Sialic Acid-Siglec-E Interactions Regulate the Response of Neonatal Macrophages to Group B Streptococcus.

The mammalian Siglec receptor sialoadhesin (Siglec1, CD169) confers innate immunity against the encapsulated pathogen group B Streptococcus (GBS). Newborn lung macrophages have lower expression levels of sialoadhesin at birth compared with the postnatal period, increasing their susceptibility to GBS infection. In this study, we investigate the mechanisms regulating sialoadhesin expression in the newborn mouse lung. In both neonatal and adult mice, GBS lung infection reduced Siglec1 expression, potentially delaying acquisition of immunity in neonates. Suppression of Siglec1 expression required interactions between sialic acid on the GBS capsule and the inhibitory host receptor Siglec-E. The Siglec1 gene contains multiple STAT binding motifs, which could regulate expression of sialoadhesin downstream of innate immune signals. Although GBS infection reduced STAT1 expression in the lungs of wild-type newborn mice, we observed increased numbers of STAT1+ cells in Siglece-/- lungs. To test if innate immune activation could increase sialoadhesin at birth, we first demonstrated that treatment of neonatal lung macrophages ex vivo with inflammatory activators increased sialoadhesin expression. However, overcoming the low sialoadhesin expression at birth using in vivo prenatal exposures or treatments with inflammatory stimuli were not successful. The suppression of sialoadhesin expression by GBS-Siglec-E engagement may therefore contribute to disease pathogenesis in newborns and represent a challenging but potentially appealing therapeutic opportunity to augment immunity at birth.

Macrophage Polarizations in the Placenta and Lung are Associated with Bronchopulmonary Dysplasia.

The intricate interplay between macrophage polarization and placenta vascular dysfunction has garnered increasing attention in the context of placental inflammatory diseases. This study delves into the complex relationship between macrophage polarization within the placenta and its potential impact on the development of vascular dysfunction and inflammatory conditions. The placenta, a crucial organ in fetal development, relies on a finely tuned balance of immune responses for proper functioning. Disruptions in this delicate equilibrium can lead to pathological conditions, including inflammatory diseases affecting the fetus and newborn infant. We explored the interconnectedness between placental macrophage polarization and its relevance to lung macrophages, particularly in the context of early life lung development. Bronchopulmonary dysplasia (BPD), the most common chronic lung disease of prematurity, has been associated with abnormal immune responses, and understanding the role of macrophages in this context is pivotal. The investigation aims to shed light on how alterations in placental macrophage polarization may contribute to lung macrophage behavior and, consequently, influence the development of BPD. By unraveling the intricate mechanisms linking macrophage polarization, placental dysfunction and BPD, this research seeks to provide insights that could pave the way for targeted therapeutic interventions. The findings may offer novel perspectives on preventing and managing placental and lung-related pathologies, ultimately contributing to improved maternal and neonatal health outcomes.

Data-driven longitudinal characterization of neonatal health and morbidity.

Although prematurity is the single largest cause of death in children under 5 years of age, the current definition of prematurity, based on gestational age, lacks the precision needed for guiding care decisions. Here, we propose a longitudinal risk assessment for adverse neonatal outcomes in newborns based on a deep learning model that uses electronic health records (EHRs) to predict a wide range of outcomes over a period starting shortly before conception and ending months after birth. By linking the EHRs of the Lucile Packard Children's Hospital and the Stanford Healthcare Adult Hospital, we developed a cohort of 22,104 mother-newborn dyads delivered between 2014 and 2018. Maternal and newborn EHRs were extracted and used to train a multi-input multitask deep learning model, featuring a long short-term memory neural network, to predict 24 different neonatal outcomes. An additional cohort of 10,250 mother-newborn dyads delivered at the same Stanford Hospitals from 2019 to September 2020 was used to validate the model. Areas under the receiver operating characteristic curve at delivery exceeded 0.9 for 10 of the 24 neonatal outcomes considered and were between 0.8 and 0.9 for 7 additional outcomes. Moreover, comprehensive association analysis identified multiple known associations between various maternal and neonatal features and specific neonatal outcomes. This study used linked EHRs from more than 30,000 mother-newborn dyads and would serve as a resource for the investigation and prediction of neonatal outcomes. An interactive website is available for independent investigators to leverage this unique dataset: https://maternal-child-health-associations.shinyapps.io/shiny_app/.

Sp3 is essential for normal lung morphogenesis and cell cycle progression during mouse embryonic development.

Members of the Sp family of transcription factors regulate gene expression via binding GC boxes within promoter regions. Unlike Sp1, which stimulates transcription, the closely related Sp3 can either repress or activate gene expression and is required for perinatal survival in mice. Here, we use RNA-seq and cellular phenotyping to show how Sp3 regulates murine fetal cell differentiation and proliferation. Homozygous Sp3-/- mice were smaller than wild-type and Sp+/- littermates, died soon after birth and had abnormal lung morphogenesis. RNA-seq of Sp3-/- fetal lung mesenchymal cells identified alterations in extracellular matrix production, developmental signaling pathways and myofibroblast/lipofibroblast differentiation. The lungs of Sp3-/- mice contained multiple structural defects, with abnormal endothelial cell morphology, lack of elastic fiber formation, and accumulation of lipid droplets within mesenchymal lipofibroblasts. Sp3-/- cells and mice also displayed cell cycle arrest, with accumulation in G0/G1 and reduced expression of numerous cell cycle regulators including Ccne1. These data detail the global impact of Sp3 on in vivo mouse gene expression and development.

Progressive Metabolic Abnormalities Associated with the Development of Neonatal Bronchopulmonary Dysplasia.

Objective: To assess the longitudinal metabolic patterns during the evolution of bronchopulmonary dysplasia (BPD) development. Methods: A case-control dataset of preterm infants (<32-week gestation) was obtained from a multicenter database, including 355 BPD cases and 395 controls. A total of 72 amino acid (AA) and acylcarnitine (AC) variables, along with infants' calorie intake and growth outcomes, were measured on day of life 1, 7, 28, and 42. Logistic regression, clustering methods, and random forest statistical modeling were utilized to identify metabolic variables significantly associated with BPD development and to investigate their longitudinal patterns that are associated with BPD development. Results: A panel of 27 metabolic variables were observed to be longitudinally associated with BPD development. The involved metabolites increased from 1 predominant different AC by day 7 to 19 associated AA and AC compounds by day 28 and 16 metabolic features by day 42. Citrulline, alanine, glutamate, tyrosine, propionylcarnitine, free carnitine, acetylcarnitine, hydroxybutyrylcarnitine, and most median-chain ACs (C5:C10) were the most associated metabolites down-regulated in BPD babies over the early days of life, whereas phenylalanine, methionine, and hydroxypalmitoylcarnitine were observed to be up-regulated in BPD babies. Most calorie intake and growth outcomes revealed similar longitudinal patterns between BPD cases and controls over the first 6 weeks of life, after gestational adjustment. When combining with birth weight, the derived metabolic-based discriminative model observed some differences between those with and without BPD development, with c-statistics of 0.869 and 0.841 at day 7 and 28 of life on the test data. Conclusions: The metabolic panel we describe identified some metabolic differences in the blood associated with BPD pathogenesis. Further work is needed to determine whether these compounds could facilitate the monitoring and/or investigation of early-life metabolic status in the lung and other tissues for the prevention and management of BPD.

The Lung Microenvironment Instructs Gene Transcription in Neonatal and Adult Alveolar Macrophages.

Immaturity of alveolar macrophages (AMs) around birth contributes to the susceptibility of newborns to lung disease. However, the molecular features differentiating neonatal and mature, adult AMs are poorly understood. In this study, we identify the unique transcriptomes and enhancer landscapes of neonatal and adult AMs in mice. Although the core AM signature was similar, murine adult AMs expressed higher levels of genes involved in lipid metabolism, whereas neonatal AMs expressed a largely proinflammatory gene profile. Open enhancer regions identified by an assay for transposase-accessible chromatin followed by high-throughput sequencing (ATAC-seq) contained motifs for nuclear receptors, MITF, and STAT in adult AMs and AP-1 and NF-κB in neonatal AMs. Intranasal LPS activated a similar innate immune response in both neonatal and adult mice, with higher basal expression of inflammatory genes in neonates. The lung microenvironment drove many of the distinguishing gene expression and open chromatin characteristics of neonatal and adult AMs. Neonatal mouse AMs retained high expression of some proinflammatory genes, suggesting that the differences in neonatal AMs result from both inherent cell properties and environmental influences.

Antenatal Mesenchymal Stromal Cell Extracellular Vesicle Therapy Prevents Preeclamptic Lung Injury in Mice.

In preeclamptic pregnancies, a variety of intrauterine alterations lead to abnormal placentation, release of inflammatory and/or antiangiogenic factors, and subsequent fetal growth restriction with significant potential to cause a primary insult to the developing fetal lung. Thus, modulation of the maternal intrauterine environment may be a key therapeutic avenue to prevent preeclampsia-associated developmental lung injury. A biologic therapy of interest is mesenchymal stromal cell-derived extracellular vesicles (MEx), which we have previously shown to ameliorate preeclamptic physiology through intrauterine immunomodulation. To evaluate the therapeutic potential of MEx to improve developmental lung injury in experimental preeclampsia, using the heme oxygenase-1-null mouse (Hmox1-/-) model, preeclamptic pregnant dams were administered intravenous antenatal MEx treatment during each week of pregnancy followed by analysis of fetal and postnatal lung tissues, amniotic fluid protein profiles, and lung explant and amniotic fluid cocultures in comparison with control and untreated preeclamptic pregnancies. We first identified that a preeclamptic intrauterine environment had a significant adverse impact on fetal lung development, including alterations in fetal lung developmental gene profiles in addition to postnatal alveolar and bronchial changes. Amniotic fluid proteomic analysis and fetal lung explant and amniotic fluid cocultures further demonstrated that maternally administered MEx altered the expression of multiple inflammatory mediators in the preeclamptic intrauterine compartment, resulting in the normalization of fetal lung branching morphogenesis and developmental gene expression. Our evaluation of fetal and postnatal parameters overall suggests that antenatal MEx treatment may provide a highly valuable preventative therapeutic modality for amelioration of lung development in preeclamptic disease.

Developmental Immaturity of Siglec Receptor Expression on Neonatal Alveolar Macrophages Predisposes to Severe Group B Streptococcal Infection.

Streptococcus agalactiae (Group B Streptococcus, GBS) is the most common neonatal pathogen. However, the cellular and molecular mechanisms for neonatal susceptibility to GBS pneumonia and sepsis are incompletely understood. Here we optimized a mouse model of GBS pneumonia to test the role of alveolar macrophage (ΑΜΦ) maturation in host vulnerability to disease. Compared with juvenile and adult mice, neonatal mice infected with GBS had increased mortality and persistence of lung injury. In addition, neonatal mice were defective in GBS phagocytosis and killing. ΑΜΦ depletion and disruption of ΑΜΦ differentiation in Csf2-/- mice both impaired GBS clearance. AMΦ engage the heavily sialylated GBS capsule via the cell surface Siglec receptors Sn and Siglec-E. Although both newborn and adult ΑΜΦ expressed Siglec-E, newborn ΑΜΦ expressed significantly lower levels of Sn. We propose that a developmental delay in Sn expression on ΑΜΦ may prevent effective killing and clearing of GBS from the newborn lung.

Transcriptional profiling of lung macrophages identifies a predictive signature for inflammatory lung disease in preterm infants.

Lung macrophages mature after birth, placing newborn infants, particularly those born preterm, within a unique window of susceptibility to disease. We hypothesized that in preterm infants, lung macrophage immaturity contributes to the development of bronchopulmonary dysplasia (BPD), the most common serious complication of prematurity. By measuring changes in lung macrophage gene expression in preterm patients at risk of BPD, we show here that patients eventually developing BPD had higher inflammatory mediator expression even on the first day of life. Surprisingly, the ex vivo response to LPS was similar across all samples. Our analysis did however uncover macrophage signature genes whose expression increased in the first week of life specifically in patients resilient to disease. We propose that these changes describe the dynamics of human lung macrophage differentiation. Our study therefore provides new mechanistic insight into both neonatal lung disease and human developmental immunology.

Computational Approach to Identifying Universal Macrophage Biomarkers.

Macrophages engulf and digest microbes, cellular debris, and various disease-associated cells throughout the body. Understanding the dynamics of macrophage gene expression is crucial for studying human diseases. As both bulk RNAseq and single cell RNAseq datasets become more numerous and complex, identifying a universal and reliable marker of macrophage cell becomes paramount. Traditional approaches have relied upon tissue specific expression patterns. To identify universal biomarkers of macrophage, we used a previously published computational approach called BECC (Boolean Equivalent Correlated Clusters) that was originally used to identify conserved cell cycle genes. We performed BECC analysis using the known macrophage marker CD14 as a seed gene. The main idea behind BECC is that it uses massive database of public gene expression dataset to establish robust co-expression patterns identified using a combination of correlation, linear regression and Boolean equivalences. Our analysis identified and validated FCER1G and TYROBP as novel universal biomarkers for macrophages in human and mouse tissues.

Transcriptomic and epigenetic mechanisms underlying myeloid diversity in the lung.

The lung is inhabited by resident alveolar and interstitial macrophages as well as monocytic cells that survey lung tissues. Each cell type plays distinct functional roles under homeostatic and inflammatory conditions, but mechanisms establishing their molecular identities and functional potential remain poorly understood. In the present study, systematic evaluation of transcriptomes and open chromatin of alveolar macrophages (AMs), interstitial macrophages (IMs) and lung monocytes from two mouse strains enabled inference of common and cell-specific transcriptional regulators. We provide evidence that these factors drive selection of regulatory landscapes that specify distinct phenotypes of AMs and IMs and entrain qualitatively different responses to toll-like receptor 4 signaling in vivo. These studies reveal a striking divergence in a fundamental innate immune response pathway in AMs and establish a framework for further understanding macrophage diversity in the lung.

Differential Immune Activation in Fetal Macrophage Populations.

Distinct macrophage subsets populate the developing embryo and fetus in distinct waves. However little is known about the functional differences between in utero macrophage populations or how they might contribute to fetal and neonatal immunity. Here we tested the innate immune response of mouse macrophages derived from the embryonic yolk sac and from fetal liver. When isolated from liver or lung, CD11bHI fetal liver derived macrophages responded to the TLR4 agonist LPS by expressing and releasing inflammatory cytokines. However F4/80HI macrophages from the yolk sac did not respond to LPS treatment. While differences in TLR4 expression did not appear to explain these data, F4/80HI macrophages had much lower NLRP3 inflammasome expression compared to CD11bHI macrophages. Gene expression profiling also demonstrated LPS-induced expression of inflammatory genes in CD11bHI macrophages, but not in F4/80HI cells. Genes expressed in LPS-treated CD11bHI macrophages were more likely to contain predicted NF-κB binding sites in their promoter regions. Our data show that CD11bHI macrophages derived from fetal liver are the major pro-inflammatory cells in the developing fetus. These findings could have important implications in better understanding the fetal inflammatory response and the unique features of neonatal immunity.

TLR Activation Alters Bone Marrow-Derived Macrophage Differentiation.

Early exposure to inflammatory signals may have a lasting impact on immune function. Present throughout embryogenesis, macrophages are key cells providing innate immune protection to the developing fetus and newborn. Here, we have used an established model of macrophage development to test how early inflammatory signals can impact cellular differentiation and function. Bone marrow-derived macrophages were treated with Escherichia coli lipopolysaccharide (LPS) 2 days after initial isolation and culture. LPS treatment during this early stage of differentiation decreased the expression of CSF1R and increased that of the mature macrophage marker F4/80. These early changes in macrophage differentiation were also measured in cells from mice lacking IKKß, but the change in CSF1R expression after LPS treatment was blocked with MAPK inhibition. LPS-induced changes in macrophage marker expression persisted following LPS removal, suggesting that early inflammatory activation could induce a lasting developmental impact. Early LPS exposure inhibited macrophage phagocytosis of labeled E. coli while LPS had no effect on fully differentiated macrophages. Our data demonstrate that early inflammatory exposure to a microbial stimulus induce lasting phenotypic changes in macrophages.

FGF10 and Human Lung Disease Across the Life Spectrum.

Lung diseases impact patients across the lifespan, from infants in the first minutes of life through the aged population. Congenital abnormalities of lung structure can cause lung disease at birth or make adults more susceptible to chronic disease. Continuous inhalation of atmospheric components also requires the lung to be resilient to cellular injury. Fibroblast growth factor 10 (FGF10) regulates multiple stages of structural lung morphogenesis, cellular differentiation, and the response to injury. As a driver of lung airway branching morphogenesis, FGF10 signaling defects during development lead to neonatal lung disease. Alternatively, congenital airway abnormalities attributed to FGF10 mutations increase the risk of chronic airway disease in adulthood. FGF10 also maintains progenitor cell populations in the airway and promotes alveolar type 2 cell expansion and differentiation following injury. Here we review the cellular and molecular mechanisms linking FGF10 to multiple lung diseases, from bronchopulmonary dysplasia in extremely preterm neonates, cystic fibrosis in children, and chronic adult lung disorders. Understanding the connections between FGF10 and lung diseases may lead to exciting new therapeutic strategies.

IKKß Activation in the Fetal Lung Mesenchyme Alters Lung Vascular Development but Not Airway Morphogenesis.

In the immature lung, inflammation and injury disrupt the epithelial-mesenchymal interactions required for normal development. Innate immune signaling and NF-κB activation disrupt the normal expression of multiple mesenchymal genes that play a key role in airway branching and alveolar formation. To test the role of the NF-κB pathway specifically in lung mesenchyme, we utilized the mesenchymal Twist2-Cre to drive expression of a constitutively active inhibitor of NF-κB kinase subunit ß (IKKßca) mutant in developing mice. Embryonic Twist2-IKKßca mice were generated in expected numbers and appeared grossly normal. Airway branching also appeared normal in Twist2-IKKßca embryos, with airway morphometry, elastin staining, and saccular branching similar to those in control littermates. While Twist2-IKKßca lungs did not contain increased levels of Il1b, we did measure an increased expression of the chemokine-encoding gene Ccl2. Twist2-IKKßca lungs had increased staining for the vascular marker platelet endothelial cell adhesion molecule 1. In addition, type I alveolar epithelial differentiation appeared to be diminished in Twist2-IKKßca lungs. The normal airway branching and lack of Il1b expression may have been due to the inability of the Twist2-IKKßca transgene to induce inflammasome activity. While Twist2-IKKßca lungs had an increased number of macrophages, inflammasome expression remained restricted to macrophages without evidence of spontaneous inflammasome activity. These results emphasize the importance of cellular niche in considering how inflammatory signaling influences fetal lung development.

The innate immune response in fetal lung mesenchymal cells targets VEGFR2 expression and activity.

In preterm infants, soluble inflammatory mediators target lung mesenchymal cells, disrupting airway and alveolar morphogenesis. However, how mesenchymal cells respond directly to microbial stimuli remains poorly characterized. Our objective was to measure the genome-wide innate immune response in fetal lung mesenchymal cells exposed to the bacterial endotoxin lipopolysaccharide (LPS). With the use of Affymetrix MoGene 1.0st arrays, we showed that LPS induced expression of unique innate immune transcripts heavily weighted toward CC and CXC family chemokines. The transcriptional response was different between cells from E11, E15, and E18 mouse lungs. In all cells tested, LPS inhibited expression of a small core group of genes including the VEGF receptor Vegfr2 Although best characterized in vascular endothelial populations, we demonstrated here that fetal mouse lung mesenchymal cells express Vegfr2 and respond to VEGF-A stimulation. In mesenchymal cells, VEGF-A increased cell migration, activated the ERK/AKT pathway, and promoted FOXO3A nuclear exclusion. With the use of an experimental coculture model of epithelial-mesenchymal interactions, we also showed that VEGFR2 inhibition prevented formation of three-dimensional structures. Both LPS and tyrosine kinase inhibition reduced three-dimensional structure formation. Our data suggest a novel mechanism for inflammation-mediated defects in lung development involving reduced VEGF signaling in lung mesenchyme.

TLR3 signaling is downregulated by a MAVS isoform in epithelial cells.

Innate immune responses to dsRNA result in signaling through the TLR3 pathway and/or the RIG-I/MDA-5/MAVS pathway which can activate type I IFN, proinflammatory cytokines and apoptosis. It is not clear whether MAVS could play a role in TLR3-dependent responses to extracellular dsRNA. Using a model of epithelial cells that express a functional TLR3 signaling pathway, we found that TLR3-dependent responses to extracellular dsRNA are negatively regulated by MAVS, precisely "miniMAVS", a recently described 50kDa isoform of MAVS. This regulation of TLR3 by a MAVS isoform constitutes an endogenous regulatory mechanism in epithelial cells that could help prevent a potentially damaging excessive inflammatory response.

Epithelial-Derived Inflammation Disrupts Elastin Assembly and Alters Saccular Stage Lung Development.

The highly orchestrated interactions between the epithelium and mesenchyme required for normal lung development can be disrupted by perinatal inflammation in preterm infants, although the mechanisms are incompletely understood. We used transgenic (inhibitory κB kinase ß transactivated) mice that conditionally express an activator of the NF-κB pathway in airway epithelium to investigate the impact of epithelial-derived inflammation during lung development. Epithelial NF-κB activation selectively impaired saccular stage lung development, with a phenotype comprising rapidly progressive distal airspace dilation, impaired gas exchange, and perinatal lethality. Epithelial-derived inflammation resulted in disrupted elastic fiber organization and down-regulation of elastin assembly components, including fibulins 4 and 5, lysyl oxidase like-1, and fibrillin-1. Fibulin-5 expression by saccular stage lung fibroblasts was consistently inhibited by treatment with bronchoalveolar lavage fluid from inhibitory κB kinase ß transactivated mice, Escherichia coli lipopolysaccharide, or tracheal aspirates from preterm infants exposed to chorioamnionitis. Expression of a dominant NF-κB inhibitor in fibroblasts restored fibulin-5 expression after lipopolysaccharide treatment, whereas reconstitution of fibulin-5 rescued extracellular elastin assembly by saccular stage lung fibroblasts. Elastin organization was disrupted in saccular stage lungs of preterm infants exposed to systemic inflammation. Our study reveals a critical window for elastin assembly during the saccular stage that is disrupted by inflammatory signaling and could be amenable to interventions that restore elastic fiber assembly in the developing lung.