Are fetal microchimerism and circulating fetal extracellular vesicles important links between spontaneous preterm delivery and maternal cardiovascular disease risk?

Trafficking and persistence of fetal microchimeric cells (fMCs) and circulating extracellular vesicles (EVs) have been observed in animals and humans, but their consequences in the maternal body and their mechanistic contributions to maternal physiology and pathophysiology are not yet fully defined. Fetal cells and EVs may help remodel maternal organs after pregnancy-associated changes, but the cell types and EV cargos reaching the mother in preterm pregnancies after exposure to various risk factors can be distinct from term pregnancies. As preterm delivery-associated maternal complications are rising, revisiting this topic and formulating scientific questions for future research to reduce the risk of maternal morbidities are timely. Epidemiological studies report maternal cardiovascular risk as one of the major complications after preterm delivery. This paper suggests a potential link between fMCs and circulating EVs and adverse maternal cardiovascular outcomes post-pregnancies, the underlying mechanisms, consequences, and methods for and how this link might be assessed.

On antigen-specific signals, immune class regulation and energetics: Report III from the workshops on foundational concepts of immune regulation.

This is a report from a one-week workshop held in Athens, Greece in July of 2022. The workshop aimed to identify emerging concepts relevant to the fundamentals of immune regulation and areas for future research. Theories of immune regulation emphasize the role of T cell help or co-stimulation (signal 2). The workshop participants considered how new data on the characteristics of agonist antigens, the role of the antigen receptor signals (signal 1) in driving fate decisions, the effect of energetics on immunity and a better understanding of class-control in the immune response, may impact theories of immune regulation. These ideas were discussed in the context of tumour immunology, autoimmunity, pregnancy and transplantation. Here we present the discussions as a narrative of different viewpoints to allow the reader to join the conversation. These discussions highlight the evolving understanding of the nature of specific antigen recognition and how both antigen-specific and non-specific mechanisms impact immune responses.

The Effects of Pregnancy on the Pulmonary Immune Response in a Mouse Model of LPS-Induced Acute Lung Injury.

OBJECTIVE: This study evaluated the effect of pregnancy on the pulmonary innate immune response in a mouse model of acute lung injury (ALI) using nebulized lipopolysaccharide (LPS). STUDY DESIGN: Pregnant (day 14) C57BL/6NCRL mice and nonpregnant controls received nebulized LPS for 15 minutes. Twenty-four hours later, mice were euthanized for tissue harvest. Analysis included blood and bronchoalveolar lavage fluid (BALF) differential cell counts, whole-lung inflammatory cytokine transcription levels by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), and whole-lung vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and BALF albumin by western blot. Mature bone marrow neutrophils from uninjured pregnant and nonpregnant mice were examined for chemotactic response using a Boyden chamber and for cytokine response to LPS by RT-qPCR. RESULTS: In LPS-induced ALI, pregnant mice had higher BALF total cell (p < 0.001) and neutrophil counts (p < 0.001) as well as higher peripheral blood neutrophils (p < 0.01) than nonpregnant mice, but a similar increase (as compared with unexposed mice) in airspace albumin levels. Whole-lung expression of interleukin 6, tumor necrosis factor-α (TNF-α), and keratinocyte chemoattractant (CXCL1) was also similar. In vitro, marrow-derived neutrophils from pregnant and nonpregnant mice had similar chemotaxis to CXCL1 and N-formylmethionine-leucyl-phenylalanine, but neutrophils from pregnant mice expressed lower levels of TNF (p < 0.001) and CXCL1 (p < 0.01) after LPS stimulation. In uninjured mice, VCAM-1 was higher in lungs from pregnant versus nonpregnant mice (p < 0.05). CONCLUSION: In this model, pregnancy is associated with an augmented lung neutrophil response to ALI without increased capillary leak or whole-lung cytokine levels relative to the nonpregnant state. This may stem from increased peripheral blood neutrophil response and intrinsically increased expression of pulmonary vascular endothelial adhesion molecules. Differences in lung innate cell homeostasis may affect the response to inflammatory stimuli and explain severe lung disease in respiratory infection during pregnancy. KEY POINTS: · Inhalation of LPS in midgestation versus virgin mice is associated with increased neutrophilia.. · This occurs without a comparative increase in cytokine expression.. · This may be explained by pregnancy-enhanced pre-exposure expression of VCAM-1 and ICAM-1..

Stress signaler p38 mitogen-activated kinase activation: a cause for concern?

Oxidative stress (OS) induced activation of p38 mitogen-activated kinase (MAPK) and cell fate from p38 signaling was tested using the human fetal membrane's amnion epithelial cells (AEC). We created p38 KO AEC using the CRISPR/Cas9 approach and tested cell fate in response to OS on an AEC-free fetal membrane extracellular matrix (ECM). Screening using image CyTOF indicated OS causing epithelial-mesenchymal transition (EMT). Further testing revealed p38 deficiency prevented AEC senescence, EMT, cell migration, and inflammation. To functionally validate in vitro findings, fetal membrane-specific conditional KO (cKO) mice were developed by injecting Cre-recombinase encoded exosomes intra-amniotically into p38αloxP/loxP mice. Amnion membranes from p38 cKO mice had reduced senescence, EMT, and increased anti-inflammatory IL-10 compared with WT animals. Our study suggested that overwhelming activation of p38 in response to OS inducing risk exposures can have an adverse impact on cells, cause cell invasion, inflammation, and ECM degradation detrimental to tissue homeostasis.

Society for Maternal-Fetal Medicine Special Statement: Race in maternal-fetal medicine research- Dispelling myths and taking an accurate, antiracist approach.

Profound inequities in maternal and infant outcomes based on race exist, and the maternal-fetal medicine community has an important role in eliminating these disparities. Accurately employing race and ethnicity as social constructs within research that guides clinical practice is essential to achieving health equity. We must abandon commonly propagated myths that race is a surrogate for genetics or economic status and that data are exempt from potential bias. These myths can lead to harmful misconceptions that exacerbate racial disparities in maternal and infant health outcomes. Furthermore, these myths obscure racism as the true underlying etiology of racial disparities. Understanding that race is a social construct and using an antiracist approach to research are essential in combating racism and eliminating unacceptable disparities in maternal and infant health. This document provides specific suggestions to approach the research process with an antiracist framework.

The Immunology of Syncytialized Trophoblast.

Multinucleate syncytialized trophoblast is found in three forms in the human placenta. In the earliest stages of pregnancy, it is seen at the invasive leading edge of the implanting embryo and has been called primitive trophoblast. In later pregnancy, it is represented by the immense, multinucleated layer covering the surface of placental villi and by the trophoblast giant cells found deep within the uterine decidua and myometrium. These syncytia interact with local and/or systemic maternal immune effector cells in a fine balance that allows for invasion and persistence of allogeneic cells in a mother who must retain immunocompetence for 40 weeks of pregnancy. Maternal immune interactions with syncytialized trophoblast require tightly regulated mechanisms that may differ depending on the location of fetal cells and their invasiveness, the nature of the surrounding immune effector cells and the gestational age of the pregnancy. Some specifically reflect the unique mechanisms involved in trophoblast cell-cell fusion (aka syncytialization). Here we will review and summarize several of the mechanisms that support healthy maternal-fetal immune interactions specifically at syncytiotrophoblast interfaces.

To drive or be driven: the path of a mouse model of recurrent pregnancy loss.

This review is an example of the use of an animal model to try to understand the immune biology of pregnancy. A well-known model of recurrent spontaneous pregnancy loss is put in clinical, historical, and theoretical context, with emphasis on T cell biology.

Sex-specific metabolic adaptations from in utero exposure to particulate matter derived from combustion of petrodiesel and biodiesel fuels.

Maternal exposure to particulate matter derived from diesel exhaust has been shown to cause metabolic dysregulation, neurological problems, and increased susceptibility to diabetes in the offspring. Diesel exhaust is a major source of air pollution and the use of biodiesel (BD) and its blends have been progressively increasing throughout the world; however, studies on the health impact of BD vs. petrodiesel combustion-generated exhaust have been controversial in part, due to differences in the chemical and physical nature of the associated particulate matter (PM). To explore the long-term impact of prenatal exposure, pregnant mice were exposed to PM generated by combustion of petrodiesel (B0) and a 20% soy BD blend (B20) by intratracheal instillation during embryonic days 9-17 and allowed to deliver. Offspring were then followed for 52 weeks. We found that mother's exposure to B0 and B20 PM manifested in striking sex-specific phenotypes with respect to metabolic adaptation, maintenance of glucose homeostasis, and medial hypothalamic glial cell makeup in the offspring. The data suggest PM exposure limited to a narrower critical developmental window may be compensated for by the mother and/or the fetus by altered metabolic programming in a marked sex-specific and fuel-derived PM-specific manner, leading to sex-specific risk for diseases related to environmental exposure later in life.

Inflammation promotes aging-associated oncogenesis in the lung.

Background: Lung cancer is the leading cause of cancer death in the world. While cigarette smoking is the major preventable factor for cancers in general and lung cancer in particular, old age is also a major risk factor. Aging-related chronic, low-level inflammation, termed inflammaging, has been widely documented; however, it remains unclear how inflammaging contributes to increased lung cancer incidence. Aim: To establish connections between aging-associated changes in the lungs and cancer risk. Methods: We analyzed public databases of gene expression for normal and cancerous human lungs and used mouse models to understand which changes were dependent on inflammation, as well as to assess the impact on oncogenesis. Results: Analyses of GTEx and TCGA databases comparing gene expression profiles from normal lungs, lung adenocarcinoma, lung squamous cell carcinoma of subjects across age groups revealed upregulated pathways such as inflammatory response, TNFA signaling via NFκB, and interferon-gamma response. Similar pathways were identified comparing the gene expression profiles of young and old mouse lungs. Transgenic expression of alpha 1 antitrypsin (AAT) partially reverses increases in markers of aging-associated inflammation and immune deregulation. Using an orthotopic model of lung cancer using cells derived from EML4-ALK fusion-induced adenomas, we demonstrated an increased tumor outgrowth in lungs of old mice while NLRP3 knockout in old mice decreased tumor volumes, suggesting that inflammation contributes to increased lung cancer development in aging organisms. Conclusions: These studies reveal how expression of an anti-inflammatory mediator (AAT) can reduce some but not all aging-associated changes in mRNA and protein expression in the lungs. We further show that aging is associated with increased tumor outgrowth in the lungs, which may relate to an increased inflammatory microenvironment.

Generalized disturbance of DNA methylation in the uterine decidua in the CBA/J x DBA/2 mouse model of pregnancy failure.

Nonchromosomal pregnancy failure is a common but poorly understood phenomenon. Because recent data have suggested that epigenetic abnormalities such as abnormal placental DNA methylation may play a role in human pregnancy failure, we undertook experiments to test whether decidual and/or placental DNA methylation abnormalities are present in a mouse model of pregnancy failure. A large number of studies have shown that crosses between CBA/J female mice and DBA/2 males result in pregnancies with a high rate of failure/resorption, whereas other crosses with CBA/J females produce normal pregnancies. Although the CBA/J × DBA/2 mouse has frequently been used as a model for miscarriage, a detailed explanation for the pregnancy failure phenotype is lacking. We performed timed matings between CBA/J female and DBA/2 male mice as well as between DBA/2 female and CBA/J male mice. Decidual caps were isolated at Embryonic Day (E) 9.5 from both crosses, and a microarray-based method was used to comparatively assess genomic methylation at approximately 16,000 loci on mouse chromosome 7. In comparison with decidual caps from DBA/2 × CBA/J pregnancies, CBA/J × DBA/2 decidual caps were characterized by widely and apparently randomly disturbed methylation. In another set of analogous experiments, genomic methylation of placental DNA from E8.5 pregnancies was assessed using the same microarray-based method. This analysis revealed that in contrast to the decidua, placental DNA methylation from CBA/J × DBA/2 pregnancies was indistinguishable from that of normal controls. We conclude that abnormal DNA methylation in the uterine decidua likely plays a role in the CBA/J × DBA/2 model of pregnancy failure. To our knowledge, these experiments are the first to demonstrate that epigenetic abnormalities of the decidua are associated with pregnancy failure, and they set the stage for future efforts to understand the role of DNA methylation at the maternal-fetal interface.

PD-1 regulates T cell proliferation in a tissue and subset-specific manner during normal mouse pregnancy.

The regulation of T cell homeostasis during pregnancy has important implications for maternal tolerance and immunity. Evidence suggests that Programmed Death-1 (PD-1) participates in regulation of T cell homeostasis and peripheral tolerance. To examine the contribution of PD-1 signaling on T cell homeostasis during normal mouse pregnancy, we examined T cell number or proportion, PD-1 expression, proliferation, and apoptosis by flow cytometry, BrdU incorporation, and TUNEL assay in pregnant mice given anti-PD-1 blocking antibody or control on days 10, 12, and 14 of gestation. We observed tissue, treatment, and T cell-specific differences in PD-1 expression. Both pregnancy and PD-1 blockade increased T cell proliferation in the spleen, yet this effect was limited to CD4 T cells in the uterine- draining nodes. In the uterus, PD-1 blockade markedly altered the composition of the T cell pool. These studies support the idea that pregnancy is a state of dynamic T cell homeostasis and suggest that this state is partially supported by PD-1 signaling.

A Framework for Understanding Maternal Immunity.

This is an alternative and controversial framing of the data relevant to maternal immunity. It argues for a departure from classical theory to view, interrogate and interpret existing data.

Characterization of fetal microchimeric immune cells in mouse maternal hearts during physiologic and pathologic pregnancies.

Introduction: During pregnancy, fetal cells can be incorporated into maternal tissues (fetal microchimerism), where they can persist postpartum. Whether these fetal cells are beneficial or detrimental to maternal health is unknown. This study aimed to characterize fetal microchimeric immune cells in the maternal heart during pregnancy and postpartum, and to identify differences in these fetal microchimeric subpopulations between normal and pregnancies complicated by spontaneous preterm induced by ascending infection. Methods: A Cre reporter mouse model, which when mated with wild-type C57BL/6J females resulted in cells and tissues of progeny expressing red fluorescent protein tandem dimer Tomato (mT+), was used to detect fetal microchimeric cells. On embryonic day (E)15, 104 colony-forming units (CFU) E. coli was administered intravaginally to mimic ascending infection, with delivery on or before E18.5 considered as preterm delivery. A subset of pregnant mice was sacrificed at E16 and postpartum day 28 to harvest maternal hearts. Heart tissues were processed for immunofluorescence microscopy and high-dimensional mass cytometry by time-of-flight (CyTOF) using an antibody panel of immune cell markers. Changes in cardiac physiologic parameters were measured up to 60 days postpartum via two-dimensional echocardiography. Results: Intravaginal E. coli administration resulted in preterm delivery of live pups in 70% of the cases. mT + expressing cells were detected in maternal uterus and heart, implying that fetal cells can migrate to different maternal compartments. During ascending infection, more fetal antigen-presenting cells (APCs) and less fetal hematopoietic stem cells (HSCs) and fetal double-positive (DP) thymocytes were observed in maternal hearts at E16 compared to normal pregnancy. These HSCs were cleared while DP thymocytes persisted 28 days postpartum following an ascending infection. No significant changes in cardiac physiologic parameters were observed postpartum except a trend in lowering the ejection fraction rate in preterm delivered mothers. Conclusion: Both normal pregnancy and ascending infection revealed distinct compositions of fetal microchimeric immune cells within the maternal heart, which could potentially influence the maternal cardiac microenvironment via (1) modulation of cardiac reverse modeling processes by fetal stem cells, and (2) differential responses to recognition of fetal APCs by maternal T cells.

Decreased level of TREM like Transcript 1 (TLT-1) is associated with prematurity and promotes the in-utero inflammatory response to maternal lipopolysaccharide (LPS) exposure.

PROBLEM: The occurrence of preterm birth is associated with multiple factors including bleeding, infection and inflammation. Platelets are mediators of hemostasis and can modulate inflammation through interactions with leukocytes. TREM like Transcript 1 (TLT-1) is a type 1 single Ig domain receptor on activated platelets. In adults, it plays a protective role by dampening the inflammatory response and facilitating platelet aggregation at sites of vascular injury. TLT-1 is expressed in human placenta and found in cord blood. We thus hypothesized that TLT-1 deficiency is associated with prematurity and fetal inflammation. METHOD OF STUDY: To test this hypothesis, we examined cord blood levels of soluble TLT-1 (sTLT) in premature and term infants and compared the inflammatory response in C57BL/6 (WT) and TLT-1-/- (treml1-/- , KO) mice given intraperitoneal LPS mid-gestation RESULTS: The preterm infant cord blood level of sTLT was significantly lower than that found at term. On exposure to LPS, histology of KO (as compared to WT) placenta and decidua showed increased hemorrhage, and KO decidual RNA expression of IL-10 was significantly lower. KO fetal interface tissues (placenta, membranes, amniotic fluid) over time showed increased expression of inflammatory cytokines such as IL-6, IFN-γ, and TNF, but not MCP-1. However, fetal organs showed similar levels. CONCLUSION: There is a potential association between insufficient TLT-1 expression and increased fetal inflammatory responses in the setting of prematurity. The data support further study of TLT-1 in the mechanistic link between bleeding, inflammation and preterm birth, and perhaps as a biomarker in human pregnancy.

COVID-19 in pregnancy: possible mechanisms not to be discounted.

SARS-CoV-2 has infected more than 16 million people worldwide. Related complications and death from COVID-19 disease and their underlying pathophysiology are intensely investigated. Pregnant women are among the affected. Although the severity of disease in pregnancy does not appear to be increased, the effects of infection on pregnancy should not escape careful examination. The currently known receptor for the virus, ACE2, regulates the renin-angiotensin system and is increased during pregnancy. Virus-receptor interactions may have significant effects on placental function, fetal development, and maternal immunity. The manifestation of cardiovascular complications of infection produces the hypothesis that a significant effect of the virus may be its influence on the maternal vascular system. Interference with the vascular adaptations to pregnancy and the post-partum may have implications for concurrent and future pregnancies as well as for long-term cardiovascular health. We should not miss the opportunity to learn from this virus about the physiology of pregnancy.

Transient modification within a pool of CD4 T cells in the maternal spleen.

Classic models suggest maternal tolerance is dependent on regulation of fetal antigen-specific T cell responses. We hypothesize that factors unique to a particular fetal antigen-specific T cell, rather than the state of pregnancy per se, are important determinants of T cell fate during pregnancy. To investigate the fate of fetal antigen-specific CD4 T cells in the systemic circulation, we examined spleen cells in a CD4 T cell receptor transgenic mouse specific for the male antigen H-Y. We observed a transient decrease in CD4(+) Vß6(+) cell numbers and, due to transient internalization of CD4, an increase in CD4(-) Vß6(+) T cells. Antigen-specific in vitro responsiveness was not depressed by pregnancy. These data suggest that pregnancy supports fluidity in this particular CD4 T cell pool that may, in turn, help to meet competing requirements of maternal immune responsiveness and fetal tolerance.

Deficiency in CD4 T Cells Leads to Enhanced Postpartum Internal Carotid Artery Vasoconstriction in Mice: The Role of Nitric Oxide.

The risk of postpartum (PP) stroke is increased in complicated pregnancies. Deficiency in CD4 T cell subsets is associated with preeclampsia and may contribute to PP vascular disease, including internal carotid artery (ICA) stenosis and stroke. We hypothesized that CD4 T cell deficiency in pregnancy would result in ICA dysregulation, including enhanced ICA vasoconstriction. We characterized the function, mechanical behavior, and structure of ICAs from C57BL/6 (WT) and CD4 deficient (CD4KO) mice, and assessed the role of NO in the control of ICA function at pre-conception and PP. WT and CD4KO mice were housed under pathogen-free conditions, mated to same-strain males, and allowed to litter or left virgin. At 3 days or 4 weeks PP, mice were euthanized. The responses to phenylephrine (PE), high K+ and acetylcholine (ACh) were assessed in pressurized ICAs before and after NOS inhibition. Passive lumen diameters were measured at 3-140 mmHg. eNOS and iNOS expression as well as the presence of T cells were evaluated by immunohistochemistry. Constriction of WT ICAs to PE was not modified PP. In contrast, responses to PE were significantly increased in ICAs from PP as compared to virgin CD4KO mice. Constriction to high K+ was not enhanced PP. ICAs from WT and CD4KO mice were equally sensitive to ACh with a significant rightward shift of dose-response curves after L-NNA treatment. NOS inhibition enhanced PE constriction of ICAs from WT virgin and PP mice. Although a similar effect was detected in ICAs of virgin CD4KO mice, no such changes were observed in vessels from PP CD4KO mice. Passive arterial distensibility at physiological levels of pressure was not modified at PP. ICA diameters were significantly increased in PP with no change in vascular wall thickness. Comparison of eNOS expression in virgin, 3 days and 4 weeks PP revealed a reduced expression in ICA from CD4 KO vs. WT PP vessels which reached significance at 4 weeks PP. iNos expression was similar and decreased over the PP period in vessels from WT and CD4KO mice. Dysregulation of the CD4 T cell population in pregnancy may make ICA vulnerable to vasospasm due to decreased NO-dependent control of ICA constriction. This may lead to cerebral hypoperfusion and increase the risk of maternal PP stroke.

Kinetics of Postpartum Mesenteric Artery Structure and Function Relative to Pregnancy and Lactation in Mice.

Epidemiological evidence suggests that normal pregnancy in women is associated with decreased cardiovascular risk in later life. Clinical studies have provided evidence that alterations in vascular function and structure are detectable long after delivery. To understand these findings, we examined mesenteric artery reactivity at both early (3 days and 2-4 weeks) and late (12 weeks) postpartum (PP) time points in relation to late pregnancy (LP) and lactation. Vessels from virgin controls, LP, PP, and nursing and non-nursing mothers were tested for responses to phenylephrine (PE), high potassium solutions (high K+), and acetylcholine (ACh). Passive arterial distensibility, vessel dimensions, and collagen and elastin content were evaluated for the studied groups. We observed that (1) there was a significant inhibition of vascular reactivity to PE in LP, 3 days and 2 weeks PP vessels that returned to pre-pregnancy levels at 4 and 12 weeks PP; (2) inhibition of NO production in PP vessels restored PE-induced constriction to pre-pregnancy levels; (3) vasodilator responses to ACh were similar at all PP periods; (4) LP and early PP was associated with a persistent increase in arterial distensibility that correlates with a PP-induced reduction in wall collagen, and regressed to pre-conception levels at 12 weeks PP; (5) vessels from non-nursing PP mice demonstrated an increased PE reactivity, diminished responses to ACh, and reduced distensibility compared to breastfeeding mice. These studies provide a timeframe for mesenteric artery adaptations that occur during pregnancy and extend to the PP period, but which may be modified by PP events.

Evidence that CD8 T-cell homeostasis and function remain intact during murine pregnancy.

Evolving models of immune tolerance have challenged the view that the response of the maternal immune system to environmental or fetal antigens must be suppressed or deviated. CD8 T cells play a central role in the immune response to viruses and intracellular pathogens so the maintenance of both the number and function of these cells is critical to protect both the mother and fetus. We show that the numbers of maternal CD8 T cells in both the spleen and the uterine draining lymph nodes are transiently increased at mid-gestation and this correlates with enhanced CD8 T-cell proliferation and an increased relative expression of both pro-survival and pro-apoptotic molecules. In transgenic mice bearing T-cell antigen receptors specific for the male HY or allo-antigens, the transgenic CD8 T cells retain the ability to proliferate and function during pregnancy. Moreover, anti-HY T-cell receptor transgenic mice have normal numbers of male pups despite the presence of CD8 T cells at the maternal-fetal interface. These data suggest that pregnancy is a dynamic state in which CD8 T-cell turnover is increased while the function and ending size of the CD8 T-cell compartment are maintained.