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.

Rationale: Pulmonary ionocytes are a newly discovered airway epithelial cell type proposed to be a major contributor to cystic fibrosis (CF) lung disease based on observations they express the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel at a higher level than any other cell type in the airway epithelia. Moreover, genetically manipulated experimental models that lack ionocytes develop NaCl transport abnormalities and airway surface liquid (ASL) dehydration consistent with CF. However, no direct evidence indicates ionocytes engage in NaCl transport or contribute to ASL formation, questioning the relevance of ionocytes to CF lung disease. Objectives: To determine the ion transport properties of pulmonary ionocytes and club cells in genetically intact healthy and CF airway epithelia. Methods: We measured ion transport at the single-cell level using a self-referencing ion-selective microelectrode technique in primary human bronchial epithelial cell culture. Measurements and Main Results: cAMP-stimulated non-CF ionocytes do not secrete Na+ or Cl- into the ASL, but rather modulate its pH by secreting bicarbonate via CFTR-linked Cl-/bicarbonate exchange. Non-CF club cells secrete Na+ and Cl- to the lumen side after cAMP stimulation. CF ionocytes and club cells do not transport ions in response to cAMP stimulation, but incubation with CFTR modulators elexacaftor/tezacaftor/ivacaftor restores transport properties. Conclusions: We conclude that ionocytes do not contribute to ASL formation but regulate ASL pH. Club cells secrete the bulk of airway fluid. In CF, abnormal ionocyte and club cell function results in acidic and dehydrated ASL, causing reduced antimicrobial properties and mucociliary clearance. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Inhaling xenobiotics, such as tobacco smoke is a major risk factor for pulmonary diseases, e.g., COPD/emphysema, interstitial lung disease, and pre-invasive diseases. Shelterin complex or telosome provides telomeric end protection during replication. Telomere protection protein 1 (TPP1) is one of the main six subunits of the shelterin complex supporting the telomere stability and genomic integrity. Dysfunctional telomeres and shelterin complex are associated as a disease mechanism of tobacco smoke-induced pulmonary damage and disease processes. The airway epithelium is critical to maintaining respiratory homeostasis and is implicated in lung diseases. Club cells (also known as clara cells) play an essential role in the immune response, surfactant production, and metabolism. Disrupted shelterin complex may lead to dysregulated cellular function, DNA damage, and disease progression. However, it is unknown if the conditional removal of TPP1 from Club cells can induce lung disease pathogenesis caused by tobacco smoke exposure. In this study, conditional knockout of Club-cell specific TPP1 demonstrated the instability of other shelterin protein subunits, such as TRF1, dysregulation of cell cycle checkpoint proteins, p53 and downstream targets, and dysregulation of telomeric genes. This was associated with age-dependent senescence-associated genes, increased DNA damage, and upregulated RANTES/IL13/IL33 mediated lung inflammation and injury network by cigarette smoke (CS). These phenomena are also associated with alterations in cytochrome P450 and glutathione transferases, upregulated molecular pathways promoting lung lesions, bronchial neoplasms, and adenocarcinomas. These findings suggest a pivotal role of TPP1 in maintaining lung homeostasis and injurious responses in response to CS. Thus, these data TPP1 may have therapeutic value in alleviating telomere-related chronic lung diseases.

BACKGROUND: Lianhua Qingke (LHQK) is an effective traditional Chinese medicine used for treating acute tracheobronchitis. In this study, we evaluated the effectiveness of LHQK in managing airway mucus hypersecretion in the acute exacerbation of chronic obstructive pulmonary disease (AECOPD).
METHODS: The AECOPD model was established by subjecting male Wistar rats to 12 weeks of cigarette smoke (CS) exposure (80 cigarettes/day, 5 days/week for 12 weeks) and intratracheal lipopolysaccharide (LPS) exposure (200 μg, on days 1, 14, and 84). The rats were divided into six groups: control (room air exposure), model (CS + LPS exposure), LHQK (LHQK-L, LHQK-M, and LHQK-H), and a positive control group (Ambroxol). H&E staining, and AB-PAS staining were used to evaluate lung tissue pathology, inflammatory responses, and goblet cell hyperplasia. RT-qPCR, immunohistochemistry, immunofluorescence and ELISA were utilized to analyze the transcription, expression and secretion of proteins related to mucus production in vivo and in the human airway epithelial cell line NCI-H292 in vitro. To predict and screen the active ingredients of LHQK, network pharmacology analysis and NF-κB reporter system analysis were employed.
RESULTS: LHQK treatment could ameliorate AECOPD-triggered pulmonary structure damage, inflammatory cell infiltration, and pro-inflammatory cytokine production. AB-PAS and immunofluorescence staining with CCSP and Muc5ac antibodies showed that LHQK reduced goblet cell hyperplasia, probably by inhibiting the transdifferentiation of Club cells into goblet cells. RT-qPCR and immunohistochemistry of Muc5ac and APQ5 showed that LHQK modulated mucus homeostasis by suppressing Muc5ac transcription and hypersecretion in vivo and in vitro, and maintaining the balance between Muc5ac and AQP5 expression. Network pharmacology analysis and NF-κB luciferase reporter system analysis provided insights into the active ingredients of LHQK that may help control airway mucus hypersecretion and regulate inflammation.
CONCLUSIONS: LHQK demonstrated therapeutic effects in AECOPD by reducing inflammation, suppressing goblet cell hyperplasia, preventing Club cell transdifferentiation, reducing Muc5ac hypersecretion, and modulating airway mucus homeostasis. These findings support the clinical use of LHQK as a potential treatment for AECOPD.

BACKGROUND: The starry puffer fish (Arothron stellatus, Anonymous, 1798) is a poisonous tetradontidae fish inhabiting the Red sea. The skin constitutes an important defense against any external effects. The study aims to characterize the dorso-ventral skin of the juvenile and the adult starry puffer fish using light and scanning electron microscopies. Twenty specimens of juvenile and adult fresh fishes were used.
RESULTS: The scanning electron microarchitecture of the skin of the juvenile and adult fish showed delicate irregular-shaped protrusions, and well-defined bricks-like elevations on the dorsal side and interrupted folds as well as irregular-shaped protrusions on the ventral side. In adult fish, the patterned microridges of the superficial and deep epithelial cells (keratinocytes) were larger and well-defined in the dorsal skin than in the ventral side, the contrary was seen in the juvenile fish. The microridges were arranged in a fingerprint or honeycomb patterns. The openings of the mucous cells were more numerous in the dorsal skin in both age stages but more noticeable in adult. Furthermore, the sensory cells were more dominant in the juveniles than the adults. The odontic spines were only seen in adult. Histologically, few taste buds were observed in the epidermis of the dorsal skin surface of the adult fish. Both mucous and club cells were embedded in the epidermis of the juvenile and adult fish with different shapes and sizes. Melanophores were observed at the dorsal skin of both juvenile and adult fishes while fewer numbers were noticed at the ventral surfaces. Several dermal bony plates with different shapes and sizes were demonstrated in the skin of both adult and juvenile fishes.
CONCLUSIONS: The structural variations of skin of the juvenile and adult fishes may reflect the various environmental difficulties that they confront.

Innate immune responses (IIR) of the epithelium play a critical role in the initiation and progression of asthma. The core of the IIR is an intracellular signaling pathway activated by pattern recognition receptors (PRRs) to limit the spread of infectious organisms. This chapter will focus on the epithelium as the major innate sentinel cell and its role in acute exacerbations (AEs). Although the pathways of how the IIR activates the NFκB transcription factor, triggering cytokine secretion, dendritic cell activation, and Th2 polarization are well-described, recent exciting work has developed mechanistic insights into how chronic activation of the IIR is linked to mucosal adaptive responses. These adaptations include changes in cell state, now called epithelial-mesenchymal plasticity (EMP). EMP is a coordinated, genomic response to airway injury disrupting epithelial barrier function, expanding the basal lamina, and producing airway remodeling. EMP is driven by activation of the unfolded protein response (UPR), a transcriptional response producing metabolic shunting of glucose through the hexosamine biosynthetic pathway (HBP) to protein N-glycosylation. NFκB signaling and UPR activation pathways potentiate each other in remodeling the basement membrane. Understanding of injury-repair process of epithelium provides new therapeutic targets for precision approaches to the treatment of asthma exacerbations and their sequelae.

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.

The immune system of a fish has cellular and molecular defense mechanisms that are substantially retained throughout the evolution of vertebrates. The innate immune system provides biological processes, such as phagocytosis and mechanical barriers, to implement an efficient defensive response after exposure to chemical or biological contaminants, pollutants, and contact with parasites, germs, and pathogens. Club cells (CCs) are widespread in the skin of Ostariophysi. After a predator attack or exposure to toxins and parasites, these cells can produce alarming substances. Given their effectiveness against viruses, parasites, and common skin lesions, recent studies have suggested that CCs are a component of the immune system. This study aims to immunohistochemically characterize the CCs for the first time in the skin of zebrafish, using mitogen-activated protein kinase (MAPK) p38, Toll-like receptor (TLR)2, Piscidin1, and inducible nitric oxide synthase (iNOS) peptides involved in the function of all types of vertebrate immune cells. According to our analysis, the intermediate layer of the epidermis exhibited rounded, oval, and elongated CCs, with central acidophilic cytoplasm and a spherical basophilic nucleus, that are positive to the antibodies tested. Our results may confirm that CCs could be involved in the immune function, increasing our knowledge of the immune system of teleosts.

Rationale: MUC5AC (mucin 5AC, oligomeric gel-forming) and MUC5B (mucin 5B, oligomeric gel-forming) are the predominant secreted polymeric mucins in mammalian airways. They contribute differently to the pathogenesis of various muco-obstructive and interstitial lung diseases, and their genes are separately regulated, but whether they are packaged together or in separate secretory granules is not known. Objectives: To determine the packaging of MUC5AC and MUC5B within individual secretory granules in mouse and human airways under varying conditions of inflammation and along the proximal-distal axis. Methods: Lung tissue was obtained from mice stimulated to upregulate mucin production by the cytokines IL-1β and IL-13 or by porcine pancreatic elastase. Human lung tissue was obtained from donated normal lungs, biopsy samples of transplanted lungs, and explanted lungs from subjects with chronic obstructive pulmonary disease. MUC5AC and MUC5B were labeled with antibodies from different animal species or, in mice only, by transgenic chimeric mucin-fluorescent proteins and imaged using widefield deconvolution or Airyscan fluorescence microscopy. Measurements and Main Results: In both mouse and human airways, most secretory granules contained both mucins interdigitating within the granules. Smaller numbers of granules contained MUC5B alone, and even fewer contained MUC5AC alone. Conclusions: MUC5AC and MUC5B are variably stored both in the same and in separate secretory granules of both mice and humans. The high fraction of granules containing both mucins under a variety of conditions makes it unlikely that their secretion can be differentially controlled as a therapeutic strategy. This work also advances knowledge of the packaging of mucins within secretory granules to understand mechanisms of epithelial stress in the pathogenesis of chronic lung diseases.

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.