Palmitate and group B Streptococcus synergistically and differentially induce IL-1ß from human gestational membranes.

Introduction: Rupture of the gestational membranes often precedes major pregnancy complications, including preterm labor and preterm birth. One major cause of inflammation in the gestational membranes, chorioamnionitis (CAM) is often a result of bacterial infection. The commensal bacterium Streptococcus agalactiae, or Group B Streptococcus (GBS) is a leading infectious cause of CAM. Obesity is on the rise worldwide and roughly 1 in 4 pregnancy complications is related to obesity, and individuals with obesity are also more likely to be colonized by GBS. The gestational membranes are comprised of several distinct cell layers which are, from outermost to innermost: maternally-derived decidual stromal cells (DSCs), fetal cytotrophoblasts (CTBs), fetal mesenchymal cells, and fetal amnion epithelial cells (AECs). In addition, the gestational membranes have several immune cell populations; macrophages are the most common phagocyte. Here we characterize the effects of palmitate, the most common long-chain saturated fatty acid, on the inflammatory response of each layer of the gestational membranes when infected with GBS, using human cell lines and primary human tissue. Results: Palmitate itself slightly but significantly augments GBS proliferation. Palmitate and GBS co-stimulation synergized to induce many inflammatory proteins and cytokines, particularly IL-1ß and matrix metalloproteinase 9 from DSCs, CTBs, and macrophages, but not from AECs. Many of these findings are recapitulated when treating cells with palmitate and a TLR2 or TLR4 agonist, suggesting broad applicability of palmitate-pathogen synergy. Co-culture of macrophages with DSCs or CTBs, upon co-stimulation with GBS and palmitate, resulted in increased inflammatory responses, contrary to previous work in the absence of palmitate. In whole gestational membrane biopsies, the amnion layer appeared to dampen immune responses from the DSC and CTB layers (the choriodecidua) to GBS and palmitate co-stimulation. Addition of the monounsaturated fatty acid oleate, the most abundant monounsaturated fatty acid in circulation, dampened the proinflammatory effect of palmitate. Discussion: These studies reveal a complex interplay between the immunological response of the distinct layers of the gestational membrane to GBS infection and that such responses can be altered by exposure to long-chain saturated fatty acids. These data provide insight into how metabolic syndromes such as obesity might contribute to an increased risk for GBS disease during pregnancy.

Environmental Toxicant Exposure Paralyzes Human Placental Macrophage Responses to Microbial Threat.

Exposure to environmental toxicants (such as dioxins) has been epidemiologically linked to adverse reproductive health outcomes, including placental inflammation and preterm birth. However, the molecular underpinnings that govern these outcomes in gravid reproductive tissues remain largely unclear. Placental macrophages (also known as Hofbauer cells) are crucial innate immune cells that defend the gravid reproductive tract and help promote maternal-fetal tolerance. We hypothesized that exposure to environmental toxicants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) could alter placental macrophage responses to inflammatory insults such as infection. To test this, placental macrophages were cultured in the presence or absence of TCDD and then infected with the perinatal pathogen Group B Streptococcus (GBS). Our results indicate that TCDD is lethal to placental macrophages at and above a 5 nM concentration and that sublethal dioxin exposure inhibits phagocytosis and cytokine production. Taken together, these results indicate that TCDD paralyzes placental macrophage responses to bacterial infection.

The Utility of Human Milk Oligosaccharides against Group B Streptococcus Infections of Reproductive Tissues and Cognate Adverse Pregnancy Outcomes.

Preterm birth affects nearly 10% of all pregnancies in the United States, with 40% of those due, in part, to infections. Streptococcus agalactiae (Group B Streptococcus, GBS) is one of the most common perinatal pathogens responsible for these infections. Current therapeutic techniques aimed to ameliorate invasive GBS infections are less than desirable and can result in complications in both the neonate and the mother. To this end, the need for novel therapeutic options is urgent. Human milk oligosaccharides (HMOs), an integral component of human breast milk, have been previously shown to possess antiadhesive and antimicrobial properties. To interrogate these characteristics, we examined HMO-mediated outcomes in both in vivo and ex vivo models of GBS infection utilizing a murine model of ascending GBS infection, an EpiVaginal human organoid tissue model, and ex vivo human gestational membranes. Supplementation of HMOs resulted in diminished adverse pregnancy outcomes, decreased GBS adherence to gestational tissues, decreased colonization within the reproductive tract, and reduced proinflammatory immune responses to GBS infection. Taken together, these results highlight the potential of HMOs as promising therapeutic interventions in perinatal health.

Host inflammatory dynamics reveal placental immune modulation by Group B Streptococcus during pregnancy.

Group B Streptococcus (GBS) is a pathobiont that can ascend to the placenta and cause adverse pregnancy outcomes, in part through production of the toxin ß-hemolysin/cytolysin (ß-h/c). Innate immune cells have been implicated in the response to GBS infection, but the impact of ß-h/c on their response is poorly defined. We show that GBS modulates innate immune cell states by subversion of host inflammation through ß-h/c, allowing worse outcomes. We used an ascending mouse model of GBS infection to measure placental cell state changes over time following infection with a ß-h/c-deficient and isogenic wild type GBS strain. Transcriptomic analysis suggests that ß-h/c-producing GBS elicit a worse phenotype through suppression of host inflammatory signaling in placental macrophages and neutrophils, and comparison of human placental macrophages infected with the same strains recapitulates these results. Our findings have implications for identification of new targets in GBS disease to support host defense against pathogenic challenge.

Streptococcus agalactiae npx Is Required for Survival in Human Placental Macrophages and Full Virulence in a Model of Ascending Vaginal Infection during Pregnancy.

Streptococcus agalactiae, also known as group B Streptococcus (GBS), is a Gram-positive encapsulated bacterium that colonizes the gastrointestinal tract of 30 to 50% of humans. GBS causes invasive infection during pregnancy that can lead to chorioamnionitis, funisitis, preterm prelabor rupture of membranes (PPROM), preterm birth, neonatal sepsis, and maternal and fetal demise. Upon infecting the host, GBS encounters sentinel innate immune cells, such as macrophages, within reproductive tissues. Once phagocytosed by macrophages, GBS upregulates the expression of the gene npx, which encodes an NADH peroxidase. GBS mutants with an npx deletion (Δnpx) are exquisitely sensitive to reactive oxygen stress. Furthermore, we have shown that npx is required for GBS survival in both THP-1 and placental macrophages. In an in vivo murine model of ascending GBS vaginal infection during pregnancy, npx is required for invading reproductive tissues and is critical for inducing disease progression, including PPROM and preterm birth. Reproductive tissue cytokine production was also significantly diminished in Δnpx mutant-infected animals compared to that in animals infected with wild-type (WT) GBS. Complementation in trans reversed this phenotype, indicating that npx is critical for GBS survival and the initiation of proinflammatory signaling in the gravid host. IMPORTANCE This study sheds new light on the way that group B Streptococcus (GBS) defends itself against oxidative stress in the infected host. The enzyme encoded by the GBS gene npx is an NADH peroxidase that, our study reveals, provides defense against macrophage-derived reactive oxygen stress and facilitates infections of the uterus during pregnancy. This enzyme could represent a tractable target for future treatment strategies against invasive GBS infections.

Streptococcus agalactiae cadD alleviates metal stress and promotes intracellular survival in macrophages and ascending infection during pregnancy.

Perinatal infection with Streptococcus agalactiae, or Group B Streptococcus (GBS), is associated with preterm birth, neonatal sepsis, and stillbirth. Here, we study the interactions of GBS with macrophages, essential sentinel immune cells that defend the gravid reproductive tract. Transcriptional analyses of GBS-macrophage co-cultures reveal enhanced expression of a gene encoding a putative metal resistance determinant, cadD. Deletion of cadD reduces GBS survival in macrophages, metal efflux, and resistance to metal toxicity. In a mouse model of ascending infection during pregnancy, the ΔcadD strain displays attenuated bacterial burden, inflammation, and cytokine production in gestational tissues. Furthermore, depletion of host macrophages alters cytokine expression and decreases GBS invasion in a cadD-dependent fashion. Our results indicate that GBS cadD plays an important role in metal detoxification, which promotes immune evasion and bacterial proliferation in the pregnant host.

Leveraging bioengineering to assess cellular functions and communication within human fetal membranes.

The fetal membranes enclose the growing fetus and amniotic fluid. Preterm prelabor rupture of fetal membranes is a leading cause of preterm birth. Fetal membranes are composed of many different cell types, both structural and immune. These cells must coordinate functions for tensile strength and membrane integrity to contain the growing fetus and amniotic fluid. They must also balance immune responses to pathogens with maintaining maternal-fetal tolerance. Perturbation of this equilibrium can lead to preterm premature rupture of membranes without labor. In this review, we describe the formation of the fetal membranes to orient the reader, discuss some of the common forms of communication between the cell types of the fetal membranes, and delve into the methods used to tease apart this paracrine signaling within the membranes, including emerging technologies such as organ-on-chip models of membrane immunobiology.

Group B streptococcal infection of the genitourinary tract in pregnant and non-pregnant patients with diabetes mellitus: An immunocompromised host or something more?

Group B Streptococcus (GBS), also known as Streptococcus agalactiae is a Gram-positive bacterium commonly encountered as part of the microbiota within the human gastrointestinal tract. A common cause of infections during pregnancy, GBS is responsible for invasive diseases ranging from urinary tract infections to chorioamnionitis and neonatal sepsis. Diabetes mellitus (DM) is a chronic disease resulting from impaired regulation of blood glucose levels. The incidence of DM has steadily increased worldwide to affecting over 450 million people. Poorly controlled DM is associated with multiple health comorbidities including an increased risk for infection. Epidemiologic studies have clearly demonstrated that DM correlates with an increased risk for invasive GBS infections, including skin and soft tissue infections and sepsis in non-pregnant adults. However, the impact of DM on risk for invasive GBS urogenital infections, particularly during the already vulnerable time of pregnancy, is less clear. We review the evolving epidemiology, immunology, and pathophysiology of GBS urogenital infections including rectovaginal colonization during pregnancy, neonatal infections of infants exposed to DM in utero, and urinary tract infections in pregnant and non-pregnant adults in the context of DM and highlight in vitro studies examining why DM might increase risk for GBS urogenital infection.

Group B Streptococcus cpsE Is Required for Serotype V Capsule Production and Aids in Biofilm Formation and Ascending Infection of the Reproductive Tract during Pregnancy.

Group B Streptococcus (GBS) is an encapsulated Gram-positive pathogen that causes ascending infections of the reproductive tract during pregnancy. The capsule of this organism is a critical virulence factor that has been implicated in a variety of cellular processes to promote pathogenesis. Primarily comprised of carbohydrates, the GBS capsule and its synthesis is driven by the capsule polysaccharide synthesis (cps) operon. The cpsE gene within this operon encodes a putative glycosyltransferase that is responsible for the transfer of a Glc-1-P from UDP-Glc to an undecaprenyl lipid molecule. We hypothesized that the cpsE gene product is important for GBS virulence and ascending infection during pregnancy. Our work demonstrates that a GBS cpsE mutant secretes fewer carbohydrates, has a reduced capsule, and forms less biofilm than the wild-type parental strain. We show that, compared to the parental strain, the ΔcpsE deletion mutant is more readily taken up by human placental macrophages and has a significantly attenuated ability to invade and proliferate in the mouse reproductive tract. Taken together, these results demonstrate that the cpsE gene product is an important virulence factor that aids in GBS colonization and invasion of the gravid reproductive tract.

Antibacterial and Anti-biofilm Activity of the Human Breast Milk Glycoprotein Lactoferrin against Group B Streptococcus.

Group B Streptococcus (GBS) is an encapsulated Gram-positive human pathogen that causes invasive infections in pregnant hosts and neonates, as well as immunocompromised individuals. Colonization of the human host requires the ability to adhere to mucosal surfaces and circumnavigate the nutritional challenges and antimicrobial defenses associated with the innate immune response. Biofilm formation is a critical process to facilitate GBS survival and establishment of a replicative niche in the vertebrate host. Previous work has shown that the host responds to GBS infection by producing the innate antimicrobial glycoprotein lactoferrin, which has been implicated in repressing bacterial growth and biofilm formation. Additionally, lactoferrin is highly abundant in human breast milk and could serve a protective role against invasive microbial pathogens. This study demonstrates that human breast milk lactoferrin has antimicrobial and anti-biofilm activity against GBS and inhibits its adherence to human gestational membranes. Together, these results indicate that human milk lactoferrin could be used as a prebiotic chemotherapeutic strategy to limit the impact of bacterial adherence and biofilm formation on GBS-associated disease outcomes.

Vitamin D and Streptococci: The Interface of Nutrition, Host Immune Response, and Antimicrobial Activity in Response to Infection.

Streptococcus species are common causes of human infection. These Gram-positive, encapsulated bacterial pathogens infect diverse anatomic spaces, leading to infections including skin and soft tissue infection, endocarditis, pneumonia, meningitis, sinusitis, otitis media, chorioamnionitis, sepsis, and even death. Risk for streptococcal infection is highest in low- and middle-income countries where micronutrient deficiency is common. Epidemiological data reveal that vitamin D deficiency is associated with enhanced risk of streptococcal infection and cognate disease outcomes. Additionally, vitamin D improves antibacterial defenses by stimulating innate immune processes such as phagocytosis and enhancing production of reactive oxygen species (oxidative burst) and antimicrobial peptides (including cathelicidin and lactoferrin), which are important for efficient killing of bacteria. This review presents the most recent published work that studies interactions between the micronutrient vitamin D, the host immune system, and pathogenic streptococci as well as comparisons with other relevant infection models.

COVID-19-related disease severity in pregnancy.

The pandemic caused by COVID-19 is affecting populations and healthcare systems worldwide. As we gain experience managing COVID-19, more data become available on disease severity, course, and treatment in patients infected with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). However, data in pregnancy remain limited. This narrative review of COVID-19 during pregnancy underscores key knowledge gaps in our understanding of the impact of this viral infection on reproductive health. Current data suggest that pregnant people have similar disease course and outcomes compared to nonpregnant people, with the majority experiencing mild disease; however, pregnant people may have increased risk of hospitalization and intensive care unit (ICU) admission. Among patients who develop severe and critical disease, major maternal morbidity and mortality have been described including cardiomyopathy, mechanical ventilation, extracorporeal membrane oxygenation, and death. Many questions remain regarding maternal severity of disease in COVID-19. Further research is needed to better understand disease course in pregnancy. Additionally, the inclusion of pregnant patients in therapeutic trials will provide vital data on treatment options for patients. As we continue to treat more patients affected by SARS-CoV-2, multidisciplinary care and continued research are both needed to achieve optimal outcomes for mother and fetus.

Dbp5, Gle1-IP6 and Nup159: a working model for mRNP export.

Gene expression is a stepwise process involving distinct cellular processes including transcription, mRNA (mRNA) processing, mRNA export, and translation. As mRNAs are being synthesized, proteins associate with the RNA to form messenger ribonucleoprotein particles (mRNPs). Previous studies have demonstrated that the RNA-binding protein composition of these mRNPs is dynamic, changing as the mRNP moves through the different steps of gene expression, and playing a critical role in these events. An important step during this maturation process occurs at the cytoplasmic face of the nuclear pore complex (NPC) where the export protein Gle1 bound to inositol hexakisphosphate (IP 6) spatially activates the ATP-hydrolysis and mRNP-remodeling activity of the DEAD-box protein Dbp5. Recent work from our laboratory and others has provided important insights into the function and regulation of Dbp5. These include a more detailed explanation of the mechanism of Dbp5 RNP remodeling, the role of Gle1-IP6 in stimulating Dbp5 ATPase activity, and the identification of a novel paradigm for regulation of Dbp5 by Nup159. Based on in vitro biochemical assays, X-ray crystallography, and corresponding in vivo phenotypes, we propose here an updated model of the Dbp5 cycle during mRNP export through the NPC. This takes into account all available data and provides a platform for future studies.

The Dbp5 cycle at the nuclear pore complex during mRNA export I: dbp5 mutants with defects in RNA binding and ATP hydrolysis define key steps for Nup159 and Gle1.

Nuclear export of messenger RNA (mRNA) occurs by translocation of mRNA/protein complexes (mRNPs) through nuclear pore complexes (NPCs). The DEAD-box protein Dbp5 mediates export by triggering removal of mRNP proteins in a spatially controlled manner. This requires Dbp5 interaction with Nup159 in NPC cytoplasmic filaments and activation of Dbp5's ATPase activity by Gle1 bound to inositol hexakisphosphate (IP(6)). However, the precise sequence of events within this mechanism has not been fully defined. Here we analyze dbp5 mutants that alter ATP binding, ATP hydrolysis, or RNA binding. We found that ATP binding and hydrolysis are required for efficient Dbp5 association with NPCs. Interestingly, mutants defective for RNA binding are dominant-negative (DN) for mRNA export in yeast and human cells. We show that the DN phenotype stems from competition with wild-type Dbp5 for Gle1 at NPCs. The Dbp5-Gle1 interaction is limiting for export and, importantly, can be independent of Nup159. Fluorescence recovery after photobleaching experiments in yeast show a very dynamic association between Dbp5 and NPCs, averaging <1 sec, similar to reported NPC translocation rates for mRNPs. This work reveals critical steps in the Gle1-IP(6)/Dbp5/Nup159 cycle, and suggests that the number of remodeling events mediated by a single Dbp5 is limited.

The Dbp5 cycle at the nuclear pore complex during mRNA export II: nucleotide cycling and mRNP remodeling by Dbp5 are controlled by Nup159 and Gle1.

Essential messenger RNA (mRNA) export factors execute critical steps to mediate directional transport through nuclear pore complexes (NPCs). At cytoplasmic NPC filaments, the ATPase activity of DEAD-box protein Dbp5 is activated by inositol hexakisphosphate (IP(6))-bound Gle1 to mediate remodeling of mRNA-protein (mRNP) complexes. Whether a single Dbp5 executes multiple remodeling events and how Dbp5 is recycled are unknown. Evidence suggests that Dbp5 binding to Nup159 is required for controlling interactions with Gle1 and the mRNP. Using in vitro reconstitution assays, we found here that Nup159 is specifically required for ADP release from Dbp5. Moreover, Gle1-IP(6) stimulates ATP binding, thus priming Dbp5 for RNA loading. In vivo, a dbp5-R256D/R259D mutant with reduced ADP binding bypasses the need for Nup159 interaction. However, NPC spatial control is important, as a dbp5-R256D/R259D nup42Δ double mutant is temperature-sensitive for mRNA export. Further analysis reveals that remodeling requires a conformational shift to the Dbp5-ADP form. ADP release factors for DEAD-box proteins have not been reported previously and reflect a new paradigm for regulation. We propose a model wherein Nup159 and Gle1-IP(6) regulate Dbp5 cycles by controlling its nucleotide-bound state, allowing multiple cycles of mRNP remodeling by a single Dbp5 at the NPC.

Nuclear mRNA on the move.

Innovations in live-cell microscopy and single-molecule analysis have allowed a new direct view of nuclear messenger RNA dynamics. A new study extends previous analyses of mRNA-protein intranuclear transport and links this critical step to the kinetics of moving through nuclear pore complexes. Seeing nuclear mRNA on the move will impact future work on pore translocation and nuclear organization.