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Lower airway club cells (CCs) serve the dual roles of a secretory cell and a stem cell. Here, we probe how the CC fate is regulated. We find that, in response to acute perturbation of Notch signaling, CCs adopt distinct fates. Although the vast majority transdifferentiate into multiciliated cells, a "variant" subpopulation (v-CCs), juxtaposed to neuroepithelial bodies (NEBs; 5%-10%) and located at bronchioalveolar duct junctions (>80%), does not. Instead, v-CCs transition into lineage-ambiguous states but can revert to a CC fate upon restoration of Notch signaling and repopulate the airways with CCs and multiciliated cells. The v-CC response to Notch inhibition is dependent on localized activation of β-catenin in v-CCs. We propose that the CC fate is stabilized by canonical Notch signaling, that airways are susceptible to perturbations to this pathway, and that NEBs/terminal bronchioles comprise niches that modulate CC plasticity via β-catenin activation to facilitate airway repair post Notch inhibition., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Club cells are found in human small airways where they play an important role in immune defense, xenobiotic metabolism, and repair after injury. Over the past few years, data from single-cell RNA sequencing (scRNA-seq) studies has generated new insights into club cell heterogeneity and function. In this review, we integrate findings from scRNA-seq experiments with earlier in vitro, in vivo, and microscopy studies and highlight the many ways club cells contribute to airway homeostasis. We then discuss evidence for loss of club cells or club cell products in the airways of patients with chronic obstructive pulmonary disease (COPD) and discuss potential mechanisms through which this might occur.

Lung infections are a perennial leading cause of death worldwide. The lung epithelium comprises three main cell types: alveolar type I (AT1), alveolar type II (AT2), and bronchiolar cells. Constitutively, these three cell types express extremely low amounts of surface MHC class I (MHC I) molecules, that is, <1% of levels found on medullary thymic epithelial cells (ECs). We report that inhalation of the TLR4 ligand LPS upregulates cell surface MHC I by ∼25-fold on the three subtypes of mouse lung ECs. This upregulation is dependent on Nlrc5 , Stat1 , and Stat2 and caused by a concerted production of the three IFN families. It is nevertheless hampered, particularly in AT1 cells, by the limited expression of genes instrumental in the peptide loading of MHC I molecules. Genes involved in production and response to cytokines and chemokines were selectively induced in AT1 cells. However, discrete gene subsets were selectively downregulated in AT2 or bronchiolar cells following LPS inhalation. Genes downregulated in AT2 cells were linked to cell differentiation and cell proliferation, and those repressed in bronchiolar cells were primarily involved in cilium function. Our study shows a delicate balance between the expression of transcripts maintaining lung epithelium integrity and transcripts involved in Ag presentation in primary lung ECs., (Copyright © 2022 by The American Association of Immunologists, Inc.)

HCO 3 - secretion in distal airways is critical for airway mucosal defense. HCO 3 - /H + transport across the apical membrane of airway surface epithelial cells was studied by measuring intracellular pH in luminally microperfused freshly dissected mice bronchioles. Functional studies demonstrated that CFTR, ENaC, Cl - -HCO 3 - exchange, Na + -H + exchange, and Na + -HCO 3 - cotransport are involved in apical HCO 3 - /H + transport. RT-PCR of isolated bronchioles detected fragments from Cftr, α, β, γ subunits of ENaC, Ae2, Ae3, NBCe1, NBCe2, NBCn1, NDCBE, NBCn2, Nhe1, Nhe2, Nhe4, Nhe5, Slc26a4, Slc26a6, and Slc26a9. We assume that continuous decline of intracellular pH following alkaline load demonstrates time course of HCO 3 - secretion into the lumen which is perfused with a HCO 3 - -free solution. Forskolin-stimulated HCO 3 - secretion was substantially inhibited by luminal application of CFTR inh -172 (5 μM), H 2 DIDS (200 μM), and amiloride (1 μM). In bronchioles from a cystic fibrosis mouse model, basal and acetylcholine-stimulated HCO 3 - secretion was substantially impaired, but forskolin transiently accelerated HCO 3 - secretion of which the magnitude was comparable to wild-type bronchioles. In conclusion, we have characterized apical HCO 3 - /H + transport in native bronchioles. We have demonstrated that cAMP-mediated and Ca 2+ -mediated pathways are involved in HCO 3 - secretion and that apical HCO 3 - secretion is largely mediated by CFTR and H 2 DIDS-sensitive Cl - -HCO 3 - exchanger, most likely Slc26a9. The impairment of HCO 3 - secretion in bronchioles from a cystic fibrosis mouse model may be related to the pathogenesis of early lung disease in cystic fibrosis., (© 2022. The Author(s).)

Repetitive aeroallergen exposure is linked to sensitization and airway remodeling through incompletely understood mechanisms. In this study, we examine the dynamic mucosal response to cat dander extract (CDE), a ubiquitous aero-allergen linked to remodeling, sensitization and asthma. We find that daily exposure of CDE in naïve C57BL/6 mice activates innate neutrophilic inflammation followed by transition to a lymphocytic response associated with waves of mucosal transforming growth factor (TGF) isoform expression. In parallel, enhanced bronchiolar Smad3 expression and accumulation of phospho-SMAD3 was observed, indicating paracrine activation of canonical TGFβR signaling. CDE exposure similarly triggered epithelial cell plasticity, associated with expression of mesenchymal regulatory factors (Snai1 and Zeb1), reduction of epithelial markers (Cdh1) and activation of the NFκB/RelA transcriptional activator. To determine whether NFκB functionally mediates CDE-induced growth factor response, mice were stimulated with CDE in the absence or presence of a selective IKK inhibitor. IKK inhibition substantially reduced the level of CDE-induced TGFβ1 expression, pSMAD3 accumulation, Snai1 and Zeb1 expression. Activation of epithelial plasticity was demonstrated by flow cytometry in whole lung homogenates, where CDE induces accumulation of SMA + Epcam + population. Club cells are important sources of cytokine and growth factor production. To determine whether Club cell innate signaling through NFκB/RelA mediated CDE induced TGFβ signaling, we depleted RelA in Secretoglobin (Scgb1a1)-expressing bronchiolar cells. Immunofluorescence-optical clearing light sheet microscopy showed a punctate distribution of Scgb1a1 progenitors throughout the small airway. We found that RelA depletion in Secretoglobin + cells results in inhibition of the mucosal TGFβ response, blockade of EMT and reduced subepithelial myofibroblast expansion. We conclude that the Secretoglobin-derived bronchiolar cell is central to coordinating the innate response required for mucosal TGFβ1 response, EMT and myofibroblast expansion. These data have important mechanistic implications for how aero-allergens trigger mucosal injury response and remodeling in the small airway., (© 2021. The Author(s).)

Previous work indicates that SARS-CoV-2 virus entry proteins angiotensin-converting enzyme 2 (ACE-2) and the cell surface transmembrane protease serine 2 (TMPRSS-2) are regulated by sex hormones. However, clinical studies addressing this association have yielded conflicting results. We sought to analyze the impact of sex hormones, age, and cardiovascular disease on ACE-2 and TMPRSS-2 expression in different mouse models. ACE-2 and TMPRSS-2 expression was analyzed by immunostaining in a variety of tissues obtained from FVB/N mice undergoing either gonadectomy or sham-surgery and being subjected to ischemia-reperfusion injury or transverse aortic constriction surgery. In lung tissues sex did not have a significant impact on the expression of ACE-2 and TMPRSS-2. On the contrary, following myocardial injury, female sex was associated to a lower expression of ACE-2 at the level of the kidney tubules. In addition, after myocardial injury, a significant correlation between younger age and higher expression of both ACE-2 and TMPRSS-2 was observed for lung alveoli and bronchioli, kidney tubules, and liver sinusoids. Our experimental data indicate that gonadal hormones and biological sex do not alter ACE-2 and TMPRSS-2 expression in the respiratory tract in mice, independent of disease state. Thus, sex differences in ACE-2 and TMPRSS-2 protein expression observed in mice may not explain the higher disease burden of COVID-19 among men., (© 2021. The Author(s).)

Idiopathic pulmonary fibrosis (IPF) causes progressive fibrosis and worsening pulmonary function. Prognosis is poor and no effective therapies exist. We show that programmed cell death 5 (PDCD5) expression is increased in the lungs of patients with IPF and in mouse models of lung fibrosis. Lung fibrosis is significantly diminished by club cell-specific deletion of Pdcd5 gene. PDCD5 mediates β-catenin/Smad3 complex formation, promoting TGF-β-induced transcriptional activation of matricellular genes. Club cell Pdcd5 knockdown reduces matricellular protein secretion, inhibiting fibroblast proliferation and collagen synthesis. Here, we demonstrate the club cell-specific role of PDCD5 as a mediator of lung fibrosis and potential therapeutic target for IPF.

Background: Immunosuppression therapy is ineffective at preventing bronchiolitis obliterans syndrome (BOS), primarily a disease of the small airways (SAs). Our previous reports show increased senescent CD28null T and natural killer T (NKT)-like cells in the peripheral blood of patients with BOS and increased cytotoxic, proinflammatory lymphocytes in the SAs. We hypothesized that the cytotoxic, proinflammatory lymphocytes in the SAs would be steroid-resistant senescent CD28null lymphocytes., Methods: Intracellular cytotoxic mediator granzyme B, interferon (IFN)-γ and tumor necrosis factor (TNF)-α proinflammatory cytokines, and CD28 were measured in the blood, bronchoalveolar lavage, large airway, and SA brushing T and NKT-like cells from 10 patients with BOS, 11 stable lung transplant recipients, and 10 healthy age-matched controls. SA brushings were cultured in the presence of ±1 µmol/liter prednisolone, ±5 mg/liter theophylline, and ±2.5 ng/ml cyclosporine A, and IFN-γ and TNF-α proinflammatory cytokines were assessed using flow cytometry., Results: Increased SA CD28null T and NKT-like cells were identified in patients with BOS compared with that in the controls and stable transplant recipients. Loss of CD28 was associated with increased T and NKT-like cells expressing granzyme B, IFN-γ, and TNF-α. Loss of CD28 expression by CD8+ T cells was significantly associated with forced expiratory volume in 1 sec (R = 0.655, p = 0.006) and with time after transplantation (R = -0.552, p = 0.041). Treatment with prednisolone + theophylline + cyclosporin A inhibited IFN-γ and TNF-α production by SA CD28null CD8+ T and NKT-like cells additively., Conclusions: BOS is associated with the loss of CD28 in SA cytotoxic, proinflammatory senescent T and NKT-like lymphocytes. Treatment options that target the proinflammatory nature of these cells in the SAs may improve graft survival., (Copyright © 2020 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

Idiopathic pulmonary fibrosis (IPF) is a fibrotic disease with the histology of usual interstitial pneumonia (UIP). Although the pathologist's visual inspection is central in histologic assessments, three-dimensional microcomputed tomography (microCT) assessment may complement the pathologist's scoring. We examined associations between the histopathologic features of UIP and IPF in explanted lungs and quantitative microCT measurements, including alveolar surface density, total lung volume taken up by tissue (%), and terminal bronchiolar number. Sixty frozen samples from 10 air-inflated explanted lungs with severe IPF and 36 samples from 6 donor control lungs were scanned with microCT and processed for histologic analysis. An experienced pathologist scored three major UIP criteria (patchy fibrosis, honeycomb, and fibroblastic foci), five additional pathologic changes, and immunohistochemical staining for CD68-, CD4-, CD8-, and CD79a-positive cells, graded on a 0 to 3+ scale. The alveolar surface density and terminal bronchiolar number decreased and the tissue percentage increased in lungs with IPF compared with controls. In lungs with IPF, lower alveolar surface density and higher tissue percentage were correlated with greater scores of patchy fibrosis, fibroblastic foci, honeycomb, CD79a-positive cells, and lymphoid follicles. A decreased number of terminal bronchioles was correlated with honeycomb score but not with the other scores. The three-dimensional microCT measurements reflect the pathological UIP and IPF criteria and suggest that the reduction in the terminal bronchioles may be associated with honeycomb cyst formation., (Copyright © 2020 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Bronchiolitis Obliterans Syndrome seriously reduces long-term survival of lung transplanted patients. Up to now there is no effective therapy once BOS is established. Nanomedicine introduces the possibility to administer drugs locally into lungs increasing drug accumulation in alveola reducing side effects. Imatinib was loaded in gold nanoparticles (GNP) functionalized with antibody against CD44 (GNP-HCIm). Lung fibroblasts (LFs) were derived from bronchoalveolar lavage of BOS patients. GNP-HCIm cytotoxicity was evaluated by MTT assay, apoptosis/necrosis and phosphorylated-cAbl (cAbl-p). Heterotopic tracheal transplantation (HTT) mouse model was used to evaluate the effect of local GNP-HCIm administration by Alzet pump. GNP-HCIm decreased LFs viability compared to Imatinib (44.4 ± 1.8% vs. 91.8 ± 3.2%, p < 0.001), inducing higher apoptosis (22.68 ± 4.3% vs. 6.43 ± 0.29; p < 0.001) and necrosis (18.65 ± 5.19%; p < 0.01). GNP-HCIm reduced cAbl-p (0.41 GNP-HCIm, 0.24 Imatinib vs. to control; p < 0.001). GNP-HCIm in HTT mouse model by Alzet pump significantly reduced tracheal lumen obliteration (p < 0.05), decreasing apoptosis (p < 0.05) and TGF-β-positive signal (p < 0.05) in surrounding tissue. GNP-HCIm treatment significantly reduced lymphocytic and neutrophil infiltration and mast cells degranulation (p < 0.05). Encapsulation of Imatinib into targeted nanoparticles could be considered a new option to inhibit the onset of allograft rejection acting on BOS specific features.

High mobility group box 1 (HMGB1) is involved in the induction of airway inflammation and injury in patients with chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). HMGB1 increased by transforming growth factor-β1 (TGF-β1), impairs airway epithelial barrier function in the lung. In the present study, to investigate how HMGB1 affects the barrier of normal human lung epithelial (HLE) cells, monolayer cells (2D culture) and bronchial-like spheroid cells (2.5 D Matrigel culture), which have lumen formation, were pretreated with TGF-β type I receptor kinase inhibitor EW-7197 before treatment with HMGB1. In 2D culture, treatment with HMGB1 decreased expression of angulin-1/LSR, TRIC and CLDN-1, -4, -7 and increased that of CLDN-2. Pretreatment with EW-7197 prevented the changes of all tight junction molecules induced by HMGB1. In 2.5D Matrigel culture, treatment with HMGB1 induced permeability of FITC-dextran (FD-4) into the lumen, whereas pretreatment with EW-7197 prevented the hyperpermeability of FD-4 into the lumen caused by HMGB1. In 2.5D Matrigel culture, knockdown of transcription factor p63 prevented the hyperpermeability induced by HMGB1 as well as pretreatment with EW-7197. In the 2D culture of HLE cells with HMGB1, knockdown of p63 increased the level of angulin-1/LSR and CLDN-4, while pretreatment with EW-7197 enhanced the increase of CLDN-4 induced by knockdown of p63. Immunohistochemical analysis of IPF, CLDN-2, HMGB1 and p63 revealed that their levels were higher in the regenerative epithelium of the terminal bronchial region than in normal epithelium. HMGB1 induces epithelial permeability of HLE cells via p63/TGF-β signaling in normal lung and IPF.

Evidence points to an indispensable function of macrophages in tissue regeneration, yet the underlying molecular mechanisms remain elusive. Here we demonstrate a protective function for the IL-33-ST2 axis in bronchial epithelial repair, and implicate ST2 in myeloid cell differentiation. ST2 deficiency in mice leads to reduced lung myeloid cell infiltration, abnormal alternatively activated macrophage (AAM) function, and impaired epithelial repair post naphthalene-induced injury. Reconstitution of wild type (WT) AAMs to ST2-deficient mice completely restores bronchial re-epithelialization. Central to this mechanism is the direct effect of IL-33-ST2 signaling on monocyte/macrophage differentiation, self-renewal and repairing ability, as evidenced by the downregulation of key pathways regulating myeloid cell cycle, maturation and regenerative function of the epithelial niche in ST2 -/- mice. Thus, the IL-33-ST2 axis controls epithelial niche regeneration by activating a large multi-cellular circuit, including monocyte differentiation into competent repairing AAMs, as well as group-2 innate lymphoid cell (ILC2)-mediated AAM activation.

Epithelial dysfunction in the small airways may cause the development of emphysema in chronic obstructive pulmonary disease. C/EBPα (CCAAT/enhancer binding protein-α), a transcription factor, is required for lung maturation during development, and is also important for lung homeostasis after birth, including the maintenance of serine protease/antiprotease balance in the bronchiolar epithelium. This study aimed to show the roles of C/EBPα in the distal airway during chronic cigarette smoke exposure in mice and in the small airways in smokers. In a model of chronic smoke exposure using epithelial cell-specific C/EBPα-knockout mice, significant pathological phenotypes, such as higher protease activity, impaired ciliated cell regeneration, epithelial cell barrier dysfunction via reduced zonula occludens-1 (Zo-1), and decreased alveolar attachments, were found in C/EBPα-knockout mice compared with control mice. We found that Spink5 (serine protease inhibitor kazal-type 5) gene (encoding lymphoepithelial Kazal-type-related inhibitor [LEKTI], an anti-serine protease) expression in the small airways is a key regulator of protease activity in this model. Finally, we showed that daily antiprotease treatment counteracted the phenotypes of C/EBPα-knockout mice. In human studies, CEBPA (CCAAT/enhancer binding protein-α) gene expression in the lung was downregulated in patients with emphysema, and six smokers with centrilobular emphysema (CLE) showed a significant reduction in LEKTI in the small airways compared with 22 smokers without CLE. LEKTI downregulation in the small airways was associated with disease development during murine small airway injury and CLE in humans, suggesting that LEKTI might be a key factor linking small airway injury to the development of emphysema.

The Non-Ciliated Bronchiolar Cell (NCBC) is responsible for the defense and maintenance of the bronchiolar epithelium. Several cellular defense mechanisms have been associated with an increase in the secretion of CC16 and changes in the phenotype of the cell; these mechanisms could be linked to tolerance to the damage due to exposure to inhaled Particulate Matter (PM) of the epithelium. These defense mechanisms have not been sufficiently explored. In this article, we studied the response of the NCBC to inhaled vanadium, an element which adheres to PM. This response was measured by the changes in the phenotype of the NCBC and the secretion of CC16 in a mouse model. Mice were exposed in two phases to different vanadium concentrations; 1.27 mg/m³ in the first phase and 2.56 mg/m³ in the second phase. Mice were sacrificed on the 2nd, 4th, 5th, 6th and 8th weeks. In the second phase, we observed the following: sloughing of the NCBC, hyperplasia and small inflammatory foci remained without changes and that the expression of CC16 was higher in this phase than in phase I. We also observed a change in the phenotype with a slow decrease in both phases. The increase in the secretion of CC16 and the phenotype reversion could be due to the anti-inflammatory activity of CC16. The changes observed in the second phase could be attributed to the tolerance to inhaled vanadium.

Bronchiolar adenoma (BA) of the lung is a rare benign neoplasm. Because of a chest abnormal shadow indicated by health checkup, a 77-year-old female nonsmoker underwent computed tomography, revealing an 8 mm ground glass nodule in the peripheral field of the right lower lobe. Wedge resection of the nodule was performed, with a frozen diagnosis of primary lung adenocarcinoma. The localized, 8 × 4 × 3 mm-sized, jelly-like mass microscopically revealed a lepidic-growing lesion composed of ciliated columnar cells, mucous cells and basal cells surrounded by mucin pool. Neither nuclear atypia nor mitotic activity was noted. Immunohistochemically, the ciliated, mucous and basal cells were positive for TTF-1 and p16 INK4a . Mucous cells were positive for napsin A and focally expressed MUC5AC. MUC6 was negative. Basal cells were positive for CK5/6, p40, p63 and podoplanin. Human papillomavirus genome was undetectable by in situ hybridization. Ultrastructurally, the bronchiolar epithelial tubules consisted of two layers, the inner nonciliated microvillous cells and the outer basal-like cells, and some of the inner cells were filled with mucin granules in cytoplasm. Molecular analysis of the tumor failed to show driver mutations. The final diagnosis was distal-type BA. The postoperative course was uneventful for 6 months., (© 2020 The Authors. Pathology International published by Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

Among nanomaterials (NMs), titanium dioxide (TiO 2 ) is one of the most manufactured NMs and can be found in many consumers' products such as skin care products, textiles and food (as E171 additive). Moreover, due to its most attractive property, a photoactivation upon non-ionizing UVA radiation, TiO 2 NMs is widely used as a decontaminating agent. Uncontrolled contaminations by TiO 2 NMs during their production (professional exposure) or by using products (consumer exposure) are rather frequent. So far, TiO 2 NMs cytotoxicity is still a matter of controversy depending on biological models, types of TiO 2 NMs, suspension preparation and biological endpoints. TiO 2 NMs photoactivation has been widely described for UV light radiation exposure, it could lead to reactive oxygen species production, known to be both cyto- and genotoxic on human cells. After higher photon energy exposition, such as X-rays used for radiotherapy and for medical imaging, TiO 2 NMs photoactivation still occurs. Importantly, the question of its hazard in the case of body contamination of persons receiving radiotherapy was never addressed, knowing that healthy tissues surrounding the tumor are indeed exposed. The present work focuses on the analysis of human normal bronchiolar cell response after co-exposition TiO 2 NMs (with different coatings) and ionizing radiation. Our results show a clear synergistic effect, in terms of cell viability, cell death and oxidative stress, between TiO 2 NMS and radiation., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results

A normal counterpart and precancerous lesion that non-terminal respiratory unit (TRU) lung adenocarcinomas (LADCs) develop from have not been clarified. Non-TRU LADCs specifically express hepatocyte nuclear factor 4α (HNF4α). Thus, we have been interested in airway epithelial cells that express HNF4α as the potential precursor of non-TRU LADC. The purposes of the present study are to report the frequency and distribution of HNF4α-expressing cells at the different airway levels, and to investigate the potential significance of the expression of HNF4α in the histogenesis of non-TRU LADC with a special reference to the relationship to bronchiolar metaplasia in idiopathic interstitial pneumonia. We herein identified a minor subpopulation of epithelial cells that express HNF4α in a physiological state. This subpopulation was mainly located in the terminal bronchioles and had the appearance of ciliated cells, which were mutually exclusive from Clara cells and others that strongly expressed thyroid transcription factor 1. Furthermore, the expression of HNF4α was similar in bronchiolar metaplastic lesions and the terminal bronchioles, and some of the metaplastic lesions showed an unequivocally higher frequency and expression level of HNF4α, which was comparable to non-TRU LADC. In summary, this is the first study to describe a subpopulation of ciliated cells that express HNF4α as a potential normal counterpart for non-TRU LADCs and suggests that bronchiolar metaplastic lesions that strongly express HNF4α are a precancerous lesion for non-TRU LADCs.

Epithelial cells that line lung airways produce and secrete proteins with important roles in barrier function and host defense. Secretion of airway goblet cells is controlled by autophagy proteins during inflammatory conditions, resulting in accumulation of mucin proteins. We hypothesized that autophagy proteins would also be important in the function of club cells, dominant secretory airway epithelial cells that are dysregulated in chronic lung disease. We found that in the absence of an inflammatory stimulus, mice with club cells deficient for the autophagy protein Atg5 had a markedly diminished expression of secreted host defense proteins secretoglobulin family 1A, member 1 (Scgb1a1) and surfactant proteins A1 and D (Sftpa1 and Sftpd), as well as abnormal club cell morphology. Adult mice with targeted loss of Atg5 also showed diminished levels of host defense proteins in regenerating cells following ablation with naphthalene. A mouse strain with global deficiency of Atg16-like 1 (Atg16l1), an Atg5 binding partner, had a similar loss of host defense proteins and abnormal club cell morphology. Cigarette smoke exposure reduced levels of Scgb1a1 in wild-type mice as expected. Smoke exposure was not required to trigger club cell abnormalities in mice bearing the human ATG16 variant Atg16l1 T300A/T300A , which had low Scgb1a1 levels independent of this environmental stress. Evaluation of lung tissues from former smokers with severe chronic obstructive pulmonary disease showed evidence of reduced autophagy and SCGB1A1 expression in club cells. Thus, autophagy proteins are required for the function of club cells, independent of the cellular stress of cigarette smoke, with roles that appear to be distinct from those of other secretory cell types.

Although small airways account for the largest fraction of the total conducting airway surfaces, the epithelial fluid and electrolyte transport in small, native airway epithelia has not been well characterized. Investigations have been limited, no doubt, by the complex tissue architecture as well as by its inaccessibility, small dimensions, and lack of applicable assays, especially in human tissues. To better understand how the critically thin layer of airway surface liquid (ASL) is maintained, we applied a "capillary"-Ussing chamber (area ≈1 mm 2 ) to measure ion transport properties of bronchioles with diameters of ~2 mm isolated from resected specimens of excised human lungs. We found that the small human airway, constitutively and concurrently, secretes and absorbs fluid as observed in porcine small airways (50). We found that the human bronchiolar epithelium is also highly anion selective and constitutively secretes bicarbonate ( HCO 3 - ), which can be enhanced pharmacologically by cAMP as well as Ca 2+ -mediated agonists. Concurrent secretion and absorption of surface liquid along with HCO 3 - secretion help explain how the delicate volume of the fluid lining the human small airway is physiologically buffered and maintained in a steady state that avoids desiccating or flooding the small airway with ASL.

Current therapeutic interventions for the treatment of respiratory infections are hampered by the evolution of multidrug resistance in pathogens as well as the lack of effective cellular targets. Despite the identification of multiple region-specific lung progenitor cells, the identity of molecules that might be therapeutically targeted in response to infections to promote activation of progenitor cell types remains elusive. Here, we report that loss of Abl1 specifically in SCGB1A1-expressing cells leads to a significant increase in the proliferation and differentiation of bronchiolar epithelial cells, resulting in dramatic expansion of an SCGB1A1+ airway cell population that coexpresses SPC, a marker for type II alveolar cells that promotes alveolar regeneration following bacterial pneumonia. Furthermore, treatment with an Abl-specific allosteric inhibitor enhanced regeneration of the alveolar epithelium and promoted accelerated recovery of mice following pneumonia. These data reveal a potential actionable target that may be exploited for efficient recovery after pathogen-induced infections., Competing Interests: The authors declare no conflict of interest.

Recent comprehensive next-generation genome and transcriptome analyses in lung cancer patients, several clinical observations, and compelling evidence from mouse models of lung cancer have uncovered a critical role for Notch signaling in the initiation and progression of non-small-cell lung cancer (NSCLC). Notably, Rumi is a "protein O-glucosyltransferase" that regulates Notch signaling through O-glucosylation of Notch receptors, and is the only enzymatic regulator whose activity is required for both ligand-dependent and ligand-independent activation of Notch. We have conducted a detailed study on RUMI's involvement in NSCLC development and progression, and have further explored the therapeutic potential of its targeting in NSCLC. We have determined that Rumi is highly expressed in the alveolar and bronchiolar epithelia, including club cells and alveolar type II cells. Remarkably, RUMI maps to the region of chromosome 3q that corresponds to the major signature of neoplastic transformation in NSCLC, and is markedly amplified and overexpressed in NSCLC tumors. Notably, RUMI expression levels are predictive of poor prognosis and survival in NSCLC patients. Our data indicates that RUMI modulates Notch activity in NSCLC cells, and that its silencing dramatically decreases cell proliferation, migration, and survival. RUMI downregulation causes severe cell cycle S-phase arrest, increases genome instability, and induces late apoptotic-nonapoptotic cell death. Our studies demonstrate that RUMI is a novel negative prognostic factor with significant therapeutic potential in NSCLC, which embodies particular relevance especially when considering that, while current Notch inhibitory strategies target only ligand-dependent Notch activation, a large number of NSCLCs are driven by ligand-independent Notch activity., (© 2018 Wiley Periodicals, Inc.)

Introduction: Hyaluronan (HA) and the receptor for hyaluronan-mediated motility (RHAMM) may play an important role in lung development. We examined the expression of HA content and RHAMM during postnatal lung development by analyzing human lung specimens from newborn infants with a variety of lung diseases at different gestational (GA) and postnatal (PNA) ages., Materials and Methods: Ninety-four patients were evaluated. Immunohistochemical RHAMM expression was studied with digital image analysis, followed by hierarchical cluster analysis of both these data and clinical data to define subgroups. The air content of the lung was determined by computerized analysis. HA content was estimated by radiometric assay., Results: Cluster analysis defined six distinct patient groups (Group 1-2: 34-41 weeks GA; Group 3-5: 23-27 weeks GA; Group 6: mixed population). Group 1-5 showed individual patterns in RHAMM expression and HA content (Group 1: high RHAMM/low HA; Group 2: low RHAMM/low HA; Group 3: low RHAMM/low HA; Group 4: low RHAMM/high HA; Group 5: high RHAMM/high HA). HA content decreased with increasing PNA independently of GA. Negative correlation was observed between air content and RHAMM expression in the bronchiolar epithelium irrespective of clustered groups. Lung hypoplasia appeared in two distinctive groups, with significant differences in lung development and RHAMM expression., Conclusions: RHAMM expression may show dynamic changes during pathological processes in the neonatal lung. The distribution of RHAMM in the lung tissue is heterogeneous with a predominance to the bronchiolar epithelium. We found a negative correlation between lung air content and RHAMM expression in bronchiolar epithelium., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Aims: l-Alpha-lysophosphatidylinositol (LPI) is an endogenous agonist of G protein-coupled receptor 55 (GPR55) which relaxes mesenteric arteries on activation. The aim of the present study was to determine the influence and underlying mechanisms of LPI-induced relaxation in human pulmonary arteries (hPAs)., Main Methods: Functional studies were performed in isolated hPAs using organ bath technique. The expression of GPR55 in hPAs and bronchioles was determined by real-time qPCR, Western blot analysis, and immunohistochemistry., Key Findings: LPI induced a concentration-dependent vasorelaxation in endothelium-intact hPAs. This effect was attenuated by the GPR55 antagonist CID16020046, the peroxisome proliferator-activated receptor-γ (PPARγ) antagonist GW9662, the putative endothelial cannabinoid receptor (CB e ) antagonist O-1918 and the inhibitor of nitric oxide (NO) synthase (L-NAME). In addition, vasorelaxation was also attenuated by the presence of a high KCl concentration, selective blockers of small (K Ca 2.3; UCL1684), intermediate (K Ca 3.1; TRAM-34) and large conductance (K Ca 1.1; iberiotoxin) calcium-activated potassium channels and by endothelium denudation. However, vasorelaxation was not attenuated by the cannabinoid CB 1 receptor antagonist AM251 or by the cyclooxygenase inhibitor indomethacin., Significance: The study showed that the LPI-induced vasorelaxation was endothelium-dependent and mediated by GPR55, PPARγ and CB e receptors, occurred in a NO- and calcium-activated potassium channel-dependent manner in isolated hPAs. LPI seems to possess positive, hypotensive properties in pulmonary vascular bed., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Cell division cycle 42 (CDC42) plays important roles in polarity establishment and maintenance as well as cell cycle progression and cell division. Although disruption of cell polarity is a prerequisite in epithelial tumor initiation, the roles of CDC42 in tumorigenesis are still poorly understood. Here we find that Cdc42 deficiency inhibits the Kras G12D -induced lung alveoli tumor formation, while conversely promotes bronchiole tumor formation in mice. Bronchial Cdc42 loss destroys contact inhibition potentially through cell polarity disruption, and results in increased tumor formation. In contrast, deletion of Cdc42 in alveoli cells prevents Kras G12D -induced cell proliferation, which leads to reduced tumor formation. Further analyses of clinical specimens uncover a significant positive correlation between CDC42 and type II alveolar epithelial cells marker SP-A, indicating the potential importance of CDC42 in this specific subset of lung cancer. Collectively, we identify the lineage-specific function of CDC42 in lung tumorigenesis potentially through the regulation of cell polarity integrity.

The small airways of the lungs are under constant assault from the pathogens and debris in the air that they must conduct to alveoli. Although hygiene is of paramount importance for respiratory health, the underlying principles of airway clearance have not been well integrated or established. Newly emerging concepts of simultaneous absorption and secretion of airway surface liquid (ASL) and the role of [Formula: see text] in the maturation of mucins have advanced from experimental evidence as well as observations from the congenital disease cystic fibrosis (CF) to present a novel model that integrates microanatomy with organ physiology to meet the constant challenge of cleaning small airways., (Copyright © 2017 the American Physiological Society.)

Lower respiratory tract infections from respiratory syncytial virus (RSV) are due, in part, to secreted signals from lower airway cells that modify the immune response and trigger airway remodeling. To understand this process, we applied an unbiased quantitative proteomics analysis of the RSV-induced epithelial secretory response in cells representative of the trachea versus small airway bronchiolar cells. A workflow was established using telomerase-immortalized human epithelial cells that revealed highly reproducible cell type-specific differences in secreted proteins and nanoparticles (exosomes). Approximately one third of secretome proteins are exosomal; the remainder are from lysosomal and vacuolar compartments. We applied this workflow to three independently derived primary human cultures from trachea versus bronchioles. A total of 577 differentially expressed proteins from control supernatants and 966 differentially expressed proteins from RSV-infected cell supernatants were identified at a 1% false discovery rate. Fifteen proteins unique to RSV-infected primary human cultures from trachea were regulated by epithelial-specific ets homologous factor. A total of 106 proteins unique to RSV-infected human small airway epithelial cells was regulated by the transcription factor NF-κB. In this latter group, we validated the differential expression of CCL20/macrophage-inducible protein 3α, thymic stromal lymphopoietin, and CCL3-like 1 because of their roles in Th2 polarization. CCL20/macrophage-inducible protein 3α was the most active mucin-inducing factor in the RSV-infected human small airway epithelial cell secretome and was differentially expressed in smaller airways in a mouse model of RSV infection. These studies provide insights into the complexity of innate responses and regional differences in the epithelial secretome participating in RSV lower respiratory tract infection-induced airway remodeling., (Copyright © 2017 by The American Association of Immunologists, Inc.)

There is evidence that certain club cells (CCs) in the murine airways associated with neuroepithelial bodies (NEBs) and terminal bronchioles are resistant to the xenobiotic naphthalene (Nap) and repopulate the airways after Nap injury. The identity and significance of these progenitors (variant CCs, v-CCs) have remained elusive. A recent screen for CC markers identified rare Uroplakin3a (Upk3a)-expressing cells (U-CCs) with a v-CC-like distribution. Here, we employ lineage analysis in the uninjured and chemically injured lungs to investigate the role of U-CCs as epithelial progenitors. U-CCs proliferate and generate CCs and ciliated cells in uninjured airways long-term and, like v-CCs, after Nap. U-CCs have a higher propensity to generate ciliated cells than non-U-CCs. Although U-CCs do not contribute to alveolar maintenance long-term, they generate alveolar type I and type II cells after Bleomycin (Bleo)-induced alveolar injury. Finally, we report that Upk3a + cells exist in the NEB microenvironment of the human lung and are aberrantly expanded in conditions associated with neuroendocrine hyperplasias., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

3-Mercaptopyruvate sulfurtransferase (MST) is one of the principal enzymes for the production of hydrogen sulfide and polysulfides in mammalians, and emerging evidence supports the physiological significance of MST. As a fundamental study of the physiology and pathobiology of MST, it is necessary to establish the tissue distribution of MST in mice. In the present study, the expression of MST in various organs of adult and fetal mice was analyzed by Western blotting and enzyme-immunohistochemistry. Moreover, the histology of MST gene-deficient mice was examined. Western blotting revealed that all organs examined had MST. The brain, liver, kidneys testes, and endocrine organs contained large amounts of MST, but the lungs, spleen, thymus, and small intestine did not. Immunohistochemically, the MST expression pattern varies in a cell-specific manner. In the brain, neural and glial cells are positively stained; in the lung, bronchiolar cells are preferentially stained; in the liver, hepatocytes around central veins are more strongly stained; renal convoluted cells are strongly stained; and pancreatic islets are strongly stained. Fetal tissues were studied, and MST expression was found to be similar before and after birth. Histological observation revealed no remarkable findings in MST gene-deficient mice. The present study revealed fundamental information regarding the MST expression of various organs in adult and fetal mice, and the morphological phenotype of MST gene-deficient mice.

Hydrogen sulfide (H 2 S) is a gasotransmitter employed for intra- and inter-cellular communication in almost all organ systems. This study investigates the role of endogenous H 2 S in nerve-evoked relaxation of pig terminal bronchioles with 260 μm medium internal lumen diameter. High expression of the H 2 S synthesis enzyme cystathionine γ-lyase (CSE) in the bronchiolar muscle layer and strong CSE-immunoreactivity within nerve fibers distributed along smooth muscle bundles were observed. Further, endogenous H 2 S generated in bronchiolar membranes was reduced by CSE inhibition. In contrast, cystathionine β-synthase expression, another H 2 S synthesis enzyme, however was not consistently detected in the bronchiolar smooth muscle layer. Electrical field stimulation (EFS) and the H 2 S donor P-(4-methoxyphenyl)-P-4-morpholinylphosphinodithioic acid (GYY4137) evoked smooth muscle relaxation. Inhibition of CSE, nitric oxide (NO) synthase, soluble guanylyl cyclase (sGC) and of ATP-dependent K + , transient receptor potential A1 (TRPA 1 ) and transient receptor potential vanilloid 1 (TRPV 1 ) channels reduced the EFS relaxation but failed to modify the GYY4137 response. Raising extracellular K + concentration inhibited the GYY4137 relaxation. Large conductance Ca 2+ -activated K + channel blockade reduced both EFS and GYY4137 responses. GYY4137 inhibited the contractions induced by histamine and reduced to a lesser extent the histamine-induced increases in intracellular [Ca 2+ ]. These results suggest that relaxation induced by EFS in the pig terminal bronchioles partly involves the H 2 S/CSE pathway. H 2 S response is produced via NO/sGC-independent mechanisms involving K + channels and intracellular Ca 2+ desensitization-dependent pathways. Thus, based on our current results H 2 S donors might be useful as bronchodilator agents for the treatment of lung diseases with persistent airflow limitation, such as asthma and chronic obstructive lung disease., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The tobacco smoking habit interferes with the innate host defence system against infections. Recurrent infections accelerated the functional respiratory decline. The present study assessed the effects of ceftaroline on TLR2 and TLR4 and on pro-inflammatory responses in airway epithelial cells (16HBE cell line and primary bronchial epithelial cells) with or without cigarette smoke extracts (CSE 10%). TLR2, TLR4, LPS binding and human beta defensin 2 (HBD2) were assessed by flow cytometry, NFkB nuclear translocation by western blot analysis, IL-8 and HBD2 mRNA by Real Time PCR; the localization of NFkB on the HBD2 and IL-8 promoters by ChiP Assay. CSE increased TLR4, TLR2 expression, LPS binding and IL-8 mRNA; CSE decreased HBD2 (protein and mRNA), activated NFkB and promoted the localization of NFkB on IL-8 promoter and not on HBD2 promoter. Ceftaroline counteracted the CSE effect on TLR2 expression, on LPS binding, on IL-8 mRNA, HBD2 and NFkB in 16HBE. The effects of ceftaroline on HBD2 protein and on IL-8 mRNA were confirmed in primary bronchial epithelial cells. In conclusion, ceftaroline is able to counteract the effects of CSE on the innate immunity and pro-inflammatory responses modulating TLR2, LPS binding, NFkB activation and activity, HBD2 and IL-8 expression in bronchial epithelial cells., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background: The long-term outcome of lung transplantation is impeded by the development of obliterative bronchiolitis (OB). We sought to investigate the relationship between the apoptosis of tracheal epithelial cells and bone marrow-derived myofibroblasts in the process of OB., Methods: The mouse orthotopic tracheal transplant model was established. The allografts and syngrafts were harvested at 1, 2, 3, and 4 weeks after tracheal transplant. The percentage of tracheal lumen occlusion was assayed via histology and morphometry. Immunofluorescence staining was used to detect the apoptotic epithelial cells and recipient-derived myofibroblasts. The expression of SDF-1α and TGF-β and the infiltrations of CD4 and CD8 T cells in the grafts were detected using immunohistochemical staining., Results: We found that there were more apoptotic epithelia in the allograft group than in the syngraft group and that the level of tracheal lumen occlusion was higher at different time points. Moreover, the increase in the apoptosis of the tracheal epithelium occurred earlier than that of occlusion of the tracheal lumen. There were more myofibroblasts derived from the recipient's bone marrow and more CD4 and CD8 T cells in the allografts. The expression of SDF-1α and TGF-β was higher in the epithelium from allografts than in those of the syngrafts., Conclusions: Our study indicated that the apoptotic tracheal epithelia in the OB model might increase the amount of myofibroblasts derived from the recipient's bone marrow. Therapeutic methods aimed at preventing apoptosis of the tracheal epithelium may improve the outcome of lung transplantation.

Club (Clara) cells are nonciliated secretory epithelial cells present in bronchioles of distal pulmonary airways. So far, no information is available on the postnatal differentiation of club cells by a combination of molecular biological, biochemical, and stereological approaches in the murine lung. Therefore, the present study was designed to investigate the changes in the club cell secretory proteins (CC10, surfactant proteins A, B and D) and club cell abundance within the epithelium of bronchioles of distal airways during the postnatal development of the mouse lung. Perfusion-fixed murine lungs of three developmental stages (newborn, 15-day-old and adult) were used. Frozen, unfixed lungs were used for cryosectioning and subsequent laser-assisted microdissection of bronchiolar epithelial cells and RT-PCR analyses. High resolution analyses of the three-dimensional structures and composition of lung airways were obtained by scanning electron microscopy. Finally, using design-based stereology, the total and average club cell volume and the volume of secretory granules were quantified by light and transmission electron microscopy. Our results reveal that murine club cells are immature at birth and differentiate postnatally. Further, increase of the club cell volume and number of intracellular granules are closely correlated to the total lung volume enlargement. However, secretory granule density was only increased within the first 15 days of postnatal development. The differentiation is accompanied by a decrease in glycogen content, and a close positive relationship between CC10 expression and secretory granule abundance. Taken together, our data are consistent with the concept that the morphological and functional differentiation of club cells is a postnatal phenomenon.

We report that p73 is expressed in multiciliated cells (MCCs), is required for MCC differentiation, and directly regulates transcriptional modulators of multiciliogenesis. Loss of ciliary biogenesis provides a unifying mechanism for many phenotypes observed in p73 knockout mice including hydrocephalus; hippocampal dysgenesis; sterility; and chronic inflammation/infection of lung, middle ear, and sinus. Through p73 and p63 ChIP-seq using murine tracheal cells, we identified over 100 putative p73 target genes that regulate MCC differentiation and homeostasis. We validated Foxj1, a transcriptional regulator of multiciliogenesis, and many other cilia-associated genes as direct target genes of p73 and p63. We show p73 and p63 are co-expressed in a subset of basal cells and suggest that p73 marks these cells for MCC differentiation. In summary, p73 is essential for MCC differentiation, functions as a critical regulator of a transcriptome required for MCC differentiation, and, like p63, has an essential role in development of tissues., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Context: Surgical and pathologic handling of lung physically affects lung tissue. This leads to artifacts that alter the morphologic appearance of pulmonary parenchyma., Objective: To describe and illustrate mechanisms of ex vivo artifacts that may lead to diagnostic pitfalls., Design: In this study 4 mechanisms of ex vivo artifacts and corresponding diagnostic pitfalls are described and illustrated., Results: The 4 patterns of artifacts are: (1) surgical collapse, due to the removal of air and blood from pulmonary resections; (2) ex vivo contraction of bronchial and bronchiolar smooth muscle; (3) clamping edema of open lung biopsies; and (4) spreading of tissue fragments and individual cells through a knife surface. Morphologic pitfalls include diagnostic patterns of adenocarcinoma, asthma, constrictive bronchiolitis, and lymphedema., Conclusion: Four patterns of pulmonary ex vivo artifacts are important to recognize in order to avoid morphologic misinterpretations.

Background: To date there is emerging clinical evidence to add long-acting anti-muscarinic agents (LAMAs) with inhaled corticosteroid (ICSs) in asthma, but the pharmacological rationale that supports the use of such a combination has not yet been explained. The aim of this study was to pharmacologically investigate the interaction between the ICS beclomethasone and the LAMA glycopyrronium on the human airway smooth muscle (ASM) tone., Methods: We investigated the rapid non-genomic bronchorelaxant effect of beclomethasone and glycopyrronium, administered alone and in combination, in human isolated bronchi and bronchioles. Experiments were carried out also in passively sensitized airways and the pharmacological analysis of drug interaction was performed by Bliss Independence method., Results: The acute administration of beclomethasone and glycopyrronium induced a significant relaxation of passively sensitized ASM pre-contracted with histamine, by causing submaximal/maximal inhibition of the contractile tone in both medium bronchi and bronchioles. Beclomethasone was characterized by a rapid non-genomic and epithelium independent bronchorelaxant effect. In passively sensitized airways, this effect seemed to be dependent by the activation of a Gsα--cyclic adenosine monophosphate (cAMP)--protein kinase A cascade. While no synergistic interaction was detected in non-sensitized bronchi, the beclomethasone/glycopyrronium combination synergistically enhanced the relaxation of passively sensitized medium and small bronchi. The synergistic interaction between beclomethasone and glycopyrronium was associated with an increase of cAMP concentrations., Conclusions: Our study provides for the first time the pharmacological rationale for combining low doses of an ICS plus a LAMA., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Mycoplasma hyopneumoniae (Mh) is a bacterium that specifically infects the surface of bronchi and bronchioles of pigs without invading the host cells, and it is considered to be the primary agent of porcine enzootic pneumonia (PEN). The present study investigates the morphological and immunohistological changes induced in bronchiolar epithelium by Mh infection. Lungs from 20 pigs with naturally occurring Mh pneumonia were compared with those from 10 uninfected controls. Bronchiolar epithelial height, inflammatory infiltration, hyperplasia of bronchus-associated lymphoid tissue (BALT) and mucin subtype MUC5AC-producing cells significantly increased in all infected animals. Mh antigen was detected in association with the cilia of the bronchial and bronchiolar epithelium. Interleukin (IL)-5 and IL-13 were expressed consistently by epithelial and mononuclear cells of the airways of infected animals. The expression of these cytokines in the bronchial and bronchiolar tissues is related to the histological changes of PEN., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The use of lung progenitors for regenerative medicine appears promising, but their biology is not fully understood. Here, we found anti-inflammatory attributes in bronchiolar progenitors that were sorted as a multipotent subset of mouse club cells and found to express secretory leukocyte protease inhibitor (SLPI). Notably, the impaired expression of SLPI in mice increased the number of bronchiolar progenitors and decreased the lung inflammation. We determined a transcriptional profile for the bronchiolar progenitors of Slpi-deficient mice and identified syndecan 4, whose expression was markedly elevated as compared to that of wild-type mice. Systemic administration of recombinant syndecan 4 protein caused a substantial increase in the number of bronchiolar progenitors with concomitant attenuation of both airway and alveolar inflammation. The syndecan 4 administration also resulted in activation of the Keap1-Nrf2 antioxidant pathway in lung cells, which is critically involved in the therapeutic responses to the syndecan 4 treatment. Moreover, in 3D culture, the presence of syndecan 4 induced differentiated club cells to undergo Nrf2-dependent transition into bronchiolar progenitors. Our observations reveal that differentiative switches between bronchiolar progenitors and club cells are under the Nrf2-mediated control of SLPI and syndecan 4, suggesting the possibility of new therapeutic approaches in inflammatory lung diseases.

Aim: Premature infants are often exposed to hyperoxia to maintain adequate oxygenation, which may lead to the development of bronchopulmonary dysplasia (BPD). Sex-specific differences exist in the development and severity of BPD. Only a few studies have examined the mechanisms underlying these sex-related differences. The aim of the present study is to examine the sex-related long-term effects of neonatal hyperoxia on the lungs of adult mice., Materials and Methods: Newborn mice were exposed to 95% oxygen (hyperoxia) for 96 hours and were allowed to recover in room air to adulthood (8 weeks of age). Lung tissues were excised at 4 days, 14 days, or 8 weeks of age. Short-term effects of neonatal hyperoxia on the mouse lung and sex-related differences in pulmonary function, airway hyper-responsiveness, and lung structure in adult mice were assessed., Results: Neonatal hyperoxia was found to have no differential effect on body weight, muscarinic acetylcholine receptor gene expression, or bronchiolar epithelial thickness in adult mice. Respiratory resistance was increased and sensitivity to methacholine was decreased in male adult mice following exposure to neonatal hyperoxia, whereas delayed alveolarization was observed in female adult mice following exposure to neonatal hyperoxia., Conclusions: The findings of the present study demonstrate that neonatal hyperoxia differentially affects pulmonary outcome in female and male adult mice.

Lung alveolar regeneration occurs in adult human lungs as a result of proliferation, differentiation and alveolar morphogenesis of stem cells. It is increasingly being believed that bronchial epithelial cells (BECs) have a potential as stem cells, because they are potent to differentiate into multiple central and peripheral lung cell types in three-dimensional (3D) cultures, and they develop multiple foci with well-differentiated histogenesis after transformed into neoplastic cells. In this study, we investigated morphogenic abilities of HBE135 human BECs immortalized by E6/E7 oncogene in 3D cultures. When HBE135 cells were cultured alone or co-cultured with endothelial cells, the cells formed spherical colonies without branching. However, in co-culture with lung fibroblast MRC-9 cells, HBE135 cells formed colonies with bronchioalveolar-like complex branching, suggesting that MRC-9-derived soluble factor(s) are responsible for the branching formation. MRC-9 cells, not endothelial cells, were found to highly express hepatocyte growth factor (HGF), a soluble molecule involved in liver and kidney regeneration. An anti-HGF neutralizing antibody severely suppressed the complex branching formation, but addition of HGF could not sufficiently compensate the morphogenic effects of MRC-9 cells, suggesting that MCR-9-derived HGF was necessary but insufficient for the bronchioalveolar structure formation. Immunohistochemistry revealed that Met, a cognate receptor for HGF, was highly expressed and phosphorylated in neoplastic BECs from lung adenocarcinomas with well-differentiated, not poorly differentiated, histogenesis. These results are consistent with the notion that BECs have an aspect of stem cells. This aspect appears to become manifest through HGF-Met signalling pathway activation., (© 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Understanding the mechanisms of drug transport in the human lung is an important issue in pulmonary drug discovery and development. For this purpose, there is an increasing interest in immortalized lung cell lines as alternatives to primary cultured lung cells. We recently reported the protein expression in human lung tissues and pulmonary epithelial cells in primary culture, (Sakamoto A, Matsumaru T, Yamamura N, Uchida Y, Tachikawa M, Ohtsuki S, Terasaki T. 2013. J Pharm Sci 102(9):3395-3406) whereas comprehensive quantification of protein expressions in immortalized lung cell lines is sparse. Therefore, the aim of the present study was to clarify the drug transporter protein expression of five commercially available immortalized lung cell lines derived from tracheobronchial cells (Calu-3 and BEAS2-B), bronchiolar-alveolar cells (NCI-H292 and NCI-H441), and alveolar type II cells (A549), by liquid chromatography-tandem mass spectrometry-based approaches. Among transporters detected, breast cancer-resistance protein in Calu-3, NCI-H292, NCI-H441, and A549 and OCTN2 in BEAS2-B showed the highest protein expression. Compared with data from our previous study,(Sakamoto A, Matsumaru T, Yamamura N, Uchida Y, Tachikawa M, Ohtsuki S, Terasaki T. 2013. J Pharm Sci 102(9):3395-3406) NCI-H441 was the most similar with primary lung cells from all regions in terms of protein expression of organic cation/carnitine transporter 1 (OCTN1). In conclusion, the protein expression profiles of transporters in five immortalized lung cell lines were determined, and these findings may contribute to a better understanding of drug transport in immortalized lung cell lines., (© 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Nitrogen-doped multi-walled carbon nanotubes (ND-MWCNTs) are modified multi-walled carbon nanotubes (MWCNTs) with enhanced electrical properties that are used in a variety of applications, including fuel cells and sensors; however, the mode of toxic action of ND-MWCNT has yet to be fully elucidated. In the present study, we compared the interaction of ND-MWCNT or pristine MWCNT-7 with human small airway epithelial cells (SAEC) and evaluated their subsequent bioactive effects. Transmission electron microscopy, X-ray photoelectron spectroscopy, Raman spectroscopy, and X-ray diffraction suggested the presence of N-containing defects in the lattice of the nanotube. The ND-MWCNTs were determined to be 93.3% carbon, 3.8% oxygen, and 2.9% nitrogen. A dose-response cell proliferation assay showed that low doses of ND-MWCNT (1.2μg/ml) or MWCNT-7 (0.12μg/ml) increased cellular proliferation, while the highest dose of 120μg/ml of either material decreased proliferation. ND-MWCNT and MWCNT-7 appeared to interact with SAEC at 6h and were internalized by 24h. ROS were elevated at 6 and 24h in ND-MWCNT exposed cells, but only at 6h in MWCNT-7 exposed cells. Significant alterations to the cell cycle were observed in SAEC exposed to either 1.2μg/ml of ND-MWCNT or MWCNT-7 in a time and material-dependent manner, possibly suggesting potential damage or alterations to cell cycle machinery. Our results indicate that ND-MWCNT induce effects in SAEC over a time and dose-related manner which differ from MWCNT-7. Therefore, the physicochemical characteristics of the materials appear to alter their biological effects., (Published by Elsevier Ireland Ltd.)

Murine γ-herpesvirus 68 (MHV-68) infection of Mus musculus-derived strains of mice is an established model of γ-herpesvirus infection. We have previously developed an alternative system using a natural host, the wood mouse (Apodemus sylvaticus), and shown that the MHV-68 M3 chemokine-binding protein contributes significantly to MHV-68 pathogenesis. Here we demonstrate in A. sylvaticus using high-density micro-arrays that M3 influences the expression of genes involved in the host response including Scgb1a1 and Bpifa1 that encode potential innate defense proteins secreted into the respiratory tract. Further analysis of MHV-68-infected animals showed that the levels of both protein and RNA for SCGB1A1 and BPIFA1 were decreased at day 7 post infection (p.i.) but increased at day 14 p.i. as compared with M3-deficient and mock-infected animals. The modulation of expression was most pronounced in bronchioles but was also present in the bronchi and trachea. Double staining using RNA in situ hybridization and immunohistology demonstrated that much of the BPIFA1 expression occurs in club cells along with SCGB1A1 and that BPIFA1 is stored within granules in these cells. The increase in SCGB1A1 and BPIFA1 expression at day 14 p.i. was associated with the differentiation of club cells into mucus-secreting cells. Our data highlight the role of club cells and the potential of SCGB1A1 and BPIFA1 as innate defense mediators during respiratory virus infection.

GPRC5A is a G-protein-coupled receptor expressed in lung tissue but repressed in most human lung cancers. Studies in Gprc5a(-/-) mice have established its role as a tumor-suppressor function in this setting, but the basis for its role has been obscure. Here, we report that GPRC5A functions as a negative modulator of EGFR signaling. Mouse tracheal epithelial cells (MTEC) from Gprc5a(-/-) mice exhibited a relative increase in EGFR and downstream STAT3 signaling, whereas GPRC5A expression inhibited EGFR and STAT3 signaling. GPRC5A physically interacted with EGFR through its transmembrane domain, which was required for its EGFR inhibitory activity. Gprc5a(-/-) MTEC were much more susceptible to EGFR inhibitors than wild-type MTEC, suggesting their dependence on EGFR signaling for proliferation and survival. Dysregulated EGFR and STAT3 were identified in the normal epithelia of small and terminal bronchioles as well as tumors of Gprc5a(-/-) mouse lungs. Moreover, in these lungs EGFR inhibitor treatment inhibited EGFR and STAT3 activation along with cell proliferation. Finally, overexpression of ectopic GPRC5A in human non-small cell lung carcinoma cells inhibited both EGF-induced and constitutively activated EGFR signaling. Taken together, our results show how GPRC5A deficiency leads to dysregulated EGFR and STAT3 signaling and lung tumorigenesis. Cancer Res; 75(9); 1801-14. ©2015 AACR., (©2015 American Association for Cancer Research.)

Purpose: Aspiration pneumonia is infection of the respiratory tract resulting from accumulation of sputum in the larynx. N-acetyl-L-cysteine (NAC) might regulate mucin (MUC) expression and activate inherent anti-infective system in bronchiolar epithelial cells after cellular uptake, and therefore, serve as the preventative agent for chronic lung disease including aspiration pneumonia. The purpose of this in vitro study was to evaluate the effect of uptake of NAC by human bronchiolar epithelial cells on bacterial infection and regulations of mucin expression in association with cellular redox status under co-culture with a representative pathogen for hospital- and community-acquired pneumonia, Streptococcus pneumoniae., Materials and Methods: Human bronchiolar epithelial cells preincubated with or without 20 mM NAC for 3 h were co-cultured with or without bacteria for 8 h and evaluated with respect to cellular redox balance, expressions of various types of MUC, proinflammatory cytokines and mediators, and bacterial infection state by biochemical, genetic, and immunofluorescent assays., Results: Markedly increased intracellular reactive oxygen species and oxidized glutathione levels plus increased release and expression of proinflammatory cytokines and mediators were observed in cells co-cultured with bacteria. These bacteria-induced cellular redox disturbance and proinflammatory events were prevented and alleviated by pretreatment with NAC. Cells co-cultured with bacteria did not increase expression of anti-infective membranous MUC4 but exhibited increases in gel-forming MUC5AC expression and bacterial infection. However, NAC-pretreated cells avoided bacterial infection along with enhancement of MUC4, but not MUC5AC, expression., Conclusion: Uptake of NAC by human bronchiolar epithelial cells prevented bacterial infection and upregulated membranous, but not gel-forming, MUC expression along with the increase in intracellular antioxidant level under co-culture conditions with S. pneumoniae.

Magnetic Resonance Imaging (MRI) and Computed Tomography (CT) scan are the two ubiquitous imaging sources that physicians use to diagnose patients with Cystic Fibrosis (CF) or any other Chronic Obstructive Pulmonary Disease (COPD). Unfortunately the cost constraints limit the frequent usage of these medical imaging procedures. In addition, even though both CT scan and MRI provide mesoscopic details of a lung, in order to obtain microscopic information a very high resolution is required. Neither MRI nor CT scans provide micro level information about the location of infection in a binary tree structure the binary tree structure of the human lung. In this paper we present an algorithm that enhances the current imaging results by providing estimated micro level information concerning the location of the infection. The estimate is based on a calculation of the distribution of possible mucus blockages consistent with available information using an offline Metropolis-Hastings algorithm in combination with a real-time interpolation scheme. When supplemented with growth rates for the pockets of mucus, the algorithm can also be used to estimate how lung functionality as manifested in spirometric tests will change in patients with CF or COPD.