils with GBS strains, namely, WT COH1 or isogenic mutants (hyperpigmented ΔcovR or nonpigmented strains, i.e., ΔcovRΔcylE or ΔcylE), at various multiplicities of infection (MOIs: 1, 10, and 100) for 4 hours, and cytotoxicity was measured by LDH release as previously 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.

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 are the average of three independent experiments performed in triplicate (error bars ± SEM). n = 3. ****P < 0.0001, ***P < 0.001, Tukey’s multiple comparison test following analysis of variance (ANOVA). (B) Neutrophils were treated with GBS (WT COH1, hyperpigmented ΔcovR, nonpigmented ΔcylE, or ΔcovRΔcylE). Cytotoxicity was measured by LDH release, and data are the average of three independent experiments performed in triplicate (error bars ± SEM). n = 3. ****P < 0.0001, Sidak’s multiple comparison test following ANOVA. (C) (Top) Scanning electron micrographs of neutrophils treated with either GBS pigment (0.5 μM) or controls (Media, ΔcylE extract) for 10 min. (Bottom) Scanning electron micrographs of neutrophils treated with hyperpigmented GBSΔcovR or nonpigmented ΔcovRΔcylE at an MOI of 100 for 10 min. A representative image from two independent experiments is shown. A minimum of 30 cells was examined in a blinded fashion.

The hemolytic GBS pigment and hyperpigmented GBS induce morphological changes in neutrophils

We next performed scanning electron microscopy to determine whether 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 extract for 10 min. Untreated neutrophils were included as controls. The results shown in Fig. 3C (top) 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 with untreated neutrophils or those treated with the control ΔcylE extract (Fig. 3C, top). Consistent with these observations, neutrophils exposed to the nonpigmented GBSΔcovRΔcylE strain also had a greater number of rounded or partially rounded cells when compared with neutrophils exposed to the hyperpigmented GBSΔcovR strain (Fig. 3C, bottom). 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

Because 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 treated with either GBS pigment (0.5 μM) or controls (ΔcylE extract or buffer DTS). Then, we measured uptake of the membrane-impermeable dye propidium iodide (PI) at various times after treatment. A significant increase in the number of fluorescent (PI-positive) cells was observed in neutrophils at 30 and 60 min after treatment with GBS pigment, indicating loss of membrane integrity, which was not seen in controls (ΔcylE or buffer DTS; see fig. S7, A to C). To determine whether the neutrophil membrane damage induced by pigment was preceded by classical events in apoptosis (extracellular exposure of phosphatidylserine), we examined binding of fluorescent annexin V. We observed that uptake of fluorescent PI (fig. S7, A to C) preceded annexin V staining (fig. S7, D to F) in pigment-treated neutrophils. These data suggest that pigment-induced neutrophil cell death was independent of apoptosis.

We next examined whether 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). Because 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, and ΔcylE) or pigment and controls for 4 hours (see Materials and Methods). The levels of cytokines IL-1β, IL-6, TNF-α, GRO-α, interferon-γ, and IL-10 were all below the limit of detection. Only WT and nonpigmented/nonhemolytic GBS strains (ΔcovRΔcylE and 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 activaion of caspase 3/7 was not observed in pigment-treated neutrophils when compared with 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

Neutrophil activation is associated with generation of reactive oxygen species (ROS) (36). To determine whether exposure to the hemolytic GBS pigment induced the generation of ROS, we pretreated neutrophils with dihydrorhodamine 123 (DHR) and exposed them 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 and B).

To confirm that GBS strains also induced ROS production, we pretreated neutrophils with DHR and exposed them to GBS strains (WT, hyperhemolytic ΔcovR or nonhemolytic ΔcovRΔcylE, and ΔcylE) at an MOI of 100. Hyperhemolytic GBS accelerated the production of ROS in neutrophils within 15 min (fig. S9, C and D). 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), to understand the role of the hemolytic pigment, independent of complement opsonization. We observed that hyperpigmented GBSΔcovR exhibits increased survival in the presence of neutrophils when compared with nonhemolytic or acapsular GBS strains (fig. S9E). These 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 NETs

The extrusion of neutrophil contents has been associated with the formation of NETs (38, 39). The formation of NETs is dependent on neutrophil components that include NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase, generation of ROS, myeloperoxidase, neutrophil elastase, and histone deamination (3941). Although 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). Because GBS-infected neutrophils in vitro exhibited DNA extrusion (45), we were interested to determine whether 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 (5 μM), equivalent amount of control (ΔcylE extract), or GBS (hyperhemolytic ΔcovR or nonhemolytic ΔcovRΔcylE) at an MOI of ~10 for 4 hours at 37°C. Phorbol 12-myristate 13-acetate (PMA; 20 nM) was included as a positive control. Scanning electron microscopy was then performed, and the results are 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, bottom). Because 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 indicate that, consistent with the scanning electron microscopy images, increased NET-associated neutrophil elastase activity was observed with pigment and hyperhemolytic GBSΔcovR compared with controls or nonpigmented GBSΔcovRΔcylE strain, respectively.

Fig. 4 Increased expression of the hemolytic pigment enables GBS to resist killing by NETs.

(A) (Top) Scanning electron micrographs of NETs in neutrophils treated with GBS pigment (5 μM), negative control (ΔcylE extract), or positive control PMA (20 nM). (Bottom) Scanning electron micrographs of NETS due to hyperpigmented GBSΔcovR or nonpigmented ΔcovRΔcylE. (B) NET-associated neutrophil elastase activity was measured after induction of NETs. Data are the average of three independent experiments performed in duplicate (error bars ± SEM). **P < 0.01, Student’s t test. (C) Neutrophils were incubated with cytochalasin B and D (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 and ΔcylE, and capsule-deficient ΔcpsK) was compared. To inhibit NET-mediated bacterial killing, we treated control wells with DNase I. Data are the average of three independent experiments performed in duplicate (error bars ± SEM). *P < 0.05, Bonferroni’s multiple comparison test following ANOVA.

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, and nonhemolytic ΔcovRΔcylE) to resist killing by NETs was evaluated. Because 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 indicate that the hyperhemolytic GBSΔcovR strain was resistant to killing by NETs unlike the nonhemolytic ΔcovRΔcylE strain or the control capsule-deficient ΔcpsK. Together, our observations suggest that the increased virulence of the hyperhemolytic GBSΔcovR strain is primarily be due to its ability to induce neutrophil cell death and may be augmented by its resistance to NETs.

To confirm that NET formation occurs during GBS infection in vivo, we stained the chorioamniotic membranes of NHPs infected with either GBSΔcovR or GBSΔcovRΔcylE for neutrophil-derived elastase and extracellular DNA, as previously described (33, 49). Membranes from saline controls were also included. The results shown in Fig. 5 demonstrate increased NETs (i.e., colocalization of extracellular DNA with neutrophil elastase) in chorioamniotic membranes of NHPs infected with GBSΔcovR (animal from Fig. 1B) when compared with membranes infected with GBSΔcovRΔcylE (animal from Fig. 1C) or the saline control. Overall, increased NETs were observed in chorioamniotic membranes of ΔcovR-infected samples when compared with GBSΔcovRΔcylE (fig. S10). Collectively, these data indicate that NETs are formed in the chorioamniotic membranes during ascending GBS infection and that the expression of the hemolytic pigment enables GBS to circumvent extracellular killing by NETs.

Fig. 5 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 NHPs inoculated with either hyperpigmented GBSΔcovR, nonpigmented GBSΔcovRΔcylE, or saline. Data are representative of five animals from each group that were examined for NETs. Neutrophil elastase staining is shown in grayscale mode for ease of visualization. PBS, phosphate-buffered saline; DAPI, 4′,6-diamidino-2-phenylindole.

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 with maternal neutrophils. To test this hypothesis, we obtained maternal and cord blood from nonlaboring pregnant women undergoing elective cesarean section at term. Neutrophils were isolated from maternal and cord blood pairs, and flow 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). Both maternal and fetal neutrophils were similar in their sensitivity to the GBS pigment 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 with maternal neutrophils (compare panels ii and iii with panels 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. Although survival of the nonhemolytic GBSΔcovRΔcylE was significantly attenuated when compared with 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 indicate that fetal neutrophils showed significantly lower NET-associated neutrophil elastase activity than did 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.

Fig. 6 Decreased ROS generation in fetal neutrophils compared with 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 are the average of three independent experiments performed in triplicate (error bars ± SEM). ****P < 0.0001, Tukey’s multiple comparison test following ANOVA. NS, not significant. (B) Maternal or fetal neutrophils were pretreated with 84 μM DHR. Subsequently, the neutrophils were treated with either 0.5 μM pigment or controls, and oxidation to fluorescent MHR was monitored. Panels (i), (ii), and (iii) show fetal neutrophils at 0, 30, and 60 min after treatment with pigment and controls, respectively. Panels (iv), (v), and (vi) show maternal neutrophils at 0, 30, and 60 min after treatment with pigment and controls, respectively. Gates shown reflect the percent cell number with various treatments. Data are representative of three independent experiments.

Fig. 7 Fetal neutrophils are more sensitive to nonpigmented GBS strains and exhibit decreased neutrophil elastase activity when compared with maternal neutrophils.

(A) Maternal and fetal neutrophils were incubated with either WT GBS COH1 or isogenic mutants (hyperpigmented ΔcovR and 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 neutrophils to CFU recovered in the absence of neutrophils. Data are the average of four independent experiments with neutrophils isolated from different donors, and each experiment was performed in duplicate (error bars ± SEM). n = 3. **P < 0.01, Sidak’s multiple comparison test following ANOVA. (B) NET-associated neutrophil elastase activity was measured after induction of NETs in maternal and fetal neutrophils treated with GBS strains (WT ΔcovR, ΔcylE, and ΔcovRΔcylE). Data are the average of three independent experiments with neutrophils isolated from different donors (error bars ± SEM). *P < 0.05, **P < 0.01, Student’s t test.

Collectively, the results presented in this study indicate that GBS uses a unique rhamnolipid pigment toxin to circumvent neutrophils and NETs in the chorioamnion to facilitate microbial trafficking into the amniotic cavity and fetus during pregnancy.

DISCUSSION

GBS infections during pregnancy remain an important public health concern (8). Although current methods of prevention of GBS transmission from mother to fetus during labor and delivery have made remarkable progress in decreasing early-onset disease, intrapartum antibiotic prophylaxis does not address or decrease ascending GBS infection, leading to 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.

Here, we used a highly relevant, chronically catheterized pregnant NHP 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 chorioamniotic membranes and infect the AF and fetal organs. 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 min to within a few hours after infection. GBS invasion of the amniotic activity induced increased expression of neutrophil-recruiting cytokines and chemokines in the chorioamniotic membranes and AF. Consistent with these findings, we observed significant recruitment of neutrophils in the chorioamnion of NHPs infected with hyperpigmented GBS. These results indicate that neutrophils comprise the initial and primary host defense mechanism used by the chorioamnion to combat invasive pathogens such as GBS. However, despite the recruitment of neutrophils, the hyperhemolytic GBS strain could traffic across the chorioamniotic membranes in three of the five animals, and bacteria were recovered from multiple fetal organs. These observations suggest that although neutrophils are recruited to the site of 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 the expression of the GBS hemolytic pigment is typically associated with the generation of a proinflammatory response and neutrophil recruitment, this appears ineffective in curtailing bacterial burden, as observed by others (13, 52, 57) and this study.

We then examined how increased expression of the hemolytic pigment may enable GBS to subvert neutrophils. Our studies revealed that the hemolytic pigment and hyperpigmented GBS induced neutrophil cell death in a dose-dependent manner within 4 hours. The neutrophil cell death observed with the GBS pigment is independent of apoptosis or even pyroptosis. Although we previously noted that macrophages lacking the NLRP3 inflammasome could 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. Scanning electron micrographs of neutrophils exposed to the GBS toxin revealed severe morphological changes, and 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. Before our study, NET formation in placental membranes was only reported in association with a noninfectious condition, that is, 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 NHPs infected with hyperpigmented GBS, both live and dying neutrophils contribute to NET formation in vivo. Whether NETs themselves contribute to placental dysfunction as previously 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 with the nonhemolytic strain. This may, in part, be due to the ability of the unsaturated polyene chain in the hemolytic pigment toxin to quench ROS. Previous work by Liu et 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 the expression of an extracellular nuclease (nuclease A) in GBS was shown to degrade NETs (60), previous work by others and us showed that the 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 deoxyribonuclease (DNase) activity. Our observations suggest 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 to wall off infection without actually inducing bacterial cell death (62, 63). Addition of DNase at the end of the NET killing assay is thought to relieve the clumping effect and to provide accurate results for discrimination between ensnaring of bacteria by NETs and bacterial killing by NETs (62). Although we repeated these experiments with the addition of DNase at the end of the experiment to confirm increased sensitivity of GBSΔcovRΔcylE to NETS, it remains plausible that GBS entrapment by NETs rather than NET-associated killing regulates MIAC in vivo.

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 AF and fetus. Recently, using a mouse vaginal colonization model, we showed that mast cell degranulation can promote neutrophil recruitment and enable eradication of hypervirulent GBS from the vaginal tract (32). Although the mast cell response of humans and NHPs 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). Together, these observations suggest that when neutrophils escape 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 with 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 lipopolysaccharide (65, 66). Furthermore, fetal neutrophils were not as efficient as maternal neutrophils in curtailing nonhemolytic GBS strains. Whereas previous studies reported that pregnancy can be associated with diminished oxidative burst function in maternal neutrophils (67), our data suggest that, compared with fetal neutrophils, maternal neutrophils play a critical role in restraining lower genital tract pathogens such as GBS from accessing fetal compartments and tissues.

Here, although 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. 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 used by this pathogen to cause fetal injury, preterm birth, or stillbirth.

In summary, we show that GBS uses the hemolytic pigment to invade the AF 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.

MATERIALS AND METHODS

For the detailed Materials and Methods, please see the Supplementary Materials.

Correction: In Figures 3 and 4, the scale bars incorrectly read 1 millimeter (mm) instead of 1 micrometer (μm). The PDF and HTML (full text) have been corrected.

SUPPLEMENTARY MATERIALS

immunology.sciencemag.org/cgi/content/full/1/4/eaah4576/DC1

Materials and Methods

Fig. S1. Uterine contractions, AF cytokines, prostaglandin, and bacterial CFU from choriodecidual inoculations of hyperpigmented GBSΔcovR in four chronically catheterized pregnant M. nemestrina.

Fig. S2. Uterine contractions, AF cytokines, prostaglandin, and bacterial CFU from choriodecidual inoculations of nonpigmented GBSΔcovRΔcylE in four chronically catheterized pregnant M. nemestrina.

Fig. S3. Choriodecidual inoculation of hyperpigmented GBSΔcovR in pregnant NHPs induced bacterial invasion of fetal tissues and increased levels of TNF-α.

Fig. S4. Heat map demonstrating differential gene expression in chorioamniotic membranes from pregnant NHPs.

Fig. S5. Changes in chorioamniotic gene expression.

Fig. S6. Fluorescence-activated cell sorting (FACS) of neutrophils purified from human blood.

Fig. S7. The hemolytic pigment toxin induces membrane permeability in neutrophils.

Fig. S8. The hemolytic pigment induces neutrophil cell death independent of pyroptosis or apoptosis.

Fig. S9. Hyperhemolytic GBS induce ROS but are resistant to neutrophil killing.

Fig. S10. Increased NETs in the chorioamnion of NHPs infected with hyperpigmented GBSΔcovR.

Fig. S11. FACS of neutrophils purified from human maternal and fetal (cord) blood.

Fig. S12. The hemolytic pigment toxin induces membrane permeability in maternal and fetal neutrophils.

Table S1. Detailed pregnancy outcomes by animal.

Table S2. Selected genes showing differential expression in NHP chorioamniotic membranes infected with GBS strains or saline.

Table S3. qPCR probes and primers.

References (6886)

REFERENCES AND NOTES

Acknowledgments: We are grateful to the participants who participated in our study. We thank A. Gest, J. Hamanishi, G. Heidel, D. Power, J. Karlinsey, and C. Hughes for their assistance. We acknowledge C. Rubens and M. Gravett for study design related to the performance of the original NHP experiments. Funding: This work was supported by the NIH (grant R01AI100989 to L.R. and K.M.A.W.; grants R56AI070749, R01AI112619, and R21AI109222 to L.R.; and grant P30HD002274). The NIH training grants T32 HD007233 (principal investigator: L. Frenkel) and T32 AI07509 (principal investigator: L. A. 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 NIH or other funders. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author contributions: E.B., C.G., J.V., L.R., and K.M.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.M.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.M.A.W. analyzed the results. E.B., C.G., J.V., T.K.B., H.D.L., L.R., and K.M.A.W. wrote the paper. Competing interests: The authors declare that they have no competing interests. Data and materials availability: The microarray data for this study have been deposited in the GEO database and are available through GEO accession number GSE80248.

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