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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.

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/ .

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.

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.

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.

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 IKKβ, 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.

Summary 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., Graphical Abstract, Highlights • Newborn mice fail to kill GBS, developing persistent lung injury • Mature AMΦ detect the Sialic acid capsule on GBS to mediate bacterial clearance • Immature newborn AMΦ lack mature Siglec expression required for killing GBS • GBS engages the inhibitory Siglec-E on newborn AMΦ to suppress innate immunity, Medical Microbiology; Reproductive Medicine; Microbiome

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., Debashis Sahoo et al. study the gene expression profiles in the lung macrophages of preterm patients. They identify an expression signature that contributes to increased susceptibility or resistance to inflammatory lung disease. Their work provides insights to human developmental immunology.

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 β (IKKβca) mutant in developing mice. Embryonic Twist2-IKKβca mice were generated in expected numbers and appeared grossly normal. Airway branching also appeared normal in Twist2-IKKβca embryos, with airway morphometry, elastin staining, and saccular branching similar to those in control littermates. While Twist2-IKKβca lungs did not contain increased levels of Il1b , we did measure an increased expression of the chemokine-encoding gene Ccl2 . Twist2-IKKβca 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-IKKβca lungs. The normal airway branching and lack of Il1b expression may have been due to the inability of the Twist2-IKKβca transgene to induce inflammasome activity. While Twist2-IKKβca 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.

Macrophages are a type of white blood cell, of the immune system, that engulfs and digests cellular debris, cancer cells, and anything else that does not have the type of proteins specific to healthy body cells on its surface. Understanding gene expression dynamics in macrophages are crucial for studying human diseases. Recent advances in high-throughput technologies have enabled the collection of immense amounts of biological data. A reliable marker of macrophage is essential to study their function. Traditional approaches use a number of markers that may have tissue specific expression patterns. To identify universal biomarker of macrophage, we used a previously published computational approach called BECC (Boolean Equivalent Correlated Clusters) that was originally used to identify universal cell cycle genes. We performed BECC analysis on a seed gene CD14, a known macrophage marker. FCER1G and TYROBP were among the top candidates which were validated as strong candidates for universal biomarkers for macrophages in human and mouse tissues. To our knowledge, such a finding is first of its kind.CONTRIBUTIONS TO THE FIELDWe have developed a computational approach to identify universal biomarkers of different entities in a biological system. We applied this approach to study macrophages and identified universal biomarkers of this particular cell type. FCER1G and TYROBP were among the top candidates which were validated as strong candidates for universal biomarkers for macrophages in human and mouse tissues. The expression patterns of TYROBP and FCER1G are found to be more homogeneous compared to currently used biomarkers such as ITGAM, EMR1 (F4/80), and CD68. Further, we demonstrated that this homogeneity extends to all the tissues currently profiled in the public domain in multiple species including human and mouse. FCER1G and TYROBP expression patterns were also found to be extremely specific to macrophages found in various tissues. They are strongly co-expressed together. We believe that these two genes are the most reliable candidates of universal biomarker for macrophages.

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.

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.

Alveolar macrophages (AMs) perform specialized functions in the lung including protection against inhaled microbes and repair following injury. Neonates are particularly susceptible to lung infection and injury, suggesting immaturity of AM function. In mice, AMs differentiate from fetal myeloid precursors within the first week following birth. To measure the molecular and functional differences between neonatal and adult AMs, we performed RNA-seq and ATAC-seq on sorted AMs. Based on RNA-seq data, adult AMs expressed higher levels of genes associated with fatty acid metabolism (Fabp1 and Abcg1), while neonatal AMs expressed higher levels of proinflammatory genes (including Il1b, Il6, Tnf, and S100a8). Consistent with higher proinflammatory gene expression in neonatal AMs, ATAC-seq revealed enrichment of accessible AP1, NF-kB, and IRF binding motifs. In contrast, adult AMs contained open chromatin enriched in motifs for KLF, AR, and STAT. To test if these differences contributed to differential innate immune function, we treated both neonatal and adult mice in vivo with inhaled LPS. LPS-exposed AMs demonstrated robust innate immune responses in both neonatal and adult mice. Interestingly, many of the LPS-induced genes in neonatal AMs had higher basal levels of expression, suggesting constitutive innate immune activation in the neonatal lung. These differences appeared to be driven by the lung microenvironment, as isolating and culturing AMs resulted in rapid loss of differences in gene expression between neonatal and adult cells. Our data therefore suggest the developing environment of the neonatal lung drives unique molecular and functional differences in AM mediated innate immunity.

Group B Streptococcus (GBS) is a major neonatal pathogen but rarely causes disease in adults. We previously showed in mice that GBS escapes killing in the neonatal lung via its heavily sialylated capsule. Immune cells detect sialic acid moieties via expression of a repertoire of Siglec receptors. Combinatory expression of proinflammatory and anti-inflammatory Siglec receptors allows differentiation between host and pathogenic microbial sialic acid modifications. We test here the hypothesis that neonatal alveolar macrophages (AMs) fail to detect and kill GBS due to developmental immaturity of Siglec receptor expression. Adult AMs express the proinflammatory sialic acid receptor Sialoadhesin (Sn, Siglec-1) and the anti-inflammatory receptors Siglec-E and Siglec-F. However, real time PCR, immunofluorescence, and FACS detected only Siglec-E in neonatal lung macrophage populations. Sn expression increased soon after birth and was restricted to AMs. The timing of increased Sn expression in newborn mice correlated with susceptibility to GBS. Mice infected with GBS on day 1 suffered early onset mortality, while mice infected on day 2 displayed late onset disease. Mice infected on day 3 survived GBS infection. Further implicating AM immaturity, Csf2−/− mice, which have defects in AM differentiation, lacked Sn expression and had reduced GBS clearance following infection. The presence of Siglec-E but absence of Sn in newborn AMs appeared to promote tolerance to GBS. Newborn SigE−/− mice had increased GBS phagocytosis and killing compared to WT controls. We therefore conclude that GBS exploits developmental immaturity of Siglec expression in AMs via its sialic acid capsule in causing neonatal disease.

Characterization of markers that identify activated macrophages could advance understanding of inflammatory lung diseases and facilitate development of novel methodologies for monitoring disease activity. We investigated whether folate receptor β (FRβ) expression could be used to identify and quantify activated macrophages in the lungs during acute inflammation induced by Escherichia coli LPS. We found that FRβ expression was markedly increased in lung macrophages at 48 hours after intratracheal LPS. In vivo molecular imaging with a fluorescent probe (cyanine 5 polyethylene glycol folate) showed that the fluorescence signal over the chest peaked at 48 hours after intratracheal LPS and was markedly attenuated after depletion of macrophages. Using flow cytometry, we identified the cells responsible for uptake of cyanine 5-conjugated folate as FRβ(+) interstitial macrophages and pulmonary monocytes, which coexpressed markers associated with an M1 proinflammatory macrophage phenotype. These findings were confirmed using a second model of acute lung inflammation generated by inducible transgenic expression of an NF-κB activator in airway epithelium. Using CC chemokine receptor 2-deficient mice, we found that FRβ(+) macrophage/monocyte recruitment was dependent on the monocyte chemotactic protein-1/CC chemokine receptor 2 pathway. Together, our results demonstrate that folate-based molecular imaging can be used as a noninvasive approach to detect classically activated monocytes/macrophages recruited to the lungs during acute inflammation.

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.

Neonates are uniquely susceptible to lung infection and injury. Alveolar (AM) and interstitial (IM) lung macrophages are the major cell populations protecting the lung against inhaled microbes and facilitating wound repair. AM and IM express unique cell surface markers and transcriptional profiles. We therefore hypothesized that neonatal and adult lung environments lead to differences in macrophage function. When lung macrophages were isolated by flow cytometry and stimulated with LPS in vitro for 2 h, neonatal AM expressed higher levels of Il1b and Il6 compared to adult AM. We next tested our hypothesis in vivo, challenging neonatal and adult mice with intranasal LPS. Contrary to our in vitro experiments, LPS induced higher cytokine expression in AM compared to IM. In addition, the adult AM response was higher than neonatal AM. To specifically test how the lung microenvironment regulated macrophage response, we stimulated neonatal and adult AM and IM with LPS either immediately after isolation or after 10–20 h of tissue culture. Consistent with our hypothesis, expression of key AM transcription factors fell quickly during cell culture. While neonatal AM and IM had similar LPS responses immediately after isolation, adult IM had higher cytokine expression compared to adult AM. Intriguingly, for both adults and neonates, longer culture time enhanced cytokine expression in activated AM while dampening the expression in IM. Our data suggest that the developmentally regulated lung microenvironment regulates the macrophage innate immune response.

Group B Streptococcus (GBS) is the most common pathogen in neonates but rarely causes disease in adults. GBS causes pneumonia in newborns, suggesting a defect in lung innate immunity at birth. Using a novel neonatal mouse model of GBS pneumonia, we showed that newborn mice have reduced killing of GBS in the lung and more severe and persistent lung injury. Here we test the hypothesis that defects in alveolar macrophage (AM) maturation in newborns leads to GBS susceptibility. Clodronate depletion of AM from adult mice prevented GBS killing, supporting the requirement of AM for GBS protection. AM maturation and function requires GM-CSF, which is abundantly expressed in the lung. While AM maturation occurred on day 4 in WT mice, GM-CSF knockout mice (Csf2−/−) failed to develop AM. Consistent with the role of AM in GBS immunity, neonatal Csf2−/− mice had reduced GBS killing and lung macrophages failed to phagocytose GBS. Csf2−/− macrophages also had reduced expression of multiple Siglecs, cell surface receptors implicated in protection against encapsulated bacteria. Newborn lung macrophages expressed primarily Siglec-E (SigE), whereas expression of other Siglecs were only detected in more mature AM. We used SigE−/− mice to test its role in neonatal GBS pneumonia. GBS killing in SigE−/− neonates was similar to WT. However SigE−/− macrophages appeared to phagocytose GBS more efficiently in vivo, suggesting GBS may employ SigE to avoid detection by neonatal macrophages. Our data show that mature alveolar macrophages are required for protection against pulmonary GBS infection and potentially implicate the developmental regulation of macrophage Siglec expression in neonatal lung immunity.

Inflammation inhibits normal lung morphogenesis in preterm infants. Soluble inflammatory mediators present in the lungs of patients developing bronchopulmonary dysplasia disrupt expression of multiple genes critical for development. However, the mechanisms linking innate immune signaling and developmental programs are not clear. NF-κB activation inhibits expression of the critical morphogen FGF-10. Here, we show that interactions between the RELA subunit of NF-κB and SP3 suppress SP1-mediated FGF-10 expression. SP3 co-expression reduced SP1-mediated Fgf-10 promoter activity, suggesting antagonistic interactions between SP1 and SP3. Chromatin immunoprecipitation of LPS-treated primary mouse fetal lung mesenchymal cells detected increased interactions between SP3, RELA, and the Fgf-10 promoter. Expression of a constitutively active IκB kinase β mutant not only decreased Fgf-10 promoter activity but also increased RELA-SP3 nuclear interactions. Expression of a dominant-negative IκB, which blocks NF-κB nuclear translocation, prevented inhibition of FGF-10 by SP3. The inhibitory functions of SP3 required sequences located in the N-terminal region of the protein. These data suggested that inhibition of FGF-10 by inflammatory signaling involves the NF-κB-dependent interactions between RELA, SP3, and the Fgf-10 promoter. NF-κB activation may therefore lead to reduced gene expression by recruiting inhibitory factors to specific gene promoters following exposure to inflammatory stimuli.

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 50 kDa 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.

Background: The regulation of epithelial cell shape and orientation during lung branching morphogenesis is not clearly understood. Nonmuscle myosins regulate cell size, morphology, and planar cell polarity. Here, we test the hypothesis that nonmuscle myosin II (NM II) regulates lung epithelial morphology in a spatially restricted manner. Results: Epithelial cell orientation at airway tips in fetal mouse lungs underwent a significant transformation at embryonic day (E) E17. Treatment of E15 lung explants with the NM II inhibitor blebbistatin increased airway branching, epithelial cell size, and the degree of anisotropy in epithelial cells lining the airway stalks. In cultured MLE-12 lung epithelial cells, blebbistatin increased cell velocity, but left the migratory response to FGF-10 unchanged. Conclusions: In the developing lung, NM II acts to constrain cell morphology and orientation, but may be suppressed at sites of branching and cell migration. The regulation of epithelial orientation may therefore undergo dynamic variations from E15 to E17. Developmental Dynamics, 2012. © 2012 Wiley Periodicals, Inc.

Aims Pulmonary arterial endothelial cells (PAECs) express the enzymes needed for generation of l-arginine from intracellular l-citrulline but do not express the enzymes needed for de novo l-citrulline synthesis. Hence, l-citrulline levels in PAECs are dependent on l-citrulline transport. Once generated, l-arginine can be converted to l-citrulline and nitric oxide (NO) by the enzyme NO synthase. We sought to determine whether hypoxia, a condition aetiologically linked to pulmonary hypertension, alters the transport of l-citrulline and the expression of the sodium-coupled neutral amino acid transporters (SNATs) in PAECs from newborn piglets. Methods and results PAECs isolated from newborn piglets were cultured under normoxic and hypoxic conditions and used to measure SNAT1, 2, 3, and 5 protein expression and 14C-l-citrulline uptake. SNAT1 protein expression was increased, while SNAT2, SNAT3, and SNAT5 expression was unaltered in hypoxic PAECs. 14C-l-citrulline uptake was increased in hypoxic PAECs. Studies with inhibitors of System A (SNAT1/2) and System N (SNAT3/5) revealed that the increased 14C-l-citrulline uptake was largely due to System A-mediated transport. Additional studies were performed to evaluate SNAT protein expression and l-citrulline levels in lungs of piglets with chronic hypoxia-induced pulmonary hypertension and comparable age controls. Lungs from piglets raised in chronic hypoxia exhibited greater SNAT1 expression and higher l-citrulline levels than lungs from controls. Conclusion Increased SNAT1 expression and the concomitant enhanced ability to transport l-citrulline in PAECs could represent an important regulatory mechanism to counteract NO signalling impairments known to occur during the development of chronic hypoxia-induced pulmonary hypertension in newborns.

The mechanisms by which chronic inflammatory lung diseases, particularly chronic obstructive pulmonary disease, confer enhanced risk for lung cancer are not well-defined. To investigate whether nuclear factor (NF)-κB, a key mediator of immune and inflammatory responses, provides an interface between persistent lung inflammation and carcinogenesis, we utilized tetracycline-inducible transgenic mice expressing constitutively active IκB kinase β in airway epithelium (IKTA (IKKβ trans-activated) mice). Intraperitoneal injection of ethyl carbamate (urethane), or 3-methylcholanthrene (MCA) and butylated hydroxytoluene (BHT) was used to induce lung tumorigenesis. Doxycycline-treated IKTA mice developed chronic airway inflammation and markedly increased numbers of lung tumors in response to urethane, even when transgene expression (and therefore epithelial NF-κB activation) was begun after exposure to carcinogen. Studies using a separate tumor initiator/promoter model (MCA+BHT) indicated that NF-κB functions as an independent tumor promoter. Enhanced tumor formation in IKTA mice was preceded by increased proliferation and reduced apoptosis of alveolar epithelium, resulting in increased formation of premalignant lesions. Investigation of inflammatory cells in lungs of IKTA mice revealed a substantial increase in macrophages and lymphocytes, including functional CD4+/CD25+/FoxP3+ regulatory T lymphocytes (Tregs). Importantly, Treg depletion using repetitive injections of anti-CD25 antibodies limited excessive tumor formation in IKTA mice. At 6 weeks following urethane injection, antibody-mediated Treg depletion in IKTA mice reduced the number of premalignant lesions in the lungs in association with an increase in CD8 lymphocytes. Thus, persistent NF-κB signaling in airway epithelium facilitates carcinogenesis by sculpting the immune/inflammatory environment in the lungs.

Bronchopulmonary dysplasia is a common pulmonary complication of extreme prematurity. Arrested lung development leads to bronchopulmonary dysplasia, but the molecular pathways that cause this arrest are unclear. Lung injury and inflammation increase disease risk, but the cellular site of the inflammatory response and the potential role of localized inflammatory signaling in inhibiting lung morphogenesis are not known. In this study, we show that tissue macrophages present in the fetal mouse lung mediate the inflammatory response to LPS and that macrophage activation inhibits airway morphogenesis. Macrophage depletion or targeted inactivation of the NF-κB signaling pathway protected airway branching in cultured lung explants from the effects of LPS. Macrophages also appear to be the primary cellular site of IL-1β production following LPS exposure. Conversely, targeted NF-κB activation in transgenic macrophages was sufficient to inhibit airway morphogenesis. Macrophage activation in vivo inhibited expression of multiple genes critical for normal lung development, leading to thickened lung interstitium, reduced airway branching, and perinatal death. We propose that fetal lung macrophage activation contributes to bronchopulmonary dysplasia by generating a localized inflammatory response that disrupts developmental signals critical for lung formation.

Group B Streptococcus (Streptococcus agalactiae, GBS) causes severe infections in neonates. While not a common adult pathogen, GBS is the leading cause of congenital pneumonia. To determine the molecular mechanisms of neonatal susceptibility, we developed a murine GBS pneumonia model. Neonatal, juvenile, and adult C57BL/6 mice were inoculated intranasally with 2800 CFU/g of GBS (COH1). After 7 d, survival was 100 % in adults and juveniles, with 10 % of neonates dying within 3 d after infection. Histopathological assessments showed severe lung injury scores 24 h after infection, with adult and juvenile mice demonstrating resolution of injury at 3 d post infection and normal histology after 7 d. In contrast, neonatal lungs had persistent lung injury up to 7 d post infection. By FACS, GBS induced rapid neutrophil recruitment into adult lungs and increased CD11b expression on adult lung macrophages. In neonatal lungs, GBS neither increased neutrophil numbers nor altered macrophage marker expression. Confocal imaging showed GBS in adult lungs almost entirely localized to alveolar macrophage phagolysosomes. However in neonatal lungs, GBS was only rarely phagocytosed by macrophages. These data were consistent with GBS killing data, which showed complete GBS killing within 24 h in adult and juvenile mice, but persistent GBS viability within the lung for up to 3 d in neonates. Defective GBS killing in neonates appeared to require the GBS capsule. Neonatal mice were much more efficient in killing the ΔcpsD capsule mutant compared to wild type GBS. Adult mice showed equal killing of ΔcpsD or wild type GBS. Our data suggest that the GBS capsule prevents killing by the newborn lung innate immune system, leading to neonatal disease susceptibility.

It is unknown whether or not tight junction formation plays any role in morula to blastocyst transformation that is associated with development of polarized trophoblast cells and fluid accumulation. Tight junctions are a hallmark of polarized epithelial cells and zonula occludens-1 (ZO-1) is a known key regulator of tight junction formation. Here we show that ZO-1 protein is first expressed during compaction of 8-cell embryos. This stage-specific appearance of ZO-1 suggests its participation in morula to blastocyst transition. Consistent with this idea, we demonstrate that ZO-1 siRNA delivery inside the blastomeres of zona-weakened embryos using electroporation not only knocks down ZO-1 gene and protein expressions, but also inhibits morula to blastocyst transformation in a concentration-dependent manner. In addition, ZO-1 inactivation reduced the expression of Cdx2 and Oct-4, but not ZO-2 and F-actin. These results provide the first evidence that ZO-1 is involved in blastocyst formation from the morula by regulating accumulation of fluid and differentiation of nonpolar blastomeres to polar trophoblast cells.

Inflammation in the developing preterm lung leads to disrupted airway morphogenesis and chronic lung disease in human neonates. However, the molecular mechanisms linking inflammation and the pathways controlling airway morphogenesis remain unclear. In this article, we show that IL-1β released by activated fetal lung macrophages is the key inflammatory mediator that disrupts airway morphogenesis. In mouse lung explants, blocking IL-1β expression, posttranslational processing, and signaling protected the formation of new airways from the inhibitory effects of Escherichia coli LPS. Consistent with a critical role for IL-1β, mice expressing a gain-of-function Nlrp3 allele and subsequent overactive inflammasome activity displayed abnormal saccular-stage lung morphogenesis and died soon after birth. Although the early-stage fetal lung appeared capable of mounting an NF-κB–mediated immune response, airway formation became more sensitive to inflammation later in development. This period of susceptibility coincided with higher expression of multiple inflammasome components that could increase the ability to release bioactive IL-1β. Macrophages from Nlrp3 gain-of-function mice also expressed higher levels of more mature cell surface markers, additionally linking inflammasome activation with macrophage maturation. These data identify developmental expression of the inflammasome and IL-1β release by fetal lung macrophages as key mechanisms and potential therapeutic targets for neonatal lung disease.

Integrin-dependent interactions between cells and extracellular matrix regulate lung development; however, specific roles for β1-containing integrins in individual cell types, including epithelial cells, remain incompletely understood. In this study, the functional importance of β1 integrin in lung epithelium during mouse lung development was investigated by deleting the integrin from E10.5 onwards using surfactant protein C promoter-driven Cre. These mutant mice appeared normal at birth but failed to gain weight appropriately and died by 4 months of age with severe hypoxemia. Defects in airway branching morphogenesis in association with impaired epithelial cell adhesion and migration, as well as alveolarization defects and persistent macrophage-mediated inflammation were identified. Using an inducible system to delete β1 integrin after completion of airway branching, we showed that alveolarization defects, characterized by disrupted secondary septation, abnormal alveolar epithelial cell differentiation, excessive collagen I and elastin deposition, and hypercellularity of the mesenchyme occurred independently of airway branching defects. By depleting macrophages using liposomal clodronate, we found that alveolarization defects were secondary to persistent alveolar inflammation. β1 integrin-deficient alveolar epithelial cells produced excessive monocyte chemoattractant protein 1 and reactive oxygen species, suggesting a direct role for β1 integrin in regulating alveolar homeostasis. Taken together, these studies define distinct functions of epithelial β1 integrin during both early and late lung development that affect airway branching morphogenesis, epithelial cell differentiation, alveolar septation and regulation of alveolar homeostasis.

Division of large, immature alveolar structures into smaller, more numerous alveoli increases the surface area available for gas exchange. Alveolar division requires precise epithelial-mesenchymal interactions. However, few experimental models exist for studying how these cell-cell interactions produce changes in 3-dimensional structure. Here we report an epithelial-mesenchymal cell co-culture model where 3-dimensional peaks form with similar cellular orientation as alveolar structures in vivo. Co-culturing fetal mouse lung mesenchyme with A549 epithelial cells produced tall peaks of cells covered by epithelia with cores of mesenchymal cells. These structures did not form when using adult lung fibroblasts. Peak formation did not require localized areas of cell proliferation or apoptosis. Mesenchymal cells co-cultured with epithelia adopted an elongated cell morphology closely resembling myofibroblasts within alveolar septa in vivo. Because inflammation inhibits alveolar formation, we tested the effects of E. coli lipopolysaccharide on 3-dimensional peak formation. Confocal and time-lapse imaging demonstrated that lipopolysaccharide reduced mesenchymal cell migration, resulting in fewer, shorter peaks with mesenchymal cells present predominantly at the base. This epithelial-mesenchymal co-culture model may therefore prove useful in future studies of mechanisms regulating alveolar morphogenesis.

In preterm infants, exposure to inflammation increases the risk of bronchopulmonary dysplasia, a chronic, developmental lung disease. Although macrophages are the key cells that initiate lung inflammation, less is known about lung macrophage phenotype and maturation. We hypothesized that fetal lung macrophages mature into distinct subpopulations during mouse development, and that activation could influence macrophage maturation. Expression of the fetal macrophage markers CD68, CD86, CD206, Ym1, fibrinogen-like protein 2, and indolamine-2, 3-dioxygenase was developmentally regulated, with each marker having different temporal patterns. Flow cytometry analysis showed macrophages within the fetal lung were less diverse than the distinctly separate subpopulations in newborn and adult lungs. Similar to adult alveolar macrophages, fetal lung macrophages responded to the TLR4 agonist LPS and the alternative activation cytokines IL-4 and IL-13. Using a macrophage-specific constitutively active IκB Kinase transgenic model (IKFM), we demonstrated that macrophage activation increased proinflammatory gene expression and reduced the response of fetal lung macrophages to IL-4 and IL-13. Activation also increased fetal lung macrophage proliferation. Fetal IKFM lungs contained increased percentages of more mature, CD11blowF4/80high cells that also expressed higher levels of the alternative activation markers CD204 and CD206. Development of fetal lung macrophages into mature alveolar macrophages may therefore include features of both proinflammatory and alternative activation paradigms.

In preterm infants, exposure to inflammation increases the risk of chronic, developmental lung disease bronchopulmonary dysplasia. While macrophages are the key cells that initiate lung inflammation, little is known about lung macrophage phenotype and maturation. We hypothesized that fetal lung macrophages mature into distinct subpopulations during mouse development, and that activation could influence macrophage maturation. Expression of the fetal macrophage markers CD68, CD86, CD206, Ym1, fibrinogen-like protein 2 (FGL2), and indolamine-2, 3-dioxygenase (Ido1) was developmentally regulated. Flow cytometry analysis measured a narrow spectrum of CD11b expression in fetal lung macrophages, with separation into CD11bloF4/80hi and CD11bhiF4/80lo subpopulations in newborn and adult lung. Similar to adult alveolar macrophages, fetal lung macrophages responded to the TLR4 agonist LPS and the alternative activation cytokines IL-4 and IL-13. Using a macrophage-specific constitutively active IKKβeta transgenic mod...

Infection and inflammation inhibit normal lung development. We have previously measured multiple alterations in growth factor expression and signaling that may link inflammation with abnormal devel...

Keratinocyte growth factor (KGF) regulates several functions in adult and developing lung epithelia; it causes proliferation, stimulates secretion of fluid and electrolytes, enhances repair, and may minimize injury. To gain insight into the molecular processes influenced by KGF, we applied KGF to primary cultures of well-differentiated human airway epithelia and used microarray hybridization to assess the abundance of gene transcripts. Of 7,069 genes tested, KGF changed expression levels of 910. Earlier studies showed that KGF causes epithelial proliferation, and as expected, treatment altered expression of numerous genes involved in cell proliferation. We found that KGF stimulated transepithelial Cl−transport, but the number of cystic fibrosis (CF) transmembrane conductance regulator (CFTR) transcripts fell. Although transcripts for ClC-1 and ClC-7 Cl−channels increased, KGF failed to augment transepithelial Cl−transport in CF epithelia, suggesting that KGF-stimulated Cl−transport in differentiated airway epithelia depends on the CFTR Cl−channel. Interestingly, KGF decreased transcripts for many interferon (IFN)-induced genes. IFN causes trafficking of Stat dimers to the nucleus, where they activate transcription of IFN-induced genes. We found that KGF prevented the IFN-stimulated trafficking of Stat1 from the cytosol to the nucleus, suggesting a molecular mechanism for KGF-mediated suppression of the IFN-signaling pathway. These results suggest that in addition to stimulating proliferation and repair of damaged airway epithelia, KGF stimulates Cl−transport and may dampen the response of epithelial cells to inflammatory mediators.

The epithelial Na+channel (ENaC) is comprised of three homologous subunits: α, β, and γ, all of which are required for formation of the fully functional channel. This channel is responsible for salt reabsorption in the kidney, the airway, and the large bowel. Mutations in ENaC can cause human disease by increasing channel function in Liddle’s syndrome, a form of hereditary hypertension, or by decreasing channel function in pseudohypoaldosteronism type I, a salt-wasting disease of infancy. We previously showed that ENaC is expressed on the cell surface as a minimally glycosylated, Triton-insoluble protein. In the present study we found that ENaC existed initially as a Triton-soluble protein that contained high-mannose glycosylation, presumably in the endoplasmic reticulum. This form of the protein disappeared as the Triton-insoluble, minimally glycosylated form became the more prevalent species. In pulse-chase studies of individually expressed subunits, we found that the Triton-soluble form of β-ENaC accumulated initially, whereas the Triton-soluble form of α-ENaC decreased throughout the time course. However, when all three subunits were coexpressed, the α- and β-subunits showed a similar pattern. The complex became Triton insoluble at some point after the endoplasmic reticulum, as incubation at 15°C blocked the conversion to the insoluble form. Deletion of the carboxy-terminal tail of β-ENaC causes Liddle’s syndrome. This mutation increased the amount of newly synthesized Triton-insoluble ENaC heteromultimers but did not affect the half-life of insoluble protein. Therefore, subunit composition and mutations in individual subunits can influence biosynthesis of the ENaC complex.

We previously demonstrated that the cystic fibrosis transmembrane conductance regulator (CFTR) is rapidly endocytosed in epithelial cells (Prince, L. S., Workman, R. B., Jr., and Marchase, R. B. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5192-5196). To determine the structural features of CFTR required for endocytosis, we prepared chimeric molecules consisting of the amino-terminal (residues 2-78) and carboxyl-terminal tail regions (residues 1391-1476) of CFTR, each fused to the transmembrane and extracellular domains of the transferrin receptor. Functional analysis of the CFTR-(2-78) and CFTR-(1391-1476) indicated that both chimeras were rapidly internalized. Deletion of residues 1440-1476 had no effect on chimera internalization. Mutations of potential internalization signals in both cytoplasmic domains reveal that only one mutation inhibits internalization, Y1424A. Using a surface biotinylation reaction, we also examined internalization rates of wild type and mutant CFTRs expressed in COS-7 cells. We found that both wild type and A1440X CFTR were rapidly internalized, whereas the Y1424A CFTR mutant, like the chimeric protein, had approximately 40% reduced internalization activity. Deletions in the amino-terminal tail region of CFTR resulted in defective trafficking of CFTR out of the endoplasmic reticulum to the cell surface, suggesting that an intact amino terminus is critical for biosynthesis. In summary, our results suggest that both tail regions of CFTR are sufficient to promote rapid internalization of a reporter molecule and that tyrosine 1424 is required for efficient CFTR endocytosis.

The epithelial Na+ channel (ENaC) complex is composed of three homologous subunits: α, β and γ. Mutations in ENaC subunits can increase the number of channels on the cell surface, causing a hereditary form of hypertension called Liddle's syndrome, or can decrease channel activity, causing pseudohypoaldosteronism type I, a salt-wasting disease of infancy. To investigate surface expression, we studied ENaC subunits expressed in COS-7 and HEK293 cells. Using surface biotinylation and protease sensitivity, we found that when individual ENaC subunits are expressed alone, they traffic to the cell surface. The subunits are glycosylated with high-mannose oligosaccharides, but seem to have the carbohydrate removed before they reach the cell surface. Moreover, subunits form a complex that cannot be disrupted by several non-ionic detergents. The pattern of glycosylation and detergent solubility/insolubility persists when the N-teminal and C-terminal cytoplasmic regions of ENaC are removed. With co-expression of all three ENaC subunits, the insoluble complex is the predominant species. These results show that ENaC and its family members are unique in their trafficking, biochemical characteristics and post-translational modifications.

Three subunits, alpha, beta, and gamma, contribute to the formation of the epithelial Na+ channel. To investigate the oligomeric assembly of the channel complex, we used sucrose gradient sedimentation analysis to determine the sedimentation properties of individual subunits and heteromultimers comprised of multiple subunits. When the alpha subunit was expressed alone, it first formed an oligomeric complex with a sedimentation coefficient of 11 S, and then generated a higher order multimer of 25 S. In contrast, individual beta and gamma subunits predominately assembled into 11 S complexes. We obtained similar results with expression in cells and in vitro. When we co-expressed beta with alpha or with alpha plus gamma, the beta subunit assembled into a 25 S complex. Glycosylation of the alpha subunit was not required for assembly into a 25 S complex. We found that the alpha subunit formed intra-chain disulfide bonds. Although such bonds were not required to generate an oligomeric complex, under nonreducing conditions the alpha subunit formed a complex that migrated more homogeneously at 25 S. This suggests that intra-chain disulfide bonds may stabilize the complex. These data suggest that the epithelial Na+ channel subunits form high order oligomeric complexes and that the alpha subunit contains the information that facilitates such formation. Interestingly, the ability of the alpha, but not the beta or gamma, subunit to assemble into a 25 S homomeric complex correlates with the ability of these subunits to generate functional channels when expressed alone.

Development of the pulmonary system is essential for terrestrial life. The molecular pathways that regulate this complex process are beginning to be defined, and such knowledge is critical to our understanding of congenital and acquired lung diseases. A recent workshop was convened by the National Heart, Lung, and Blood Institute to discuss the developmental principles that regulate the formation of the pulmonary system. Emerging evidence suggests that key developmental pathways not only regulate proper formation of the pulmonary system but are also reactivated upon postnatal injury and repair and in the pathogenesis of human lung diseases. Molecular understanding of early lung development has also led to new advances in areas such as generation of lung epithelium from pluripotent stem cells. The workshop was organized into four different topics, including early lung cell fate and morphogenesis, mechanisms of lung cell differentiation, tissue interactions in lung development, and environmental impact on early lung development. Critical points were raised, including the importance of epigenetic regulation of lung gene expression, the dearth of knowledge on important mesenchymal lineages within the lung, and the interaction between the developing pulmonary and cardiovascular system. This manuscript describes the summary of the discussion along with general recommendations to overcome the gaps in knowledge in lung developmental biology.

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a rare and lethal developmental disorder of the lung defined by a constellation of characteristic histopathological features. Non-pulmonary anomalies involving organs of gastrointestinal, cardiovascular, and genitourinary systems have been identified in approximately 80% of patients with ACD/MPV. We have collected DNA and pathological samples from more than 90 infants with ACD/MPV and their family members. Since the publication of our initial report of four point mutations and ten deletions, we have identified an additional thirty eight novel nonsynonymous mutations of FOXF1 (nine nonsense, seven frameshift, one inframe deletion, twenty missense, and one no stop). This report represents an up to date list of all known FOXF1 mutations to the best of our knowledge. Majority of the cases are sporadic whereas four familial cases with three showing maternal inheritance, consistent with paternal imprinting of the gene. Twenty five mutations (60%) are located within the putative DNA binding domain, indicating its plausible role in gene regulation. Five mutations map to the second exon. We identified two additional genic and eight genomic deletions upstream to FOXF1. These results corroborate and extend our previous observations and further establish involvement of FOXF1 in ACD/MPV and lung organogenesis.

Summary Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in premature infants. During NEC pathogenesis, bacteria are able to penetrate innate immune defenses and invade the intestinal epithelial layer causing subsequent inflammation and tissue necrosis. Normally the Paneth cells appear in the intestinal crypts during the first trimester of human pregnancy. Paneth cells constitute a major component of the innate immune system by producing multiple antimicrobial peptides and pro-inflammatory mediators. To better understand the possible role of Paneth cell disruption in NEC, we quantified the number of Paneth cells present in infants with NEC and found they were significantly decreased compared to age matched controls. We were able to model this loss in the intestine of P14-16 day old mice by treating them with the zinc chelator dithizone. Intestines from dithizone treated animals retained approximately half the number of Paneth cells as controls. Furthermore, by combining dithizone treatment with exposure to Klebsiella pneumoniae we were able to induce intestinal injury and inflammatory induction that resembles human NEC. Additionally, this novel Paneth cell ablation model produces NEC-like pathology consistent with other currently used animal models, but is simpler to use, can be used in older animals that have been dam fed, and represents a novel line of investigation to study NEC pathogenesis and treatment.