Connective tissue disease-associated lung disease in children.

Connective tissue diseases are a heterogeneous group of autoimmune diseases that can affect a variety of organ systems. Lung parenchymal involvement is an important contributor to morbidity and mortality in children with connective tissue disease. Connective tissue disease-associated lung disease in children often manifests as one of several radiologic-pathologic patterns of disease, with certain patterns having a propensity to occur in association with certain connective tissue diseases. In this article, key clinical, histopathologic, and computed tomography (CT) features of typical patterns of connective tissue disease-associated lung disease in children are reviewed, with an emphasis on radiologic-pathologic correlation, to improve recognition of these patterns of lung disease at CT and to empower the pediatric radiologist to more fully contribute to the care of pediatric patients with these conditions.

Flexible bronchoscopy in pediatric lung transplantation.

Pediatric lung transplantation represents a treatment option for children with advanced lung disease or pulmonary vascular disorders who are deemed an appropriate candidate. Pediatric flexible bronchoscopy is an important and evolving field that is highly relevant in the pediatric lung transplant population. It is thus important to advance our knowledge to better understand how care for children after lung transplant can be maximally optimized using pediatric bronchoscopy. Our goals are to continually improve procedural skills when performing bronchoscopy and to decrease the complication rate while acquiring adequate samples for diagnostic evaluation. Attainment of these goals is critical since allograft assessment by bronchoscopic biopsy is required for histological diagnosis of acute cellular rejection and is an important contributor to establishing chronic lung allograft dysfunction, a common complication after lung transplant. Flexible bronchoscopy with bronchoalveolar lavage and transbronchial lung biopsy plays a key role in lung transplant graft assessment. In this article, we discuss the application of bronchoscopy in pediatric lung transplant evaluation including historical approaches, our experience, and future directions not only in bronchoscopy but also in the evolving pediatric lung transplantation field. Pediatric flexible bronchoscopy has become a vital modality for diagnosing lung transplant complications in children as well as assessing therapeutic responses. Herein, we review the value of flexible bronchoscopy in the management of children after lung transplant and discuss the application of novel techniques to improve care for this complex pediatric patient population and we provide a brief update about new diagnostic techniques applied in the growing lung transplantation field.

A toxicology study of Csf2ra complementation and pulmonary macrophage transplantation therapy of hereditary PAP in mice.

Pulmonary macrophage transplantation (PMT) is a gene and cell transplantation approach in development as therapy for hereditary pulmonary alveolar proteinosis (hPAP), a surfactant accumulation disorder caused by mutations in CSF2RA/B (and murine homologs). We conducted a toxicology study of PMT of Csf2ra gene-corrected macrophages (mGM-Rα+Mϕs) or saline-control intervention in Csf2raKO or wild-type (WT) mice including single ascending dose and repeat ascending dose studies evaluating safety, tolerability, pharmacokinetics, and pharmacodynamics. Lentiviral-mediated Csf2ra cDNA transfer restored GM-CSF signaling in mGM-Rα+Mϕs. Following PMT, mGM-Rα+Mϕs engrafted, remained within the lungs, and did not undergo uncontrolled proliferation or result in bronchospasm, pulmonary function abnormalities, pulmonary or systemic inflammation, anti-transgene product antibodies, or pulmonary fibrosis. Aggressive male fighting caused a similarly low rate of serious adverse events in saline- and PMT-treated mice. Transient, minor pulmonary neutrophilia and exacerbation of pre-existing hPAP-related lymphocytosis were observed 14 days after PMT of the safety margin dose but not the target dose (5,000,000 or 500,000 mGM-Rα+Mϕs, respectively) and only in Csf2raKO mice but not in WT mice. PMT reduced lung disease severity in Csf2raKO mice. Results indicate PMT of mGM-Rα+Mϕs was safe, well tolerated, and therapeutically efficacious in Csf2raKO mice, and established a no adverse effect level and 10-fold safety margin.

Antibody-mediated rejection (AMR) in pediatric lung transplantation-Current state and future directions.

Lung transplantation is considered as the ultimate therapy for children with advanced pulmonary disease. International data show a median conditional 1-year post-transplantation survival of 9.1 years. Recently, antibody-mediated rejection (AMR) has increasingly been recognized as an important cause of allograft dysfunction although pediatric reports are still scarce. Donor-specific anti-human leukocyte antigen (HLA) antibodies (DSA) are known to play a role in AMR development post-transplant but AMR pathogenesis is still poorly understood. Central to the concept of pulmonary AMR is immune activation with the production of allo-specific B-cells and plasma cells directed against donor lung antigens. The frequency of pulmonary AMR in children is currently unknown. Due to the lack of AMR data in children, the diagnostic approach for pediatric pulmonary AMR is solely based on adult literature. This personal viewpoint article evaluates the rational for the creation of age-based thresholds for different diagnostic categories of pulmonary AMR and data on the management of pulmonary AMR in children. To the authors' knowledge, there have been no randomized controlled trials comparing different management regimes in pulmonary AMR, and thus, management and treatment algorithms for pulmonary AMR in children are only extrapolated from adults. To advance the knowledge of AMR in children, the authors propose that children be included in collaborative, multi-center trials. It is vital that future decisions on internationally agreed upon guidelines for pulmonary AMR take its impact on children into consideration. Research is needed to fill the current knowledge gaps in the field of pulmonary AMR in children focused on optimizing outcomes.

Single Cell Multiomics Identifies Cells and Genetic Networks Underlying Alveolar Capillary Dysplasia.

Rationale: Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal developmental disorder of lung morphogenesis caused by insufficiency of FOXF1 (forkhead box F1) transcription factor function. The cellular and transcriptional mechanisms by which FOXF1 deficiency disrupts human lung formation are unknown. Objectives: To identify cell types, gene networks, and cell-cell interactions underlying the pathogenesis of ACDMPV. Methods: We used single-nucleus RNA and assay for transposase-accessible chromatin sequencing, immunofluorescence confocal microscopy, and RNA in situ hybridization to identify cell types and molecular networks influenced by FOXF1 in ACDMPV lungs. Measurements and Main Results: Pathogenic single-nucleotide variants and copy-number variant deletions involving the FOXF1 gene locus in all subjects with ACDMPV (n = 6) were accompanied by marked changes in lung structure, including deficient alveolar development and a paucity of pulmonary microvasculature. Single-nucleus RNA and assay for transposase-accessible chromatin sequencing identified alterations in cell number and gene expression in endothelial cells (ECs), pericytes, fibroblasts, and epithelial cells in ACDMPV lungs. Distinct cell-autonomous roles for FOXF1 in capillary ECs and pericytes were identified. Pathogenic variants involving the FOXF1 gene locus disrupt gene expression in EC progenitors, inhibiting the differentiation or survival of capillary 2 ECs and cell-cell interactions necessary for both pulmonary vasculogenesis and alveolar type 1 cell differentiation. Loss of the pulmonary microvasculature was associated with increased VEGFA (vascular endothelial growth factor A) signaling and marked expansion of systemic bronchial ECs expressing COL15A1 (collagen type XV α 1 chain). Conclusions: Distinct FOXF1 gene regulatory networks were identified in subsets of pulmonary endothelial and fibroblast progenitors, providing both cellular and molecular targets for the development of therapies for ACDMPV and other diffuse lung diseases of infancy.

Guided construction of single cell reference for human and mouse lung.

Accurate cell type identification is a key and rate-limiting step in single-cell data analysis. Single-cell references with comprehensive cell types, reproducible and functionally validated cell identities, and common nomenclatures are much needed by the research community for automated cell type annotation, data integration, and data sharing. Here, we develop a computational pipeline utilizing the LungMAP CellCards as a dictionary to consolidate single-cell transcriptomic datasets of 104 human lungs and 17 mouse lung samples to construct LungMAP single-cell reference (CellRef) for both normal human and mouse lungs. CellRefs define 48 human and 40 mouse lung cell types catalogued from diverse anatomic locations and developmental time points. We demonstrate the accuracy and stability of LungMAP CellRefs and their utility for automated cell type annotation of both normal and diseased lungs using multiple independent methods and testing data. We develop user-friendly web interfaces for easy access and maximal utilization of the LungMAP CellRefs.

Single-cell multiomic analysis identifies a HOX-PBX gene network regulating the survival of lymphangioleiomyomatosis cells.

Lymphangioleiomyomatosis (LAM) is a rare, progressive lung disease that predominantly affects women. LAM cells carry TSC1/TSC2 mutations, causing mTORC1 hyperactivation and uncontrolled cell growth. mTORC1 inhibitors stabilize lung function; however, sustained efficacy requires long-term administration, and some patients fail to tolerate or respond to therapy. Although the genetic basis of LAM is known, mechanisms underlying LAM pathogenesis remain elusive. We integrated single-cell RNA sequencing and single-nuclei ATAC-seq of LAM lungs to construct a gene regulatory network controlling the transcriptional program of LAM cells. We identified activation of uterine-specific HOX-PBX transcriptional programs in pulmonary LAMCORE cells as regulators of cell survival depending upon HOXD11-PBX1 dimerization. Accordingly, blockage of HOXD11-PBX1 dimerization by HXR9 suppressed LAM cell survival in vitro and in vivo. PBX1 regulated STAT1/3, increased the expression of antiapoptotic genes, and promoted LAM cell survival in vitro. The HOX-PBX gene network provides promising targets for treatment of LAM/TSC mTORC1-hyperactive cancers.

Single cell transcriptomic analysis of HPV16-infected epithelium identifies a keratinocyte subpopulation implicated in cancer.

Persistent HPV16 infection is a major cause of the global cancer burden. The viral life cycle is dependent on the differentiation program of stratified squamous epithelium, but the landscape of keratinocyte subpopulations which support distinct phases of the viral life cycle has yet to be elucidated. Here, single cell RNA sequencing of HPV16 infected compared to uninfected organoids identifies twelve distinct keratinocyte populations, with a subset mapped to reconstruct their respective 3D geography in stratified squamous epithelium. Instead of conventional terminally differentiated cells, an HPV-reprogrammed keratinocyte subpopulation (HIDDEN cells) forms the surface compartment and requires overexpression of the ELF3/ESE-1 transcription factor. HIDDEN cells are detected throughout stages of human carcinogenesis including primary human cervical intraepithelial neoplasias and HPV positive head and neck cancers, and a possible role in promoting viral carcinogenesis is supported by TCGA analyses. Single cell transcriptome information on HPV-infected versus uninfected epithelium will enable broader studies of the role of individual keratinocyte subpopulations in tumor virus infection and cancer evolution.

Insights into pulmonary phosphate homeostasis and osteoclastogenesis emerge from the study of pulmonary alveolar microlithiasis.

Pulmonary alveolar microlithiasis is an autosomal recessive lung disease caused by a deficiency in the pulmonary epithelial Npt2b sodium-phosphate co-transporter that results in accumulation of phosphate and formation of hydroxyapatite microliths in the alveolar space. The single cell transcriptomic analysis of a pulmonary alveolar microlithiasis lung explant showing a robust osteoclast gene signature in alveolar monocytes and the finding that calcium phosphate microliths contain a rich protein and lipid matrix that includes bone resorbing osteoclast enzymes and other proteins suggested a role for osteoclast-like cells in the host response to microliths. While investigating the mechanisms of microlith clearance, we found that Npt2b modulates pulmonary phosphate homeostasis through effects on alternative phosphate transporter activity and alveolar osteoprotegerin, and that microliths induce osteoclast formation and activation in a receptor activator of nuclear factor-κB ligand and dietary phosphate dependent manner. This work reveals that Npt2b and pulmonary osteoclast-like cells play key roles in pulmonary homeostasis and suggest potential new therapeutic targets for the treatment of lung disease.

Upregulation of acid ceramidase contributes to tumor progression in tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is characterized by multisystem, low-grade neoplasia involving the lung, kidneys, brain, and heart. Lymphangioleiomyomatosis (LAM) is a progressive pulmonary disease affecting almost exclusively women. TSC and LAM are both caused by mutations in TSC1 and TSC2 that result in mTORC1 hyperactivation. Here, we report that single-cell RNA sequencing of LAM lungs identified activation of genes in the sphingolipid biosynthesis pathway. Accordingly, the expression of acid ceramidase (ASAH1) and dihydroceramide desaturase (DEGS1), key enzymes controlling sphingolipid and ceramide metabolism, was significantly increased in TSC2-null cells. TSC2 negatively regulated the biosynthesis of tumorigenic sphingolipids, and suppression of ASAH1 by shRNA or the inhibitor ARN14976 (17a) resulted in markedly decreased TSC2-null cell viability. In vivo, 17a significantly decreased the growth of TSC2-null cell-derived mouse xenografts and short-term lung colonization by TSC2-null cells. Combined rapamycin and 17a treatment synergistically inhibited renal cystadenoma growth in Tsc2+/- mice, consistent with increased ASAH1 expression and activity being rapamycin insensitive. Collectively, the present study identifies rapamycin-insensitive ASAH1 upregulation in TSC2-null cells and tumors and provides evidence that targeting aberrant sphingolipid biosynthesis pathways has potential therapeutic value in mechanistic target of rapamycin complex 1-hyperactive neoplasms, including TSC and LAM.

Pulmonary osteoclast-like cells in silica induced pulmonary fibrosis.

The pathophysiology of silicosis is poorly understood, limiting development of therapies for those who have been exposed to the respirable particle. We explored the mechanisms of silica-induced pulmonary fibrosis in a mouse model using multiple modalities including whole-lung single-nucleus RNA sequencing. These analyses revealed that in addition to pulmonary inflammation and fibrosis, intratracheal silica challenge induced osteoclast-like differentiation of alveolar macrophages and recruited monocytes, driven by induction of the osteoclastogenic cytokine, receptor activator of nuclear factor-κB ligand (RANKL) in pulmonary lymphocytes and alveolar type II cells. Furthermore, anti-RANKL monoclonal antibody treatment suppressed silica-induced osteoclast-like differentiation in the lung and attenuated silica-induced pulmonary fibrosis. We conclude that silica induces osteoclast-like differentiation of distinct recruited and tissue resident monocyte populations, leading to progressive lung injury, likely due to sustained elaboration of bone resorbing proteases and hydrochloric acid. Interrupting osteoclast-like differentiation may therefore constitute a promising avenue for moderating lung damage in silicosis.

Marked Improvement in Soft Tissue and CNS Manifestations of Adult Langerhans Cell Histiocytosis on Targeted MEK Inhibitor Therapy.

Multiple trials have demonstrated the efficacy of therapies targeting the RAS/MAPK pathway in children with Langerhans cell histiocytosis (LCH), but less is known about the success of this strategy in adults or in LCH that is the result of mutations other than BRAF V600E. A 53-year-old woman who has never smoked presented to our clinic with multisystem, multifocal LCH that resulted from an uncommon BRAF N486_P490del mutation. Low dose, and even intermittent, MEK inhibitor (trametinib) therapy was associated with rapid improvement in almost all of her disease manifestations, including regression of masses in her groin and neck, reduction in seizure frequency and intensity, improvement in white matter lesions on MRI, diabetes insipidus, dyspnea, and cognitive and memory functions. We conclude that MEK inhibitor therapy was effective for BRAF mutation-associated adult multisystem LCH, including CNS manifestations, in this patient.

In vivo development of immune tissue in human intestinal organoids transplanted into humanized mice.

Human intestinal organoids (HIOs) derived from pluripotent stem cells provide a valuable model for investigating human intestinal organogenesis and physiology, but they lack the immune components required to fully recapitulate the complexity of human intestinal biology and diseases. To address this issue and to begin to decipher human intestinal-immune crosstalk during development, we generated HIOs containing immune cells by transplanting HIOs under the kidney capsule of mice with a humanized immune system. We found that human immune cells temporally migrate to the mucosa and form cellular aggregates that resemble human intestinal lymphoid follicles. Moreover, after microbial exposure, epithelial microfold cells are increased in number, leading to immune cell activation determined by the secretion of IgA antibodies in the HIO lumen. This in vivo HIO system with human immune cells provides a framework for future studies on infection- or allergen-driven intestinal diseases.

Personalized medicine approaches in cystic fibrosis related pancreatitis.

We report a rare case of a patient with cystic fibrosis suffering from debilitating abdominal pain due to chronic pancreatitis. This 13-year-old patient was evaluated for surgical intervention to relieve pain from chronic pancreatitis and to improve quality of life. The patient carried two mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene; the most common ΔF508 variant and a second variant, p.Glu1044Gly, which has not been previously described. The patient's condition did not improve despite medical management and multiple endoscopic interventions, and therefore total pancreatectomy with islet autotransplantation and a near-total duodenectomy was offered for definitive management. Patient-derived duodenal crypts were isolated and cultured from the resected duodenum, and duodenal organoids were generated to test CFTR function. Our studies demonstrate that this novel mutation (ΔF508/p.Glu1044Gly) caused severely impaired CFTR function in vitro. The Food and Drug Administration (FDA)-approved drug ivacaftor, a CFTR potentiator, was identified to robustly improve CFTR function in the context of this novel mutation. Herein, we describe a personalized medicine approach consisting of performing drug testing on individual patient derived organoids that has potential to guide management of patients with novel CFTR genetic mutations. Identified effective medical therapeutics using this approach may avoid irreversible surgical treatments such as total pancreatectomy with islet autotransplantation in the future.

Pulmonary lymphoproliferative disorders in children: a practical review.

Pulmonary lymphoproliferative disorders represent an uncommon spectrum of proliferation of lymphoid tissue in the lung parenchyma ranging from benign hyperplasia to malignancy. They tend to occur in certain clinical situations and have typical imaging features that together can be used by the radiologist to suggest these entities as part of the differential diagnosis. We review key clinical, histopathological and computed tomography features of pulmonary lymphoproliferative disorders in children including follicular bronchiolitis, lymphoid interstitial pneumonia, granulomatous-lymphocytic interstitial lung disease, lymphoma and post-transplant lymphoproliferative disorder to familiarize the pediatric radiologist with this group of disorders.

A census of the lung: CellCards from LungMAP.

The human lung plays vital roles in respiration, host defense, and basic physiology. Recent technological advancements such as single-cell RNA sequencing and genetic lineage tracing have revealed novel cell types and enriched functional properties of existing cell types in lung. The time has come to take a new census. Initiated by members of the NHLBI-funded LungMAP Consortium and aided by experts in the lung biology community, we synthesized current data into a comprehensive and practical cellular census of the lung. Identities of cell types in the normal lung are captured in individual cell cards with delineation of function, markers, developmental lineages, heterogeneity, regenerative potential, disease links, and key experimental tools. This publication will serve as the starting point of a live, up-to-date guide for lung research at https://www.lungmap.net/cell-cards/. We hope that Lung CellCards will promote the community-wide effort to establish, maintain, and restore respiratory health.

Directed differentiation of human pluripotent stem cells into epidermal stem and progenitor cells.

BACKGROUND: Pluripotent stem cells (PSCs) produced by somatic cell reprogramming self-renew in culture and can differentiate into any cell type, representing a powerful tool for disease modeling, drug screening, regenerative medicine, and the discovery of personalized therapies to treat tissue-specific pathologies. We previously reported the directed differentiation of human PSCs into epidermal stem and progenitor cells (ESPCs) and 3D epidermis to model the inherited syndrome Fanconi anemia (FA), wherein epidermal cell-junctional defects discovered using this system were validated in patient populations. Here, we describe in detail the corresponding protocol for generating PSC-derived keratinocytes using a distinct, normal PSC line (209.2 PSC). METHODS AND RESULTS: Our approach modifies previous protocols to minimize spontaneous cell death and terminal differentiation, eliminate cell stress-inducing keratinocyte selection steps, and reduce total protocol duration and cost. Independent donor-derived PSC lines were converted into ESPCs through the addition of relevant morphogens and a ROCK inhibitor. Results for the 209.2 PSC line highlight consistencies in 2D and also variable features in 3D epidermis compared to the previously published FA-PSC lines. 209.2 PSC-derived ESPCs exhibited a basal cell phenotype while maintaining the capacity to form epidermal organotypic rafts with morphology consistent with fetal epidermis. Transcriptional analyses demonstrated 209.2 ESPCs express epidermis-selective markers and not early endoderm markers, thus supporting an immature stage of p63+ epidermal development. CONCLUSIONS: This protocol provides an accelerated path for the generation of human ESPCs and 3D epidermal models to study normal epidermal development and homeostasis, elucidate mechanisms of epidermal disease pathogenesis, and provides a platform for developing personalized therapies.

Binary pan-cancer classes with distinct vulnerabilities defined by pro- or anti-cancer YAP/TEAD activity.

Cancer heterogeneity impacts therapeutic response, driving efforts to discover over-arching rules that supersede variability. Here, we define pan-cancer binary classes based on distinct expression of YAP and YAP-responsive adhesion regulators. Combining informatics with in vivo and in vitro gain- and loss-of-function studies across multiple murine and human tumor types, we show that opposite pro- or anti-cancer YAP activity functionally defines binary YAPon or YAPoff cancer classes that express or silence YAP, respectively. YAPoff solid cancers are neural/neuroendocrine and frequently RB1-/-, such as retinoblastoma, small cell lung cancer, and neuroendocrine prostate cancer. YAP silencing is intrinsic to the cell of origin, or acquired with lineage switching and drug resistance. The binary cancer groups exhibit distinct YAP-dependent adhesive behavior and pharmaceutical vulnerabilities, underscoring clinical relevance. Mechanistically, distinct YAP/TEAD enhancers in YAPoff or YAPon cancers deploy anti-cancer integrin or pro-cancer proliferative programs, respectively. YAP is thus pivotal across cancer, but in opposite ways, with therapeutic implications.