HHS Public Access Author manuscript Author Manuscript
Sci Immunol. Author manuscript; available in PMC 2017 April 01. 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
Author Manuscript
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, 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
Author Manuscript
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
Author Manuscript
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 β-hemolytic, gram-positive bacteria, which commonly colonize the vagina but have been
#
Address correspondence to Kristina Adams Waldorf, [email protected] and Lakshmi Rajagopal, [email protected]. *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.
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Author Manuscript Author Manuscript
recovered from the amniotic fluid in preterm birth cases. To understand temporal events that occur 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 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).
Author Manuscript
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 development of preventive therapies.
Author Manuscript
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 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).
Sci Immunol. Author manuscript; available in PMC 2017 April 01.
Boldenow et al.
Page 3
Author Manuscript
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 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 (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).
Author Manuscript Author Manuscript
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 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).
Author Manuscript
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
Sci Immunol. Author manuscript; available in PMC 2017 April 01.
Boldenow et al.
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Author Manuscript
hyperpigmented GBSΔcovR (n=5) or control nonpigmented GBSΔcovRΔcylE (n=5); these results were compared with saline controls (n=5) that were previously described (21).
Author Manuscript
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 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).
Author Manuscript
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 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).
Author Manuscript
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
Sci Immunol. Author manuscript; available in PMC 2017 April 01.
Boldenow et al.
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not exhibit signs of preterm labor or MIAC. Taken 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 vs. GBSΔcovRΔcylE; P =0.0009, GBSΔcovR vs. saline). Increased amniotic fluid and fetal cytokines are observed in pregnant NHP infected with hyperpigmented GBS
Author Manuscript Author Manuscript
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
Sci Immunol. Author manuscript; available in PMC 2017 April 01. 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
Author Manuscript
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, 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
Author Manuscript
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
Author Manuscript
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 β-hemolytic, gram-positive bacteria, which commonly colonize the vagina but have been
#
Address correspondence to Kristina Adams Waldorf, [email protected] and Lakshmi Rajagopal, [email protected]. *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.
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Author Manuscript Author Manuscript
recovered from the amniotic fluid in preterm birth cases. To understand temporal events that occur 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 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).
Author Manuscript
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 development of preventive therapies.
Author Manuscript
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 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).
Sci Immunol. Author manuscript; available in PMC 2017 April 01.
Boldenow et al.
Page 3
Author Manuscript
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 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 (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).
Author Manuscript Author Manuscript
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 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).
Author Manuscript
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
Sci Immunol. Author manuscript; available in PMC 2017 April 01.
Boldenow et al.
Page 4
Author Manuscript
hyperpigmented GBSΔcovR (n=5) or control nonpigmented GBSΔcovRΔcylE (n=5); these results were compared with saline controls (n=5) that were previously described (21).
Author Manuscript
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 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).
Author Manuscript
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 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).
Author Manuscript
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
Sci Immunol. Author manuscript; available in PMC 2017 April 01.
Boldenow et al.
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not exhibit signs of preterm labor or MIAC. Taken 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 vs. GBSΔcovRΔcylE; P =0.0009, GBSΔcovR vs. saline). Increased amniotic fluid and fetal cytokines are observed in pregnant NHP infected with hyperpigmented GBS
Author Manuscript Author Manuscript
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
