Bulk RNA sequencing of human pediatric lung cell populations reveals unique transcriptomic signature associated with postnatal pulmonary development.

Postnatal lung development results in an increasingly functional organ prepared for gas exchange and pathogenic challenges. It is achieved through cellular differentiation and migration. Changes in the tissue architecture during this development process are well-documented and increasing cellular diversity associated with it are reported in recent years. Despite recent progress, transcriptomic and molecular pathways associated with human postnatal lung development are yet to be fully understood. In this study, we investigated gene expression patterns associated with healthy pediatric lung development in four major enriched cell populations (epithelial, endothelial, and nonendothelial mesenchymal cells, along with lung leukocytes) from 1-day-old to 8-yr-old organ donors with no known lung disease. For analysis, we considered the donors in four age groups [less than 30 days old neonates, 30 days to < 1 yr old infants, toddlers (1 to < 2 yr), and children 2 yr and older] and assessed differentially expressed genes (DEG). We found increasing age-associated transcriptional changes in all four major cell types in pediatric lung. Transition from neonate to infant stage showed highest number of DEG compared with the number of DEG found during infant to toddler- or toddler to older children-transitions. Profiles of differential gene expression and further pathway enrichment analyses indicate functional epithelial cell maturation and increased capability of antigen presentation and chemokine-mediated communication. Our study provides a comprehensive reference of gene expression patterns during healthy pediatric lung development that will be useful in identifying and understanding aberrant gene expression patterns associated with early life respiratory diseases.NEW & NOTEWORTHY This study presents postnatal transcriptomic changes in major cell populations in human lung, namely endothelial, epithelial, mesenchymal cells, and leukocytes. Although human postnatal lung development continues through early adulthood, our results demonstrate that greatest transcriptional changes occur in first few months of life during neonate to infant transition. These early transcriptional changes in lung parenchyma are particularly notable for functional maturation and activation of alveolar type II cell genes.

New insights into the natural history of bronchopulmonary dysplasia from proteomics and multiplexed immunohistochemistry.

Bronchopulmonary dysplasia (BPD) is a disease of prematurity related to the arrest of normal lung development. The objective of this study was to better understand how proteome modulation and cell-type shifts are noted in BPD pathology. Pediatric human donors aged 1-3 yr were classified based on history of prematurity and histopathology consistent with "healed" BPD (hBPD, n = 3) and "established" BPD (eBPD, n = 3) compared with respective full-term born (n = 6) age-matched term controls. Proteins were quantified by tandem mass spectroscopy with selected Western blot validations. Multiplexed immunofluorescence (MxIF) microscopy was performed on lung sections to enumerate cell types. Protein abundances and MxIF cell frequencies were compared among groups using ANOVA. Cell type and ontology enrichment were performed using an in-house tool and/or EnrichR. Proteomics detected 5,746 unique proteins, 186 upregulated and 534 downregulated, in eBPD versus control with fewer proteins differentially abundant in hBPD as compared with age-matched term controls. Cell-type enrichment suggested a loss of alveolar type I, alveolar type II, endothelial/capillary, and lymphatics, and an increase in smooth muscle and fibroblasts consistent with MxIF. Histochemistry and Western analysis also supported predictions of upregulated ferroptosis in eBPD versus control. Finally, several extracellular matrix components mapping to angiogenesis signaling pathways were altered in eBPD. Despite clear parsing by protein abundance, comparative MxIF analysis confirms phenotypic variability in BPD. This work provides the first demonstration of tandem mass spectrometry and multiplexed molecular analysis of human lung tissue for critical elucidation of BPD trajectory-defining factors into early childhood.NEW & NOTEWORTHY We provide new insights into the natural history of bronchopulmonary dysplasia in donor human lungs after the neonatal intensive care unit hospitalization. This study provides new insights into how the proteome and histopathology of BPD changes in early childhood, uncovering novel pathways for future study.

Proteomic Analysis of Human Lung Development.

Rationale: The current understanding of human lung development derives mostly from animal studies. Although transcript-level studies have analyzed human donor tissue to identify genes expressed during normal human lung development, protein-level analysis that would enable the generation of new hypotheses on the processes involved in pulmonary development are lacking. Objectives: To define the temporal dynamic of protein expression during human lung development. Methods: We performed proteomics analysis of human lungs at 10 distinct times from birth to 8 years to identify the molecular networks mediating postnatal lung maturation. Measurements and Main Results: We identified 8,938 proteins providing a comprehensive view of the developing human lung proteome. The analysis of the data supports the existence of distinct molecular substages of alveolar development and predicted the age of independent human lung samples, and extensive remodeling of the lung proteome occurred during postnatal development. Evidence of post-transcriptional control was identified in early postnatal development. An extensive extracellular matrix remodeling was supported by changes in the proteome during alveologenesis. The concept of maturation of the immune system as an inherent part of normal lung development was substantiated by flow cytometry and transcriptomics. Conclusions: This study provides the first in-depth characterization of the human lung proteome during development, providing a unique proteomic resource freely accessible at Lungmap.net. The data support the extensive remodeling of the lung proteome during development, the existence of molecular substages of alveologenesis, and evidence of post-transcriptional control in early postnatal development.

Neonatal Hyperoxia Activates Activating Transcription Factor 4 to Stimulate Folate Metabolism and Alveolar Epithelial Type 2 Cell Proliferation.

Oxygen supplementation in preterm infants disrupts alveolar epithelial type 2 (AT2) cell proliferation through poorly understood mechanisms. Here, newborn mice are used to understand how hyperoxia stimulates an early aberrant wave of AT2 cell proliferation that occurs between Postnatal Days (PNDs) 0 and 4. RNA-sequencing analysis of AT2 cells isolated from PND4 mice revealed hyperoxia stimulates expression of mitochondrial-specific methylenetetrahydrofolate dehydrogenase 2 and other genes involved in mitochondrial one-carbon coupled folate metabolism and serine synthesis. The same genes are induced when AT2 cells normally proliferate on PND7 and when they proliferate in response to the mitogen fibroblast growth factor 7. However, hyperoxia selectively stimulated their expression via the stress-responsive activating transcription factor 4 (ATF4). Administration of the mitochondrial superoxide scavenger mitoTEMPO during hyperoxia suppressed ATF4 and thus early AT2 cell proliferation, but it had no effect on normative AT2 cell proliferation seen on PND7. Because ATF4 and methylenetetrahydrofolate dehydrogenase are detected in hyperplastic AT2 cells of preterm infant humans and baboons with bronchopulmonary dysplasia, dampening mitochondrial oxidative stress and ATF4 activation may provide new opportunities for controlling excess AT2 cell proliferation in neonatal lung disease.

Dissociation, cellular isolation, and initial molecular characterization of neonatal and pediatric human lung tissues.

Human lung morphogenesis begins by embryonic life and continues after birth into early childhood to form a complex organ with numerous morphologically and functionally distinct cell types. Pulmonary organogenesis involves dynamic changes in cell proliferation, differentiation, and migration of specialized cells derived from diverse embryonic lineages. Studying the molecular and cellular processes underlying formation of the fully functional lung requires isolating distinct pulmonary cell populations during development. We now report novel methods to isolate four major pulmonary cell populations from pediatric human lung simultaneously. Cells were dissociated by protease digestion of neonatal and pediatric lung and isolated on the basis of unique cell membrane protein expression patterns. Epithelial, endothelial, nonendothelial mesenchymal, and immune cells were enriched by fluorescence-activated cell sorting. Dead cells and erythrocytes were excluded by 7-aminoactinomycin D uptake and glycophorin-A (CD235a) expression, respectively. Leukocytes were identified by membrane CD45 (protein tyrosine phosphatase, receptor type C), endothelial cells by platelet endothelial cell adhesion molecule-1 (CD31) and vascular endothelial cadherin (CD144), and both were isolated. Thereafter, epithelial cell adhesion molecule (CD326)-expressing cells were isolated from the endothelial- and immune cell-depleted population to enrich epithelial cells. Cells lacking these membrane markers were collected as "nonendothelial mesenchymal" cells. Quantitative RT-PCR and RNA sequencing analyses of population specific transcriptomes demonstrate the purity of the subpopulations of isolated cells. The method efficiently isolates major human lung cell populations that we announce are now available through the National Heart, Lung, and Blood Institute Lung Molecular Atlas Program (LungMAP) for their further study.

Single-Cell Transcriptomic Profiling Identifies Molecular Phenotypes of Newborn Human Lung Cells.

While animal model studies have extensively defined the mechanisms controlling cell diversity in the developing mammalian lung, there exists a significant knowledge gap with regards to late-stage human lung development. The NHLBI Molecular Atlas of Lung Development Program (LungMAP) seeks to fill this gap by creating a structural, cellular and molecular atlas of the human and mouse lung. Transcriptomic profiling at the single-cell level created a cellular atlas of newborn human lungs. Frozen single-cell isolates obtained from two newborn human lungs from the LungMAP Human Tissue Core Biorepository, were captured, and library preparation was completed on the Chromium 10X system. Data was analyzed in Seurat, and cellular annotation was performed using the ToppGene functional analysis tool. Transcriptional interrogation of 5500 newborn human lung cells identified distinct clusters representing multiple populations of epithelial, endothelial, fibroblasts, pericytes, smooth muscle, immune cells and their gene signatures. Computational integration of data from newborn human cells and with 32,000 cells from postnatal days 1 through 10 mouse lungs generated by the LungMAP Cincinnati Research Center facilitated the identification of distinct cellular lineages among all the major cell types. Integration of the newborn human and mouse cellular transcriptomes also demonstrated cell type-specific differences in maturation states of newborn human lung cells. Specifically, newborn human lung matrix fibroblasts could be separated into those representative of younger cells (n = 393), or older cells (n = 158). Cells with each molecular profile were spatially resolved within newborn human lung tissue. This is the first comprehensive molecular map of the cellular landscape of neonatal human lung, including biomarkers for cells at distinct states of maturity.

FOXO3 deficiency leads to increased susceptibility to cigarette smoke-induced inflammation, airspace enlargement, and chronic obstructive pulmonary disease.

Forkhead box class O 3a (FOXO3) is a member of the FoxO transcription factor subfamily, which regulates the expression of target genes not only through DNA binding as a transcription factor, but also through protein-protein interaction. Although FoxO3 is a well-known transcription factor involved in diverse biological processes, the role of FoxO3 in cigarette smoke (CS)-induced lung inflammation and injury has not been studied. It is, therefore, hypothesized that deficiency of FoxO3 leads to increased susceptibility to CS-induced lung inflammatory response and airspace enlargement. In this article, we show that the levels of FOXO3 are significantly decreased in lungs of smokers and patients with chronic obstructive pulmonary disease, as well as in lungs of mice exposed to CS. Genetic ablation of FoxO3 led to pulmonary emphysema and exaggerated inflammatory response in lungs of mice exposed to CS. We further showed that CS induced the translocation of FoxO3 into the nucleus where FoxO3 interacted with NF-κB and disrupted NF-κB DNA-binding ability, leading to inhibition of its activity. Targeted disruption of FoxO3 also resulted in downregulation of antioxidant genes in mouse lungs in response to CS exposure. These results suggest that FoxO3 plays a pivotal role in regulation of lung inflammatory response and antioxidant genes, and deficiency of FoxO3 results in development of chronic obstructive pulmonary disease/emphysema.

Parenchymal cell TNF receptors contribute to inflammatory cell recruitment and respiratory failure in Pneumocystis carinii-induced pneumonia.

The opportunistic organism Pneumocystis carinii (Pc) produces a life-threatening pneumonia (PcP) in patients with low CD4(+) T cell counts. Animal models of HIV-AIDS-related PcP indicate that development of severe disease is dependent on the presence of CD8(+) T cells and the TNF receptors (TNFR) TNFRsf1a and TNFRsf1b. To distinguish roles of parenchymal and hematopoietic cell TNF signaling in PcP-related lung injury, murine bone marrow transplant chimeras of wild-type, C57BL6/J, and TNFRsf1a/1b double-null origin were generated, CD4(+) T cell depleted, and inoculated with Pc. As expected, C57 --> C57 chimeras (donor marrow --> recipient) developed significant disease as assessed by weight loss, impaired pulmonary function (lung resistance and dynamic lung compliance), and inflammatory cell infiltration. In contrast, TNFRsf1a/1b(-/-) --> TNFRsf1a/1b(-/-) mice were relatively mildly affected despite carrying the greatest organism burden. Mice solely lacking parenchymal TNFRs (C57 --> TNFRsf1a/1b(-/-)) had milder disease than did C57 --> C57 mice. Both groups of mice with TNFR-deficient parenchymal cells had low bronchoalveolar lavage fluid total cell counts and fewer lavageable CD8(+) T cells than did C57 --> C57 mice, suggesting that parenchymal TNFR signaling contributes to PcP-related immunopathology through the recruitment of damaging immune cells. Interestingly, mice with wild-type parenchymal cells but TNFRsf1a/1b(-/-) hematopoietic cells (TNFRsf1a/1b(-/-) --> C57) displayed exacerbated disease characterized by increased MCP-1 and KC production in the lung and increased macrophage and lymphocyte numbers in the lavage, indicating a dysregulated immune response. This study supports a key role of parenchymal cell TNFRs in lung injury induced by Pc and a potential protective effect of receptors on radiosensitive, bone marrow-derived cells.

Alveolar Airspace Size in Healthy and Diseased Infant Lungs Measured via Hyperpolarized 3He Gas Diffusion Magnetic Resonance Imaging.

BACKGROUND: Alveolar development and lung parenchymal simplification are not well characterized in vivo in neonatal patients with respiratory morbidities, such as bronchopulmonary dysplasia (BPD). Hyperpolarized (HP) gas diffusion magnetic resonance imaging (MRI) is a sensitive, safe, nonionizing, and noninvasive biomarker for measuring airspace size in vivo but has not yet been implemented in young infants. OBJECTIVE: This work quantified alveolar airspace size via HP gas diffusion MRI in healthy and diseased explanted infant lung specimens, with comparison to histological morphometry. METHODS: Lung specimens from 8 infants were obtained: 7 healthy left upper lobes (0-16 months, post-autopsy) and 1 left lung with filamin-A mutation, closely representing BPD lung disease (11 months, post-transplantation). Specimens were imaged using HP 3He diffusion MRI to generate apparent diffusion coefficients (ADCs) as biomarkers of alveolar airspace size, with comparison to mean linear intercept (Lm) via quantitative histology. RESULTS: Mean ADC and Lm were significantly increased throughout the diseased specimen (ADC = 0.26 ± 0.06 cm2/s, Lm = 587 ± 212 µm) compared with healthy specimens (ADC = 0.14 ± 0.03 cm2/s, Lm = 133 ± 37 µm; p < 1 × 10-7); increased values reflect enlarged airspaces. Mean ADCs in healthy specimens were significantly correlated to Lm (r = 0.69, p = 0.041). CONCLUSIONS: HP gas diffusion MRI is sensitive to healthy and diseased regional alveolar airspace size in infant lungs, with good comparison to quantitative histology in ex vivo specimens. This work demonstrates the translational potential of gas MRI techniques for in vivo assessment of normal and abnormal alveolar development in neonates with pulmonary disease.

Flow-based sorting of neonatal lymphocyte populations for transcriptomics analysis.

RATIONALE: Emerging data suggest an important role for T lymphocytes in the pathogenesis of chronic lung disease in preterm infants. Comprehensive assessment of the lymphocyte transcriptome may identify biomarkers and mechanisms of disease. METHODS: Small volume peripheral blood samples were collected from premature infants enrolled with consent in the Prematurity and Respiratory Outcomes Program (PROP), at the time of discharge from the hospital. Blood samples were collected at two sites and shipped to a central laboratory for processing. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque gradient centrifugation and separated into individual lymphocyte cell types by fluorescence-activated cell sorting. Gating strategies were optimized to ensure reproducible recovery of highly purified lymphocyte populations over a multi-year recruitment period. RNA was isolated from sorted cells and characterized by high-throughput sequencing (RNASeq). RESULTS: Blood volumes averaged 2.5ml, and sufficient PBMCs were collected from 165 of the 246 samples obtained (67%) from the 277 recruited subjects to complete sorting and RNASeq analysis on the resulting sorted cells. The number of total lymphocytes per ml of blood in the neonatal subjects was approximately 4 million/ml. Total lymphocyte frequencies recovered following sort varied widely among subjects, as did the frequency of individual lymphocyte and NK cell sub-populations. RNA yield from sorted cells varied according to cell type, but RNA of sufficient quantity and quality was recovered to enable RNASeq. SUMMARY: Our results describe a validated procedure for the generation of genome-wide expression data from isolated lymphocyte sub-populations obtained from newborn blood.

Induction of chemokines by low-dose intratracheal silica is reduced in TNFR I (p55) null mice.

Previous studies suggest that tumor necrosis factor alpha (TNF-alpha) and the TNFRI (p55) and TNFRRII (p75) receptors mediate the pulmonary fibrotic response to silica. In order to further define the role of the TNFRI (p55) receptor in induction of profibrotic chemokines by low-dose silica/crystalline silica (50 micro g/50 micro l/mouse) or control diluent saline was instilled into the trachea of TNFRI gene ablated ((-/-)) and C57BL/6 (WT) control mice. Lung tissue was harvested and bronchoalveolar lavage (BAL) performed 24 h and 28 days following silica administration. Selected profibrotic chemokine mRNAs were quantified by ribonuclease protection assay, normalized to ribosomal protein L32 mRNA content and expressed relative to saline control treated lungs. Induction of MIP-1beta, MIP-1alpha, MIP-2, IP-10, and MCP-1 mRNAs was attenuated in the TNFRI(-/-) mice, in comparison to WT mice, particularly at 28 days after exposure. ELISA assays for MIP-1alpha and MIP-2 in homogenized lung tissue similarly demonstrated marked induction of both chemokines 24 h after silica treatment, which was persistent at 28 days in WT but not in TNFRI(-/-) mice. The percentage of BAL cells that was neutrophils was comparably increased in WT and RI(-/-) lungs at 24 h (49 +/- 12% vs. 46 +/- 10%) and 28 days (6.2 +/- 1.5% vs. 4.5 +/- 1%). The increase in total lavagable cells and BAL protein was also independent of strain. Histology revealed mild alveolitis without granuloma formation in both strains, slightly decreased in TNFRI(-/-). This study demonstrates an increase in pro-fibrotic chemokines in response to a single intratracheal exposure to crystalline silica that was sustained at 28 days after treatment in WT but not in TNFRI(-/-) mice. Silica dependent recruitment of neutrophils to the alveolar space and alveolar protein leak were, however, not altered by the absence of the TNF receptor.

Acute tumor necrosis factor-alpha-induced liver injury in the absence of tumor necrosis factor receptor-associated factor 1 gene expression.

Pulmonary and serum levels of tumor necrosis factor-alpha (TNF-alpha), are elevated in many lung diseases, causing local inflammation, fever, and multiorgan, including hepatic, dysfunction. Cellular responses to TNF-alpha are determined by recruitment of specific proteins to intracellular receptor signaling complexes. One of these proteins, TNF receptor-associated factor 1 (TRAF1), is highly regulated in pulmonary cells. To determine the effect of reduced pulmonary TRAF1 expression, TRAF1-null (-/-) and control, BALB/c (wild-type), mice were treated intratracheally, intraperitoneally, or intravenously, with TNF-alpha. Despite relatively mild lung injury, intratracheal TNF-alpha-treated TRAF1-/- mice exhibited marked liver injury with an approximate fivefold increase in serum liver enzyme levels as compared to wild-type mice. In addition, serum TNF-alpha levels were strikingly elevated in TRAF1-/- mice. Pretreatment with neutralizing anti-TNFRI antibody significantly reduced liver injury and serum TNF-alpha. Cells isolated by bronchoalveolar lavage from intratracheally treated TRAF1-/- mice produced more TNF-alpha than cells from treated wild-type mice, suggesting that lung cells contributed to elevated serum TNF-alpha. These studies suggest that TRAF1 provides negative feedback for TNF-alpha synthesis and limits TNFRI-mediated systemic effects of TNF-alpha originating in the lung.