Homeostatic Macrophages Prevent Preterm Birth and Improve Neonatal Outcomes by Mitigating In Utero Sterile Inflammation in Mice.

Preterm birth (PTB), often preceded by preterm labor, is a major cause of neonatal morbidity and mortality worldwide. Most PTB cases involve intra-amniotic inflammation without detectable microorganisms, termed in utero sterile inflammation, for which there is no established treatment. In this study, we propose homeostatic macrophages to prevent PTB and adverse neonatal outcomes caused by in utero sterile inflammation. Single-cell atlases of the maternal-fetal interface revealed that homeostatic maternal macrophages are reduced with human labor. M2 macrophage treatment prevented PTB and reduced adverse neonatal outcomes in mice with in utero sterile inflammation. Specifically, M2 macrophages halted premature labor by suppressing inflammatory responses in the amniotic cavity, including inflammasome activation, and mitigated placental and offspring lung inflammation. Moreover, M2 macrophages boosted gut inflammation in neonates and improved their ability to fight systemic bacterial infections. Our findings show that M2 macrophages are a promising strategy to mitigate PTB and improve neonatal outcomes resulting from in utero sterile inflammation.

Differential immunophenotype of circulating monocytes from pregnant women in response to viral ligands.

BACKGROUND: Viral infections during pregnancy can have deleterious effects on mothers and their offspring. Monocytes participate in the maternal host defense against invading viruses; however, whether pregnancy alters monocyte responses is still under investigation. Herein, we undertook a comprehensive in vitro study of peripheral monocytes to characterize the differences in phenotype and interferon release driven by viral ligands between pregnant and non-pregnant women. METHODS: Peripheral blood was collected from third-trimester pregnant (n = 20) or non-pregnant (n = 20, controls) women. Peripheral blood mononuclear cells were isolated and exposed to R848 (TLR7/TLR8 agonist), Gardiquimod (TLR7 agonist), Poly(I:C) (HMW) VacciGrade™ (TLR3 agonist), Poly(I:C) (HMW) LyoVec™ (RIG-I/MDA-5 agonist), or ODN2216 (TLR9 agonist) for 24 h. Cells and supernatants were collected for monocyte phenotyping and immunoassays to detect specific interferons, respectively. RESULTS: The proportions of classical (CD14hiCD16-), intermediate (CD14hiCD16+), non-classical (CD14loCD16+), and CD14loCD16- monocytes were differentially affected between pregnant and non-pregnant women in response to TLR3 stimulation. The proportions of pregnancy-derived monocytes expressing adhesion molecules (Basigin and PSGL-1) or the chemokine receptors CCR5 and CCR2 were diminished in response to TLR7/TLR8 stimulation, while the proportions of CCR5- monocytes were increased. Such differences were found to be primarily driven by TLR8 signaling, rather than TLR7. Moreover, the proportions of monocytes expressing the chemokine receptor CXCR1 were increased during pregnancy in response to poly(I:C) stimulation through TLR3, but not RIG-I/MDA-5. By contrast, pregnancy-specific changes in the monocyte response to TLR9 stimulation were not observed. Notably, the soluble interferon response to viral stimulation by mononuclear cells was not diminished in pregnancy. CONCLUSIONS: Our data provide insight into the differential responsiveness of pregnancy-derived monocytes to ssRNA and dsRNA, mainly driven by TLR8 and membrane-bound TLR3, which may help to explain the increased susceptibility of pregnant women to adverse outcomes resulting from viral infection as observed during recent and historic pandemics.

One-third of patients with eclampsia at term do not have an abnormal angiogenic profile.

OBJECTIVES: An abnormal angiogenic profile is present in about one-half of women with preeclampsia at term. Few studies examined the roles of angiogenic biomarkers in eclampsia. The aims of this study were to determine (1) whether the degree of an anti-angiogenic state, reflected by a low placental growth factor (PlGF) to soluble fms-like tyrosine kinase-1 (sFlt-1) ratio, in women with eclampsia differed from that of women with severe preeclampsia; and (2) the prevalence of women who had an abnormal angiogenic profile at the diagnoses of preterm and term eclampsia. METHODS: A cross-sectional study was conducted to include women in the following groups: (1) uncomplicated pregnancy (n=40); (2) severe preeclampsia (n=50); and (3) eclampsia (n=35). Maternal serum concentrations of PlGF and sFlt-1 were determined by immunoassays. RESULTS: Women with preterm, but not term, eclampsia had a more severe anti-angiogenic state than those with severe preeclampsia (lower PlGF and PlGF/sFlt-1 ratio, each p<0.05). However, the difference diminished in magnitude with increasing gestational age (interaction, p=0.005). An abnormal angiogenic profile was present in 95% (19/20) of women with preterm eclampsia but in only 67% (10/15) of women with eclampsia at term. CONCLUSIONS: Angiogenic biomarkers can be used for risk assessment of preterm eclampsia. By contrast, a normal profile of angiogenic biomarkers cannot reliably exclude patients at risk for eclampsia at term. This observation has major clinical implications given that angiogenic biomarkers are frequently used in the triage area as a test to rule out preeclampsia.

Cellular immune responses in amniotic fluid of women with preterm clinical chorioamnionitis.

OBJECTIVE: Preterm birth is the leading cause of neonatal morbidity and mortality worldwide. Some preterm births are associated with clinical chorioamnionitis; yet, this condition has been poorly investigated. Herein, we characterized the amniotic fluid cellular immune responses in women with preterm clinical chorioamnionitis. METHODS AND SUBJECTS: Amniotic fluid samples were obtained from women with preterm clinical chorioamnionitis and a positive or negative microbiological culture (n = 17). The cellular composition of amniotic fluid was evaluated using fluorescence microscopy, scanning and transmission electron microscopy, and flow cytometry. Women without preterm clinical chorioamnionitis were also examined (n = 10). RESULTS: Amniotic fluid from women with preterm clinical chorioamnionitis and a positive culture had: (1) abundant neutrophils associated with viable and non-viable bacteria, (2) neutrophils performing phagocytosis, (3) neutrophils forming NETs, (4) increased numbers of neutrophils, monocytes/macrophages, and CD4+ T cells, and (5) high expression of IL-1ß by neutrophils and monocytes/macrophages. Amniotic fluid from women with preterm clinical chorioamnionitis and proven infection tended to have fewer monocytes/macrophages and CD4+ T cells compared to those without chorioamnionitis. CONCLUSION: We provide the first morphologic and phenotypic characterization of the cellular immune responses in the amniotic cavity of women with preterm clinical chorioamnionitis, a condition associated with adverse neonatal outcomes.

Pregnancy-specific transcriptional changes upon endotoxin exposure in mice.

Objectives Pregnant women are more susceptible to certain infections; however, this increased susceptibility is not fully understood. Herein, systems biology approaches were utilized to elucidate how pregnancy modulates tissue-specific host responses to a bacterial product, endotoxin. Methods Pregnant and non-pregnant mice were injected with endotoxin or saline on 16.5 days post coitum (n=8-11 per group). The uterus, cervix, liver, adrenal gland, kidney, lung, and brain were collected 12 h after injection and transcriptomes were measured using microarrays. Heatmaps and principal component analysis were used for visualization. Differentially expressed genes between groups were assessed using linear models that included interaction terms to determine whether the effect of infection differed with pregnancy status. Pathway analysis was conducted to interpret gene expression changes. Results We report herein a multi-organ atlas of the transcript perturbations in pregnant and non-pregnant mice in response to endotoxin. Pregnancy strongly modified the host responses to endotoxin in the uterus, cervix, and liver. In contrast, pregnancy had a milder effect on the host response to endotoxin in the adrenal gland, lung, and kidney. However, pregnancy did not drastically affect the host response to endotoxin in the brain. Conclusions Pregnancy imprints organ-specific host immune responses upon endotoxin exposure. These findings provide insight into the host-response against microbes during pregnancy.

Cellular immune responses in amniotic fluid of women with a sonographic short cervix.

Objectives A sonographic short cervix is one of the strongest predictors of preterm delivery. However, the cellular immune composition of amniotic fluid in women with a short cervix has not yet been described. Herein, we determined cellular and soluble immune responses in amniotic fluid from pregnant women with a mid-trimester asymptomatic short cervix. Methods Amniotic fluid samples (n=77) were collected from asymptomatic women with a cervical length between 15 and 25 mm (n=36, short cervix) or ≤15 mm (n=41, severely short cervix) diagnosed by ultrasound. Flow cytometry and multiplex measurement of cytokines/chemokines were performed. Results (1) The cellular immune composition of amniotic fluid did not differ between women with a severely short cervix (≤15 mm) and those with a short cervix 15-25 mm; (2) amniotic fluid concentrations of multiple cytokines/chemokines were higher in women with a severely short cervix (≤15 mm) than in those with a short cervix 15-25 mm; (3) the cellular immune composition of amniotic fluid did not differ between women with a severely short cervix (≤15 mm) who ultimately underwent preterm delivery and those who delivered at term; and (4) amniotic fluid concentrations of IL-2, but not other immune mediators, were increased in women with a severely short cervix (≤15 mm) who ultimately delivered preterm compared to those who delivered at term. Conclusions Women with a severely short cervix (≤15 mm) have increased concentrations of pro-inflammatory mediators in the amniotic cavity; yet, these do not translate to changes in the cellular immune response.

Microbial burden and inflammasome activation in amniotic fluid of patients with preterm prelabor rupture of membranes.

Background Intra-amniotic inflammation, which is associated with adverse pregnancy outcomes, can occur in the presence or absence of detectable microorganisms, and involves activation of the inflammasome. Intra-amniotic inflammasome activation has been reported in clinical chorioamnionitis at term and preterm labor with intact membranes, but it has not yet been investigated in women with preterm prelabor rupture of membranes (preterm PROM) in the presence/absence of detectable microorganisms. The aim of this study was to determine whether, among women with preterm PROM, there is an association between detectable microorganisms in amniotic fluid and intra-amniotic inflammation, and whether intra-amniotic inflammasome activation correlates with microbial burden. Methods Amniotic fluids from 59 cases of preterm PROM were examined for the presence/absence of microorganisms through culture and 16S ribosomal RNA (rRNA) gene quantitative real-time polymerase chain reaction (qPCR), and concentrations of interleukin-6 (IL-6) and ASC [apoptosis-associated spec-like protein containing a caspase recruitment domain (CARD)], an indicator of inflammasome activation, were determined. Results qPCR identified more microbe-positive amniotic fluids than culture. Greater than 50% of patients with a negative culture and high IL-6 concentration in amniotic fluid yielded a positive qPCR signal. ASC concentrations were greatest in patients with high qPCR signals and elevated IL-6 concentrations in amniotic fluid (i.e. intra-amniotic infection). ASC concentrations tended to increase in patients without detectable microorganisms but yet with elevated IL-6 concentrations (i.e. sterile intra-amniotic inflammation) compared to those without intra-amniotic inflammation. Conclusion qPCR is a valuable complement to microbiological culture for the detection of microorganisms in the amniotic cavity in women with preterm PROM, and microbial burden is associated with the severity of intra-amniotic inflammatory response, including inflammasome activation.

The origin of amniotic fluid monocytes/macrophages in women with intra-amniotic inflammation or infection.

Background Monocytes, after neutrophils, are the most abundant white blood cells found in the amniotic cavity of women with intra-amniotic inflammation/infection. However, the origin of such cells has not been fully investigated. Herein, we determined (1) the origin of amniotic fluid monocytes/macrophages from women with intra-amniotic inflammation/infection, (2) the relationship between the origin of amniotic fluid monocytes/macrophages and preterm or term delivery and (3) the localization of monocytes/macrophages in the placental tissues. Methods Amniotic fluid samples (n = 16) were collected from women with suspected intra-amniotic inflammation or infection. Amniotic fluid monocytes/macrophages were purified by fluorescence-activated cell sorting, and DNA fingerprinting was performed. Blinded placental histopathological evaluations were conducted. Immunohistochemistry was performed to detect CD14+ monocytes/macrophages in the placental tissues. Results DNA fingerprinting revealed that (1) 56.25% (9/16) of amniotic fluid samples had mostly fetal monocytes/macrophages, (2) 37.5% (6/16) had predominantly maternal monocytes/macrophages and (3) one sample (6.25% [1/16]) had a mixture of fetal and maternal monocytes/macrophages. (4) Most samples with predominantly fetal monocytes/macrophages were from women who delivered early preterm neonates (77.8% [7/9]), whereas all samples with mostly maternal monocytes/macrophages or a mixture of both were from women who delivered term or late preterm neonates (100% [7/7]). (5) Most of the women included in this study presented acute maternal and fetal inflammatory responses in the placenta (85.7% [12/14]). (6) Women who had mostly fetal monocytes/macrophages in amniotic fluid had abundant CD14+ cells in the umbilical cord and chorionic plate, whereas women with mostly maternal amniotic fluid monocytes/macrophages had abundant CD14+ cells in the chorioamniotic membranes. Conclusion Amniotic fluid monocytes/macrophages can be of either fetal or maternal origin, or a mixture of both, in women with intra-amniotic inflammation or infection. These immune cells could be derived from the fetal and maternal vasculature of the placenta.

Thymic stromal lymphopoietin participates in the host response to intra-amniotic inflammation leading to preterm labor and birth.

The aim of this study was to establish the role of thymic stromal lymphopoietin (TSLP) in the intra-amniotic host response of women with spontaneous preterm labor (sPTL) and birth. Amniotic fluid and chorioamniotic membranes (CAM) were collected from women with sPTL who delivered at term (n = 30) or preterm without intra-amniotic inflammation (n = 34), with sterile intra-amniotic inflammation (SIAI, n = 27), or with intra-amniotic infection (IAI, n = 17). Amnion epithelial cells (AEC), Ureaplasma parvum, and Sneathia spp. were also utilized. The expression of TSLP, TSLPR, and IL-7Rα was evaluated in amniotic fluid or CAM by RT-qPCR and/or immunoassays. AEC co-cultured with Ureaplasma parvum or Sneathia spp. were evaluated for TSLP expression by immunofluorescence and/or RT-qPCR. Our data show that TSLP was elevated in amniotic fluid of women with SIAI or IAI and expressed by the CAM. TSLPR and IL-7Rα had detectable gene and protein expression in the CAM; yet, CRLF2 was specifically elevated with IAI. While TSLP localized to all layers of the CAM and increased with SIAI or IAI, TSLPR and IL-7Rα were minimal and became most apparent with IAI. Co-culture experiments indicated that Ureaplasma parvum and Sneathia spp. differentially upregulated TSLP expression in AEC. Together, these findings indicate that TSLP is a central component of the intra-amniotic host response during sPTL.

Maternal-fetal immune responses in pregnant women infected with SARS-CoV-2.

Pregnant women represent a high-risk population for severe/critical COVID-19 and mortality. However, the maternal-fetal immune responses initiated by SARS-CoV-2 infection, and whether this virus is detectable in the placenta, are still under investigation. Here we show that SARS-CoV-2 infection during pregnancy primarily induces unique inflammatory responses at the maternal-fetal interface, which are largely governed by maternal T cells and fetal stromal cells. SARS-CoV-2 infection during pregnancy is also associated with humoral and cellular immune responses in the maternal blood, as well as with a mild cytokine response in the neonatal circulation (i.e., umbilical cord blood), without compromising the T-cell repertoire or initiating IgM responses. Importantly, SARS-CoV-2 is not detected in the placental tissues, nor is the sterility of the placenta compromised by maternal viral infection. This study provides insight into the maternal-fetal immune responses triggered by SARS-CoV-2 and emphasizes the rarity of placental infection.

Maternal-Fetal Immune Responses in Pregnant Women Infected with SARS-CoV-2.

Pregnant women are a high-risk population for severe/critical COVID-19 and mortality. However, the maternal-fetal immune responses initiated by SARS-CoV-2 infection, and whether this virus is detectable in the placenta, are still under investigation. Herein, we report that SARS-CoV-2 infection during pregnancy primarily induced specific maternal inflammatory responses in the circulation and at the maternal-fetal interface, the latter being governed by T cells and macrophages. SARS-CoV-2 infection during pregnancy was also associated with a cytokine response in the fetal circulation (i.e. umbilical cord blood) without compromising the cellular immune repertoire. Moreover, SARS-CoV-2 infection neither altered fetal cellular immune responses in the placenta nor induced elevated cord blood levels of IgM. Importantly, SARS-CoV-2 was not detected in the placental tissues, nor was the sterility of the placenta compromised by maternal viral infection. This study provides insight into the maternal-fetal immune responses triggered by SARS-CoV-2 and further emphasizes the rarity of placental infection.

A Mutated PB1 Residue 319 Synergizes with the PB2 N265S Mutation of the Live Attenuated Influenza Vaccine to Convey Temperature Sensitivity.

Current influenza vaccines have modest efficacy. This is especially true for current live attenuated influenza vaccines (LAIV), which have been inferior to the inactivated versions in recent years. Therefore, a new generation of live vaccines may be needed. We previously showed that a mutation at PB1 residue 319 confers enhanced temperature sensitivity and attenuation in an LAIV constructed in the genetic background of the mouse-adapted Influenza A Virus (IAV) strain A/PR/8/34 (PR8). Here, we describe the origin/discovery of this unique mutation and demonstrate that, when combined with the PB2 N265S mutation of LAIV, it conveys an even greater level of temperature sensitivity and attenuation on PR8 than the complete set of attenuating mutations from LAIV. Furthermore, we show that the combined PB1 L319Q and PB2 N265S mutations confer temperature sensitivity on IAV polymerase activity in two different genetic backgrounds, PR8 and A/Cal/04/09. Collectively, these findings show that the PB2 LAIV mutation synergizes with a mutation in PB1 and may have potential utility for improving LAIVs.

Prenatal Maternal Stress Causes Preterm Birth and Affects Neonatal Adaptive Immunity in Mice.

Maternal stress is a well-established risk factor for preterm birth and has been associated with adverse neonatal outcomes in the first and subsequent generations, including increased susceptibility to disease and lasting immunological changes. However, a causal link between prenatal maternal stress and preterm birth, as well as compromised neonatal immunity, has yet to be established. To fill this gap in knowledge, we used a murine model of prenatal maternal stress across three generations and high-dimensional flow cytometry to evaluate neonatal adaptive immunity. We report that recurrent prenatal maternal stress induced preterm birth in the first and second filial generations and negatively impacted early neonatal growth. Strikingly, prenatal maternal stress induced a systematic reduction in T cells and B cells, the former including regulatory CD4+ T cells as well as IL-4- and IL-17A-producing T cells, in the second generation. Yet, neonatal adaptive immunity gained resilience against prenatal maternal stress by the third generation. We also show that the rate of prenatal maternal stress-induced preterm birth can be reduced upon cessation of stress, though neonatal growth impairments persisted. These findings provide evidence that prenatal maternal stress causes preterm birth and affects neonatal immunity across generations, adverse effects that can be ameliorated upon cessation.

Intra-Amniotic Infection with Ureaplasma parvum Causes Preterm Birth and Neonatal Mortality That Are Prevented by Treatment with Clarithromycin.

Intra-amniotic infection is strongly associated with adverse pregnancy and neonatal outcomes. Most intra-amniotic infections are due to Ureaplasma species; however, the pathogenic potency of these genital mycoplasmas to induce preterm birth is still controversial. Here, we first laid out a taxonomic characterization of Ureaplasma isolates from women with intra-amniotic infection, which revealed that Ureaplasma parvum is the most common bacterium found in this clinical condition. Next, using animal models, we provided a causal link between intra-amniotic inoculation with Ureaplasma species and preterm birth. Importantly, the intra-amniotic inoculation of Ureaplasma species induced high rates of mortality in both preterm and term neonates. The in vivo potency of U. parvum to induce preterm birth was not associated with known virulence factors. However, term-derived and preterm-derived U. parvum isolates were capable of inducing an intra-amniotic inflammatory response. Both U. parvum isolates invaded several fetal tissues, primarily the fetal lung, and caused fetal inflammatory response syndrome. This bacterium was also detected in the placenta, reproductive tissues, and most severely in the fetal membranes, inducing a local inflammatory response that was replicated in an in vitro model. Importantly, treatment with clarithromycin, a recently recommended yet not widely utilized antibiotic, prevented the adverse pregnancy and neonatal outcomes induced by U. parvum These findings shed light on the maternal-fetal immunobiology of intra-amniotic infection.IMPORTANCE Preterm birth is the leading cause of neonatal morbidity and mortality worldwide. Multiple etiologies are associated with preterm birth; however, 25% of preterm infants are born to a mother with intra-amniotic infection, most commonly due to invasion of the amniotic cavity by Ureaplasma species. Much research has focused on establishing a link between Ureaplasma species and adverse pregnancy/neonatal outcomes; however, little is known about the taxonomy of and host response against Ureaplasma species. Here, we applied a multifaceted approach, including human samples, in vivo models, and in vitro manipulations, to study the maternal-fetal immunobiology of Ureaplasma infection during pregnancy. Furthermore, we investigated the use of clarithromycin as a treatment for this infection. Our research provides translational knowledge that bolsters scientific understanding of Ureaplasma species as a cause of adverse pregnancy/neonatal outcomes and gives strong evidence for the use of clarithromycin as the recommended treatment for women intra-amniotically infected with Ureaplasma species.