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Abstract Rationale MUC5AC and MUC5B are the predominant gel-forming mucins in the mucus layer of human airways. Each mucin has distinct functions and site-specific expression. However, the regional distribution of expression and cell types that secrete each mucin in normal/healthy human airways are not fully understood. Objectives To characterize the regional distribution of MUC5B and MUC5AC in normal/healthy human airways and assess which cell types produce these mucins, referenced to the club cell secretory protein (CCSP). Methods Multiple airway regions from 16 nonsmoker lungs without a history of lung disease were studied. MUC5AC, MUC5B, and CCSP expression/colocalization were assessed by RNA in situ hybridization and immunohistochemistry in five lungs with histologically healthy airways. Droplet digital PCR and cell cultures were performed for absolute quantification of MUC5AC/5B ratios and protein secretion, respectively. Measurements and Main Results Submucosal glands expressed MUC5B, but not MUC5AC. However, MUC5B was also extensively expressed in superficial epithelia throughout the airways except for the terminal bronchioles. Morphometric calculations revealed that the distal airway superficial epithelium was the predominant site for MUC5B expression, whereas MUC5AC expression was concentrated in proximal, cartilaginous airways. RNA in situ hybridization revealed MUC5AC and MUC5B were colocalized with CCSP-positive secretory cells in proximal superficial epithelia, whereas MUC5B and CCSP-copositive cells dominated distal regions. Conclusions In normal/healthy human airways, MUC5B is the dominant secretory mucin in the superficial epithelium and glands, with distal airways being a major site of expression. MUC5B and MUC5AC expression is a property of CCSP-positive secretory cells in superficial airway epithelia.

Low serum CC16 levels are associated with accelerated lung function decline in human population studies, but it is not known whether low serum CC16 levels contribute to lung function decline, or are an epiphenomenon. We tested the hypothesis that unchallenged Cc16−/− mice develop accelerated rates of pulmonary function test abnormalities and pulmonary pathologies over time compared with unchallenged WT mice. Respiratory mechanics, airspace enlargement, and small airway fibrosis were measured in unchallenged wild‐type (WT) versus Cc16−/− mice over 6–18 months of age. Lung leukocyte counts and lung levels of metalloproteinases (Mmps), cytokines, oxidative stress, cellular senescence markers (p19 and p21), and lung cell apoptosis, and serum C‐reactive protein (CRP) levels were measured in age‐matched WT versus Cc16−/− mice. Unchallenged Cc16−/− mice developed greater increases in lung compliance, airspace enlargement, and small airway fibrosis than age‐matched WT mice over 6–18 months of age. Cc16−/− mice had greater: (1) lung leukocyte counts; (2) lung levels of Ccl2, Ccl‐5, interleukin‐10, Mmp‐2, and Mmp‐9; (3) pulmonary oxidative stress levels, (4) alveolar septal cell apoptosis and staining for p16 and p21; and (5) serum CRP levels. Unchallenged Cc16−/− mice had greater nuclear factor‐κB (NF‐κB) activation in their lungs than age‐matched WT mice, but similar lung levels of secretory phospholipase‐A2 activity. Cc16 deficiency in mice leads spontaneously to an accelerated lung aging phenotype with exaggerated pulmonary inflammation and COPD‐like lung pathologies associated with increased activation of NF‐ κB in the lung. CC16 augmentation strategies may reduce lung aging in CC16‐deficient individuals. Low serum CC16 levels are associated with accelerated lung function decline in human population studies, but it is not known whether low serum CC16 levels contribute to lung function decline, or are an epiphenomenon. Cc16 deficiency in mice leads spontaneously to an accelerated lung aging phenotype with exaggerated pulmonary inflammation and COPD‐like lung pathologies associated with increased activation of NF‐κB in the lung. Thus, Cc16 deficiency contributes to inflamm‐aging in the lungs of mice.

Little is known about human club cells, dome-shaped cells with dense cytoplasmic granules and microvilli that represent the major secretory cells of the human small airways (at least sixth-generation bronchi). To define the ontogeny and biology of the human small airway epithelium club cell. The small airway epithelium was sampled from the normal human lung by bronchoscopy and brushing. Single-cell transcriptome analysis and air-liquid interface culture were used to assess club cell ontogeny and biology. We identified the club cell population by unbiased clustering using single-cell transcriptome sequencing. Principal component gradient analysis uncovered an ontologic link between KRT5 (keratin 5) basal cells and SCGB1A1 (secretoglobin family 1A member 1) club cells, a hypothesis verified by demonstrating in vitro that a pure population of human KRT5 SCGB1A1 small airway epithelial basal cells differentiate into SCGB1A1 KRT5 club cells on air-liquid interface culture. Using SCGB1A1 as the marker of club cells, the single-cell analysis identified novel roles for these cells in host defense, xenobiotic metabolism, antiprotease, physical barrier function, monogenic lung disorders, and receptors for human viruses. These observations provide novel insights into the molecular phenotype and biologic functions of the human club cell population and identify basal cells as the human progenitor cells for club cells.

The report presents the cellular structure of the respiratory system as well as the history of club cells (Clara cells), their ultrastructure, and location in the airways and human organs. The authors discuss the biochemical structure of proteins secreted by these cells and their importance for the integrity and regeneration of the airway epithelium. Their role as progenitor cells for the airway epithelium and their involvement in the biotransformation of toxic xenobiotics introduced into the lungs during breathing is emphasized. This is followed by a discussion of the clinical aspects associated with club cells, demonstrating that tracking the serum concentration of club cell-secreted proteins is helpful in the diagnosis of a number of lung tissue diseases. Finally, suggestions are provided regarding the possible use of proteins secreted by club cells in the treatment of serious respiratory conditions.

The lungs have a remarkable ability to regenerate damaged tissues caused by acute injury. Many lung diseases, especially chronic lung diseases, are associated with a reduced or disrupted regeneration potential of the lungs. Therefore, understanding the underlying mechanisms of the regenerative capacity of the lungs offers the potential to identify novel therapeutic targets for these diseases. R-spondin2, a co-activator of WNT/β-catenin signaling, plays an important role in embryonic murine lung development. However, the role of Rspo2 in adult lung homeostasis and regeneration remains unknown. The aim of this study is to determine Rspo2 function in distal lung stem/progenitor cells and adult lung regeneration. In this study, we found that robust Rspo2 expression was detected in different epithelial cells, including airway club cells and alveolar type 2 (AT2) cells in the adult lungs. However, Rspo2 expression significantly decreased during the first week after naphthalene-induced airway injury and was restored by day 14 post-injury. In ex vivo 3D organoid culture, recombinant RSPO2 promoted the colony formation and differentiation of both club and AT2 cells through the activation of canonical WNT signaling. In contrast, Rspo2 ablation in club and AT2 cells significantly disrupted their expansion capacity in the ex vivo 3D organoid culture. Furthermore, mice lacking Rspo2 showed significant defects in airway regeneration after naphthalene-induced injury. Our results strongly suggest that RSPO2 plays a key role in the adult lung epithelial stem/progenitor cells during homeostasis and regeneration, and therefore, it may be a potential therapeutic target for chronic lung diseases with reduced regenerative capability.

Background Chronic obstructive pulmonary disease (COPD) is a prevalent respiratory condition, with its severity inversely related to the levels of Club cell 10 kDa secretory protein (CC10). The gene Lsm2 , involved in RNA metabolism and cell proliferation, has an unclear role in COPD development. Methods An in vitro COPD model was developed by stimulating 16HBE cells with cigarette smoke extract (CSE). To establish an in vivo COPD model, mice with defective Lsm2 gene expression in lung or club cells were exposed to cigarette smoke for 3 months. Multiplexed immunohistochemistry (mIHC) was employed to identify the specific cells where Lsm2 gene expression is predominant. RNA sequencing and single-nucleus RNA sequencing were conducted to investigate the role of Lsm2 in the pathogenesis of COPD. Results In this study, we found that cigarette smoke extract increases Lsm2 expression, and knocking down Lsm2 in 16HBE cells significantly reduces cell viability in vitro. mIHC showed that Lsm2 is primarily expressed in Club cells. Knockout of Lsm2 , either in the lungs or specifically in Club cells, exacerbated lung injury and inflammation caused by cigarette smoke exposure in vivo. Single-nucleus RNA sequencing analysis revealed that Club cell-specific knockout of Lsm2 leads to a reduction in the Club cell population, particularly those expressing Chia1 + /Crb1 + . This decrease in Club cells subsequently reduces the number of ciliated epithelial cells. Conclusion Knocking out Lsm2 in Club cells results in a significant decrease in Club cell numbers, which subsequently leads to a reduction in ciliated epithelial cells. This increased lung vulnerability to cigarette smoke and accelerating the progression of COPD. Our findings highlight that Lsm2 is critical to club cell proliferation and its inhibition aggravates COPD progression.

Epidermal club cells (ECCs), along with mucus cells, are present in the skin of many fishes, particularly in the well-studied Ostariophysan family Cyprinidae. Most ECC-associated literature has focused on the potential role of ECCs as a component of chemical alarm cues released passively when a predator damages the skin of its prey, alerting nearby prey to the presence of an active predator. Because this warning system is maintained by receiver-side selection (senders are eaten), there is want of a mechanism to confer fitness benefits to the individual that invests in ECCs to explain their evolutionary origin and maintenance in this speciose group of fishes. In an attempt to understand the fitness benefits that accrue from investment in ECCs, we reviewed the phylogenetic distribution of ECCs and their histochemical properties. ECCs are found in various forms in all teleost superorders and in the chondrostei inferring either early or multiple independent origins over evolutionary time. We noted that ECCs respond to several environmental stressors/immunomodulators including parasites and pathogens, are suppressed by immunomodulators such as testosterone and cortisol, and their density covaries with food ration, demonstrating a dynamic metabolic cost to maintaining these cells. ECC density varies widely among and within fish populations, suggesting that ECCs may be a convenient tool with which to assay ecoimmunological tradeoffs between immune stress and foraging activity, reproductive state, and predator–prey interactions. Here, we review the case for ECC immune function, immune functions in fishes generally, and encourage future work describing the precise role of ECCs in the immune system and life history evolution in fishes.

Abstract Telomere dysfunction is associated with multiple fibrotic lung processes, including chronic lung allograft dysfunction (CLAD)—the major limitation to long-term survival following lung transplantation. Although shorter donor telomere lengths are associated with an increased risk of CLAD, it is unknown whether short telomeres are a cause or consequence of CLAD pathology. Our objective was to test whether telomere dysfunction contributes to the pathologic changes observed in CLAD. Histopathologic and molecular analysis of human CLAD lungs demonstrated shortened telomeres in lung epithelial cells quantified by teloFISH, increased numbers of surfactant protein C immunoreactive type II alveolar epithelial cells, and increased expression of senescence markers (β-galactosidase, p16, p53, and p21) in lung epithelial cells. TRF1F/F (telomere repeat binding factor 1 flox/flox) mice were crossed with tamoxifen-inducible SCGB1a1-cre mice to generate SCGB1a1-creTRF1F/F mice. Following 9 months of tamoxifen-induced deletion of TRF1 in club cells, mice developed mixed obstructive and restrictive lung physiology, small airway obliteration on microcomputed tomography, a fourfold decrease in telomere length in airway epithelial cells, collagen deposition around bronchioles and adjacent lung parenchyma, increased type II aveolar epithelial cell numbers, expression of senescence-associated β-galactosidase in epithelial cells, and decreased SCGB1a1 expression in airway epithelial cells. These findings demonstrate that telomere dysfunction isolated to airway epithelial cells leads to airway-centric lung remodeling and fibrosis similar to that observed in patients with CLAD and suggest that lung epithelial cell telomere dysfunction may be a molecular driver of CLAD.

In the last decade, zebrafish has been used as a model for the study of several human skin diseases. The epidermis of Danio rerio is composed of keratinocytes and two types of secretory cells: mucous cells and club cells. Club cells have multiple biological functions and among them may be important in the protection against ultraviolet damage through the proliferative response or through the increased production of protective substances. Calcium-binding proteins such as calbindin D28K and calretinin are used as markers of nervous and enteric nervous systems, but they are present in numerous other cells. These proteins are involved in a wide variety of cell activities, such as cytoskeletal organization, cell motility and differentiation, cell cycle regulation and neuroprotective function. In this study we demonstrated, for the first time, the presence of calretinin and calbindin D28K in skin club cells of Danio rerio exposed to different wavelengths by immunohistochemistry analysis. Exposure to white-blue light and blue light causes the expression and colocalization of calbindin-D28K and calretinin. These proteins were moderately expressed and no colocalization was observed in the club cells of the control fish. In zebrafish exposed to continuous darkness for 10 days, in the club cells the two antibodies did not detect any proteins specifically. These results demonstrate that calbindin and calretinin could be involved in the pathophysiology of skin injury due to exposure to short-wavelength visible light spectrums.

Background Gene therapy holds great potential for treating various acquired and inherited pulmonary diseases. Adeno-associated viral (AAV) vectors have been thought to be primary candidates for gene delivery in patients with pulmonary diseases. However, the tropism of AAVs in the lungs remains largely unknown. Results Here, we investigate the tropism of twenty serotypes of AAVs by examining AAV-packed vector expression of the enhanced green fluorescent protein (eGFP) in mice. AAV1, AAV4, AAV5, AAV6, AAV6.2, AAV-PHP.B, and AAV-PHP.S exhibit high transduction rates in the airway epithelium. AAV1, AAV4, AAV5, AAV6, and AAV6.2 highly infect club cells. AAV1, AAV4, AAV5, AAV6, AAV6.2, and AAV-PHP.B efficiently infect ciliated cells. AAV8 and AAVrh10 can infect a few alveolar type I cells. AAV1, AAV5, AAV6, AAV6.2, AAV9, and AAVie can infect alveolar type II cells. AAV1, AAV5, AAVie, AAV-PHP.B, AAV-PHP.eB, and AAV-PHP.S can infect a few endothelial cells. However, none of these AAVs can efficiently infect neuroendocrine or smooth muscle cells. Conclusions Our findings provide comprehensive information about the tropism of AAVs in pulmonary epithelium in mice, which might be helpful in developing efficient AAV-mediated gene therapy strategies for pulmonary disease treatment.