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Determining how alveoli are formed and maintained is critical to understanding lung organogenesis and regeneration after injury. While technological barriers have heretofore limited real-time observation of alveologenesis, we have now used scanned oblique plane illumination microscopy of living lung slices to observe specific cellular behaviors at high resolution over several days. Contrary to the prevailing paradigm that alveoli form by airspace subdivision via ingrowing septa, we find that alveoli form by ballooning epithelial outgrowth supported by stable mesenchymal ring structures. Our systematic analysis allowed creation of a computational model of finely-timed cellular structural changes that drive alveologenesis under normal conditions or with perturbed intercellular Wnt signaling. This new paradigm and platform can be leveraged for mechanistic studies and screening for therapies to promote lung regeneration.One-Sentence SummaryLong-term live analysis of neonatal lungs supports a dynamic epithelial outgrowth model for alveologenesis.

During alveolar repair, alveolar type 2 (AT2) epithelial cell progenitors rapidly proliferate and differentiate into flat type 1 alveolar epithelial cells. Failure of normal alveolar repair mechanisms can lead to loss of alveolar structure (emphysema) or development of fibrosis, depending on the type and severity of injury. To test if β1-containing integrins are required during repair following acute injury, we administeredE. colilipopolysaccharide (LPS) by intratracheal injection to mice with a post-developmental deletion of β1 integrin in AT2 cells. While control mice recovered from LPS injury without structural abnormalities, β1-deficient mice had more severe inflammation and developed emphysema. In addition, recovering alveoli were repopulated with an abundance of rounded epithelial cells co-expressing type 2, type 1, and mixed intermediate cell state markers, with few mature type 1 cells. β1-deficient AT2 cells showed persistently increased proliferation after injury, which was blocked by inhibiting NF-κB activation in these cells. Lineage tracing experiments revealed that β1-deficient AT2 cells failed to differentiate into mature type 1 alveolar epithelial cells. Together, these findings demonstrate that functional alveolar repair after injury with terminal alveolar epithelial differentiation requires β1-containing integrins.

Rationale: Bronchopulmonary dysplasia (BPD) is a leading complication of preterm birth that affects infants born in the saccular stage of lung development at

A female infant is born at 38 weeks’ estimated gestational age to a 23-year-old, gravida 2, para 2 woman. Maternal history is significant for poorly controlled chronic hypertension and limited prenatal care. The woman reports she was returning home from vacation when she experienced painful, frequent contractions, prompting presentation to the nearest emergency department. Her initial blood pressure is 152/102 mm Hg. She has spontaneous rupture of membranes and undergoes repeat cesarean section 3 hours after arrival at the academic medical center. In the delivery room, Apgar scores are 7 at 1 minute (0 for skin color, and 1 for respiratory effort) and 9 at 5 minutes (1 for skin color). Growth parameters are consistent with a small-for–gestational age infant; the newborn’s head circumference, weight, and length are all less than the 1st percentile for gestational age. Initial physical examination is remarkable for upper limb abnormalities, including oligodactyly with the presence of only 2 formed digits on the left upper extremity, a single palpable forearm bone in the left upper extremity, a sacral dimple, significant hirsutism, and abnormal facial features, including synophrys and highly arched eyebrows. She is admitted to the NICU for further care. In the NICU, she is initiated on feeds of expressed breast milk and colostrum. She passes meconium on the first day after birth. The neonate initially tolerates small volumes of feeds, but by day 2, she experiences nonbilious emesis 15 to 30 minutes after each feed. Intravenous fluids are initiated and a Replogle tube is placed. An abdominal radiograph is obtained on the second day after …

Integrins - the principal extracellular matrix (ECM) receptors of the cell - promote cell adhesion, migration, and proliferation, which are key events for cancer growth and metastasis. To date, most integrin-targeted cancer therapeutics have disrupted integrin-ECM interactions, which are viewed as critical for integrin functions. However, such agents have failed to improve cancer patient outcomes. We show that the highly expressed integrin β1 subunit is required for lung adenocarcinoma development in a carcinogen-induced mouse model. Likewise, human lung adenocarcinoma cell lines with integrin β1 deletion failed to form colonies in soft agar and tumors in mice. Mechanistically, we demonstrate that these effects do not require integrin β1-mediated adhesion to ECM but are dependent on integrin β1 cytoplasmic tail-mediated activation of focal adhesion kinase (FAK). These studies support a critical role for integrin β1 in lung tumorigenesis that is mediated through constitutive, ECM binding-independent signaling involving the cytoplasmic tail.

SummaryLung organogenesis requires precisely timed shifts in the spatial organization and function of parenchymal cells, especially during the later stages of lung development. To investigate the mechanisms governing lung parenchymal dynamics during development, we performed a single cell RNA sequencing (scRNA-seq) time-series yielding 92,238 epithelial, endothelial, and mesenchymal cells across 8 time points from embryonic day 12 (E12) to postnatal day 14 (P14) in mice. We combined new computational analyses with RNAin situhybridization to explore transcriptional velocity, fate likelihood prediction, and spatiotemporal localization of cell populations during the transition between the saccular and alveolar stages. We interrogated this atlas to illustrate the complexity of type 1 pneumocyte function during the saccular and alveolar stages, and we demonstrate an integrated view of the cellular dynamics during lung development.

Emerging evidence indicates that early life events can increase the risk for developing chronic obstructive pulmonary disease (COPD). Using an inducible transgenic mouse model for NF-κB activation in the airway epithelium, we found that a brief period of inflammation during the saccular stage (P3–P5) but not alveolar stage (P10–P12) of lung development disrupted elastic fiber assembly, resulting in permanent reduction in lung function and development of a COPD-like lung phenotype that progressed through 24 months of age. Neutrophil depletion prevented disruption of elastic fiber assembly and restored normal lung development. Mechanistic studies uncovered a role for neutrophil elastase (NE) in downregulating expression of critical elastic fiber assembly components, particularly fibulin-5 and elastin. Further, purified human NE and NE-containing exosomes from tracheal aspirates of premature infants with lung inflammation downregulated elastin and fibulin-5 expression by saccular-stage mouse lung fibroblasts. Together, our studies define a critical developmental window for assembling the elastin scaffold in the distal lung, which is required to support lung structure and function throughout the lifespan. Although neutrophils play a well-recognized role in COPD development in adults, neutrophilic inflammation may also contribute to early-life predisposition to COPD.

The SARS-CoV-2 novel coronavirus global pandemic (COVID-19) has led to millions of cases and hundreds of thousands of deaths around the globe. While the elderly appear at high risk for severe disease, hospitalizations and deaths due to SARS-CoV-2 among children have been relatively rare. Integrating single-cell RNA sequencing (scRNA-seq) of the developing mouse lung with temporally-resolved RNA-in-situ hybridization (ISH) in mouse and human lung tissue, we found that expression of SARS-CoV-2 Spike protein primerTMPRSS2was highest in ciliated cells and type I alveolar epithelial cells (AT1), andTMPRSS2expression was increased with aging in mice and humans. Analysis of autopsy tissue from fatal COVID-19 cases revealed SARS-CoV-2 RNA was detected most frequently in ciliated and secretory cells in the airway epithelium and AT1 cells in the peripheral lung. SARS-CoV-2 RNA was highly colocalized in cells expressingTMPRSS2.Together, these data demonstrate the cellular spectrum infected by SARS-CoV-2 in the lung epithelium, and suggest that developmental regulation ofTMPRSS2may underlie the relative protection of infants and children from severe respiratory illness.

Integrins, the extracellular matrix receptors that facilitate cell adhesion and migration, are necessary for organ morphogenesis; however, their role in maintaining adult tissue homeostasis is poorly understood. To define the functional importance of β(1) integrin in adult mouse lung, we deleted it after completion of development in type 2 alveolar epithelial cells (AECs). Aged β(1) integrin–deficient mice exhibited chronic obstructive pulmonary disease–like (COPD-like) pathology characterized by emphysema, lymphoid aggregates, and increased macrophage infiltration. These histopathological abnormalities were preceded by β(1) integrin–deficient AEC dysfunction such as excessive ROS production and upregulation of NF-κB–dependent chemokines, including CCL2. Genetic deletion of the CCL2 receptor, Ccr2, in mice with β(1) integrin–deficient type 2 AECs impaired recruitment of monocyte-derived macrophages and resulted in accelerated inflammation and severe premature emphysematous destruction. The lungs exhibited reduced AEC efferocytosis and excessive numbers of inflamed type 2 AECs, demonstrating the requirement for recruited monocytes/macrophages in limiting lung injury and remodeling in the setting of a chronically inflamed epithelium. These studies support a critical role for β(1) integrin in alveolar homeostasis in the adult lung.

Alveolar type II (AT2) epithelial cells are uniquely specialized to produce surfactant in the lung and act as progenitor cells in the process of repair after lung injury. AT2 cell injury has been implicated in several lung diseases, including idiopathic pulmonary fibrosis and bronchopulmonary dysplasia. The inability to maintain primary AT2 cells in culture has been a significant barrier in the investigation of pulmonary biology. We have addressed this knowledge gap by developing a three-dimensional (3D) organotypic coculture using primary human fetal AT2 cells and pulmonary fibroblasts. Grown on top of matrix-embedded fibroblasts, the primary human AT2 cells establish a monolayer and have direct contact with the underlying pulmonary fibroblasts. Unlike conventional two-dimensional (2D) culture, the structural and functional phenotype of the AT2 cells in our 3D organotypic culture was preserved over 7 days of culture, as evidenced by the presence of lamellar bodies and by production of surfactant proteins B and C. Importantly, the AT2 cells in 3D cocultures maintained the ability to replicate, with approximately 60% of AT2 cells staining positive for the proliferation marker Ki67, whereas no such proliferation is evident in 2D cultures of the same primary AT2 cells. This organotypic culture system enables interrogation of AT2 epithelial biology by providing a reductionist in vitro model in which to investigate the response of AT2 epithelial cells and AT2 cell–fibroblast interactions during lung injury and repair.

Hyperoxia exposure in premature infants increases the risk of subsequent lung diseases, such as asthma and bronchopulmonary dysplasia. Fibroblasts help maintain bronchial and alveolar integrity. Thus, understanding mechanisms by which hyperoxia influences fibroblasts is critical. Cellular senescence is increasingly recognized as important to the pathophysiology of multiple diseases. We hypothesized that clinically relevant moderate hyperoxia (

Background Although studies involving preterm infants ≤34 weeks gestation report a decreased incidence of patent ductus arteriosus after antenatal betamethasone, studies involving younger gestation infants report conflicting results. Methods We used preterm baboons, mice and humans (≤276/7 weeks gestation) to examine betamethasone’s effects on ductus gene expression and constriction both in vitro and in vivo. Results In mice, betamethasone increased the sensitivity of the premature ductus to the contractile effects of oxygen without altering the effects of other contractile or vasodilatory stimuli. Betamethasone’s effects on oxygen sensitivity could be eliminated by inhibiting endogenous prostaglandin/nitric oxide signaling. In mice and baboons, betamethasone increased the expression of several developmentally-regulated genes that mediate oxygen-induced constriction (K+ channels) and inhibit vasodilator signaling (phosphodiesterases). In human infants, betamethasone increased the rate of ductus constriction at all gestational ages. However, in infants born ≤256/7 weeks gestation, betamethasone’s contractile effects were only apparent when prostaglandin signaling was inhibited, whereas, at 26-27 weeks gestation betamethasone’s contractile effects were apparent even in the absence of prostaglandin inhibitors. Conclusions We speculate that betamethasone’s contractile effects may be mediated through genes that are developmentally regulated. This could explain why betamethasone’s effects vary according to the infant’s developmental age at birth.

Preterm birth affects approximately 1 out of every 10 births in the United States, leading to high rates of mortality and long-term negative health consequences. To investigate the mechanisms leading to preterm birth so as to develop prevention strategies, researchers have developed numerous mouse models of preterm birth. However, the lack of standard definitions for preterm birth in mice limits our field's ability to compare models and make inferences about preterm birth in humans. In this review, we discuss numerous mouse preterm birth models, propose guidelines for experiments and reporting, and suggest markers that can be used to assess whether pups are premature or mature. We argue that adoption of these recommendations will enhance the utility of mice as models for preterm birth., To improve reporting of mouse models of preterm birth, a set of universal guidelines and simple assays of developmental markers are proposed to distinguish between mature and premature pups.

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.

Interleukin (IL)-18 is an important effector of innate and adaptive immunity, but its expression must also be tightly regulated because it can potentiate lethal systemic inflammation and death. Healthy and septic human neonates demonstrate elevated serum concentrations of IL-18 compared with adults. Thus, we determined the contribution of IL-18 to lethality and its mechanism in a murine model of neonatal sepsis. We find that IL-18-null neonatal mice are highly protected from polymicrobial sepsis, whereas replenishing IL-18 increased lethality to sepsis or endotoxemia. Increased lethality depended on IL-1 receptor 1 (IL-1R1) signaling but not adaptive immunity. In genome-wide analyses of blood mRNA from septic human neonates, expression of the IL-17 receptor emerged as a critical regulatory node. Indeed, IL-18 administration in sepsis increased IL-17A production by murine intestinal γδT cells as well as Ly6G(+) myeloid cells, and blocking IL-17A reduced IL-18-potentiated mortality to both neonatal sepsis and endotoxemia. We conclude that IL-17A is a previously unrecognized effector of IL-18-mediated injury in neonatal sepsis and that disruption of the deleterious and tissue-destructive IL-18/IL-1/IL-17A axis represents a novel therapeutic approach to improve outcomes for human neonates with sepsis.

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.

Although commonly asymptomatic, congenital CMV infection is the leading cause of nonhereditary SNHL. Other sequelae that may be evident only after the neonatal period can include chorioretinitis, neurodevelopmental delay with mental or motor impairment, and microcephaly. (13) • Congenital CMV infection is confirmed by detection of the virus in urine, blood, or saliva within the first 3 weeks of life by culture or polymerase chain reaction. A positive test does not necessarily confirm symptomatic CMV disease or need for treatment. (13) • Postnatal CMV infections transmitted through human milk have been reported and may be clinically relevant in extremely premature infants; however, the risk-benefit ratio of pasteurizing human milk for the prevention of postnatal CMV infection is unclear. • Ganciclovir, valganciclovir, foscarnet, cidofovir, and CMV hyperimmune globulin are effective in treating or preventing CMV infections in the immunocompromised host, but require close monitoring for associated toxicities. Treatment for congenital CMV is associated with significant toxicity and uncertain effectiveness. • Based on strong evidence, anticipatory guidance for congenital CMV infection should include hearing tests and neurodevelopmental assessments until school age. (3) In patients with symptomatic congenital CMV infection, lifelong ophthalmologic screening should be included. (4) • Based primarily on consensus, owing to lack of relevant clinical studies, it is not recommended to withhold human milk produced by CMV-seropositive mothers from healthy term infants. (5)(6) • Based on some research evidence, as well as consensus, treatment for congenital CMV is recommended only in symptomatic infants with central nervous system involvement. (9)

OBJECTIVE. The objective of this study was to assess the validity of parental report of influenza vaccinations. PATIENTS AND METHODS. A cross-sectional study of children who were 6 to 59 months of age and presented to a large, pediatric residency clinic from February through April 2005 was performed. A standardized, parental questionnaire ascertained the influenza vaccination status of children during the 2004–2005 influenza season and was compared with the medical chart, the criterion standard. Children were classified as being at high risk when they had a specific influenza vaccine recommendation in 2004–2005 by age (6–23 months of age) or by chronic medical condition. RESULTS. Of 218 parents approached in the pediatric residency clinic, 198 (95%) children who were 6 to 59 months of age were enrolled, and 84 (42%) were vaccinated according to the medical chart. More children who were 6 to 23 months than those who were 24 to 59 months of age were vaccinated (63% vs 21%). Children with chronic medical conditions were more likely to be vaccinated than healthy children who were 24 to 59 months of age (57% vs 11%), but no difference was observed for children who were 6 to 23 months of age (79% vs 60%). In comparison with the medical chart, parental report of influenza vaccination had a sensitivity of 88%, a specificity of 90%, and a κ coefficient of 0.78. For children who were 6 to 23 months of age or had a chronic medical condition (n = 123), parental report had a sensitivity of 89%, a specificity of 81%, and a κ coefficient of 0.71. CONCLUSIONS. Parental report of influenza vaccination among children who were 6 to 59 months of age had reasonable sensitivity, specificity, and reliability as compared with the medical chart in this study population.