Submucosal Gland Myoepithelial Cells Are Reserve Stem Cells That Can Regenerate Mouse Tracheal Epithelium

The mouse trachea is thought to contain two distinct stem cell compartments that contribute to airway repair-basal cells in the surface airway epithelium (SAE) and an unknown submucosal gland (SMG) cell type. Whether a lineage relationship exists between these two stem cell compartments remains unclear. Using lineage tracing of glandular myoepithelial cells (MECs), we demonstrate that MECs can give rise to seven cell types of the SAE and SMGs following severe airway injury. MECs progressively adopted a basal cell phenotype on the SAE and established lasting progenitors capable of further regeneration following reinjury. MECs activate Wnt-regulated transcription factors (Lef-1/TCF7) following injury and Lef-1 induction in cultured MECs promoted transition to a basal cell phenotype. Surprisingly, dose-dependent MEC conditional activation of Lef-1 in vivo promoted self-limited airway regeneration in the absence of injury. Thus, modulating the Lef-1 transcriptional program in MEC-derived progenitors may have regenerative medicine applications for lung diseases

Proteomic Analysis of Pure Human Airway Gland Mucus Reveals a Large Component of Protective Proteins

<div><p>Airway submucosal glands contribute to innate immunity and protect the lungs by secreting mucus, which is required for mucociliary clearance and which also contains antimicrobial, anti-inflammatory, anti-proteolytic and anti-oxidant proteins. We stimulated glands in tracheal trimmings from three lung donors and collected droplets of uncontaminated mucus as they formed at the gland orifices under an oil layer. We analyzed the mucus using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Analysis identified 5486 peptides and 441 proteins from across the 3 samples (269–319 proteins per subject). We focused on 269 proteins common to at least 2 0f 3 subjects, of which 102 (38%) had protective or innate immunity functions. While many of these have long been known to play such roles, for many others their cellular protective functions have only recently been appreciated in addition to their well-studied biologic functions (e.g. annexins, apolipoproteins, gelsolin, hemoglobin, histones, keratins, and lumican). A minority of the identified proteins are known to be secreted via conventional exocytosis, suggesting that glandular secretion occurs via multiple mechanisms. Two of the observed protective proteins, major vault protein and prohibitin, have not been observed in fluid from human epithelial cultures or in fluid from nasal or bronchoalveolar lavage. Further proteomic analysis of pure gland mucus may help clarify how healthy airways maintain a sterile environment.</p></div

Protein families with protective functions found in gland mucus.

<p>At least one member of these families of proteins with protective functions was found in 2–3 samples of human mucus, with (n) indicating the total number of family members found in human gland mucus. MW is shown as kDa. The full list of 102 protective proteins is found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116756#pone.0116756.s003" target="_blank">S3 Table</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116756#pone.0116756.s004" target="_blank">S4 Table</a>. Abbreviations: GBP, Galectin binding protein; DMBT-1, Deleted in malignant brain tumors 1 protein; HSC 71, Heat shock cognate 71 kDa protein; LE inhibitor, Leukocyte elastase inhibitor; PS-A2, pulmonary surfactant A2; 40S RP, 40S ribosomal proteins; and SA A-1, Serum amyloid A-1. Accession # is from the UniProt database <a href="http://www.uniprot.org/uniprot/" target="_blank">http://www.uniprot.org/uniprot/</a>.</p><p>Protein families with protective functions found in gland mucus.</p

Proteins identified in human tracheal submucosal gland mucus by LC-MS/MS.

<p><b>(A)</b> A modified Venn diagram of showing distribution of proteins among three healthy human (HN) donors. Across the 3 donors, 5486 peptides and 441 proteins were identified. Of these, 61% (269 proteins, the shaded area) and 35% (154 proteins) were found in 2–3 and all 3 HN gland mucus samples, respectively. <b>(B)</b> Among these 269 proteins, 102 (38%) were categorized as proteins with “anti”-activities: antimicrobial, antiinflammatory, antiproteolytic and antioxidant. Other biological activities include: biological regulation, metabolic/multi-organismal/developmental processes, binding activity, and establishment of localization.</p

A comparative analysis of anti-proteins of 4 airway secretions.

<p>The data represent a summary of our comparative analysis of 35 common protective HN anti-proteins in the present study to 3 other airway fluids. Abbreviations: SMG, airway submucosal gland mucus; BALF, bronchial alveolar lavage fluid; HTBE, apical fluid of primary human tracheobronchial epithelial cell culture; and NLF, nasal lavage fluid. The data from other airway fluids are referenced in the text.</p><p>A comparative analysis of anti-proteins of 4 airway secretions.</p

Detargeting Lentiviral-Mediated CFTR Expression in Airway Basal Cells Using miR-106b

Lentiviral-mediated integration of a CFTR transgene cassette into airway basal cells is a strategy being considered for cystic fibrosis (CF) cell-based therapies. However, CFTR expression is highly regulated in differentiated airway cell types and a subset of intermediate basal cells destined to differentiate. Since basal stem cells typically do not express CFTR, suppressing the CFTR expression from the lentiviral vector in airway basal cells may be beneficial for maintaining their proliferative capacity and multipotency. We identified miR-106b as highly expressed in proliferating airway basal cells and extinguished in differentiated columnar cells. Herein, we developed lentiviral vectors with the miR-106b-target sequence (miRT) to both study miR-106b regulation during basal cell differentiation and detarget CFTR expression in basal cells. Given that miR-106b is expressed in the 293T cells used for viral production, obstacles of viral genome integrity and titers were overcome by creating a 293T-B2 cell line that inducibly expresses the RNAi suppressor B2 protein from flock house virus. While miR-106b vectors effectively detargeted reporter gene expression in proliferating basal cells and following differentiation in the air&ndash;liquid interface and organoid cultures, the CFTR-miRT vector produced significantly less CFTR-mediated current than the non-miR-targeted CFTR vector following transduction and differentiation of CF basal cells. These findings suggest that miR-106b is expressed in certain airway cell types that contribute to the majority of CFTR anion transport in airway epithelium