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The main laminin-binding integrins α3β1, α6β1 and α6β4 are co-expressed in the developing kidney collecting duct system. We previously showed that deleting the integrin α3 or α6 subunit in the ureteric bud, which gives rise to the kidney collecting system, caused either a mild or no branching morphogenesis phenotype, respectively. To determine whether these two integrin subunits cooperate in kidney collecting duct development, we deleted α3 and α6 in the developing ureteric bud. The collecting system of the double knockout phenocopied the α3 integrin conditional knockout. However, with age, the mice developed severe inflammation and fibrosis around the collecting ducts, resulting in kidney failure. Integrin α3α6-null collecting duct epithelial cells showed increased secretion of pro-inflammatory cytokines and displayed mesenchymal characteristics, causing loss of barrier function. These features resulted from increased nuclear factor kappa-B (NF-κB) activity, which regulated the Snail and Slug (also known as Snai1 and Snai2, respectively) transcription factors and their downstream targets. These data suggest that laminin-binding integrins play a key role in the maintenance of kidney tubule epithelial cell polarity and decrease pro-inflammatory cytokine secretion by regulating NF-κB-dependent signaling.

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.

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.

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.

Bronchopulmonary dysplasia (BPD) is a frequent complication of preterm birth. This chronic lung disease results from arrested saccular airway development and is most common in infants exposed to inflammatory stimuli. In experimental models, inflammation inhibits expression of fibroblast growth factor-10 (FGF-10) and impairs epithelial–mesenchymal interactions during lung development; however, the mechanisms connecting inflammatory signaling with reduced growth factor expression are not yet understood. In this study we found that soluble inflammatory mediators present in tracheal fluid from preterm infants can prevent saccular airway branching. In addition, LPS treatment led to local production of mediators that inhibited airway branching and FGF-10 expression in LPS-resistant C.C3-Tlr4Lpsd/J fetal mouse lung explants. Both direct NF-κB activation and inflammatory cytokines (IL-1β and TNF-α) that activate NF-κB reduced FGF-10 expression, whereas chemokines that signal via other inflammatory pathways had no effect. Mutational analysis of the FGF-10 promoter failed to identify genetic elements required for direct NF-κB–mediated FGF-10 inhibition. Instead, NF-κB activation appeared to interfere with the normal stimulation of FGF-10 expression by Sp1. Chromatin immunoprecipitation and nuclear coimmunoprecipitation studies demonstrated that the RelA subunit of NF-κB and Sp1 physically interact at the FGF-10 promoter. These findings indicate that inflammatory signaling through NF-κB disrupts the normal expression of FGF-10 in fetal lung mesenchyme by interfering with the transcriptional machinery critical for lung morphogenesis.