Pulmonary Ionocytes Regulate Airway Surface Liquid pH in Primary Human Bronchial Epithelial Cells.

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.

An update in club cell biology and its potential relevance to chronic obstructive pulmonary disease.

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.

AAV5 delivery of CRISPR-Cas9 supports effective genome editing in mouse lung airway.

Genome editing in the lung has the potential to provide long-term expression of therapeutic protein to treat lung genetic diseases. Yet efficient delivery of CRISPR to the lung remains a challenge. The NIH Somatic Cell Genome Editing (SCGE) Consortium is developing safe and effective methods for genome editing in disease tissues. Methods developed by consortium members are independently validated by the SCGE small animal testing center to establish rigor and reproducibility. We have developed and validated a dual adeno-associated virus (AAV) CRISPR platform that supports effective editing of a lox-stop-lox-Tomato reporter in mouse lung airway. After intratracheal injection of the AAV serotype 5 (AAV5)-packaged S. pyogenes Cas9 (SpCas9) and single guide RNAs (sgRNAs), we observed ∼19%-26% Tomato-positive cells in both large and small airways, including club and ciliated epithelial cell types. This highly effective AAV delivery platform will facilitate the study of therapeutic genome editing in the lung and other tissue types.

Histological and ultrastructural characterization of the dorso-ventral skin of the juvenile and the adult starry puffer fish (Arothron stellatus, Anonymous 1798).

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. CONCLUSION: The structural variations of skin of the juvenile and adult fishes may reflect the various environmental difficulties that they confront.

Mucins MUC5AC and MUC5B Are Variably Packaged in the Same and in Separate Secretory Granules.

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-1ß 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.

Innate Immunity, Epithelial Plasticity, and Remodeling in Asthma.

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.

Ataxia telangiectasia mutated is required for efficient proximal airway epithelial cell regeneration following influenza A virus infection.

Children and young adults with mutant forms of ataxia telangiectasia mutated (ATM), a kinase involved in DNA damage signaling and mitochondrial homeostasis, suffer from recurrent respiratory infections, immune deficiencies, and obstructive airways disease associated with disorganized airway epithelium. We previously showed in mice how Atm was required to mount a protective immune memory response to influenza A virus [IAV; Hong Kong/X31 (HKx31), H3N2]. Here, Atm wildtype (WT) and knockout (Atm-null) mice were used to investigate how Atm is required to regenerate the injured airway epithelium following IAV infection. When compared with WT mice, naive Atm-null mice had increased airway resistance and reduced lung compliance that worsened during infection before returning to naïve levels by 56 days postinfection (dpi). Although Atm-null lungs appeared pathologically normal before infection by histology, they developed an abnormal proximal airway epithelium after infection that contained E-cadherin+, Sox2+, and Cyp2f2+ cells lacking secretoglobin family 1 A member 1 (Scgb1a1) protein expression. Patchy and low expression of Scgb1a1 were eventually observed by 56 dpi. Genetic lineage tracing in HKx31-infected mice revealed club cells require Atm to rapidly and efficiently restore Scgb1a1 expression in proximal airways. Since Scgb1a1 is an immunomodulatory protein that protects the lung against a multitude of respiratory challenges, failure to efficiently restore its expression may contribute to the respiratory diseases seen in individuals with ataxia telangiectasia.

Using intracellular SCGB1A1-sorted, formalin-fixed club cells for successful transcriptomic analysis.

As opposed to surface marker staining, certain cell types can only be recognized by intracellular markers. Intracellular staining for use in cell sorting remains challenging. Fixation and permeabilization steps for intracellular staining and the presence of RNases notably affect preservation of high-quality mRNA. We report the work required for the optimization of a successful protocol for microarray analysis of intracellular target-sorted, formalin-fixed human bronchial club cells. Cells obtained from differentiated air-liquid interface cultures were stained with the most characteristic intracellular markers for club cell (SCGB1A1+) sorting. A benchmarked intracellular staining protocol was carried out before flow cytometry. The primary outcome was the extraction of RNA sufficient quality for microarray analysis as assessed by Bioanalyzer System. Fixation with 4% paraformaldehyde coupled with 0.1% Triton/0.1% saponin permeabilization obtained optimal results for SCGB1A1 staining. Addition of RNase inhibitors throughout the protocol and within the appropriate RNA extraction kit (Formalin-Fixed-Paraffin-Embedded) dramatically improved RNA quality, resulting in samples eligible for microarray analysis. The protocol resulted in successful cell sorting according to specific club cell intracellular marker without using cell surface marker. The protocol also preserved RNA of sufficient quality for subsequent microarray transcriptomic analysis, and we were able to generate transcriptomic signature of club cells.

Novel dynamics of human mucociliary differentiation revealed by single-cell RNA sequencing of nasal epithelial cultures.

The upper airway epithelium, which is mainly composed of multiciliated, goblet, club and basal cells, ensures proper mucociliary function and can regenerate in response to assaults. In chronic airway diseases, defective repair leads to tissue remodeling. Delineating key drivers of differentiation dynamics can help understand how normal or pathological regeneration occurs. Using single-cell transcriptomics and lineage inference, we have unraveled trajectories from basal to luminal cells, providing novel markers for specific populations. We report that: (1) a precursor subgroup of multiciliated cells, which we have entitled deuterosomal cells, is defined by specific markers, such as DEUP1, FOXN4, YPEL1, HES6 and CDC20B; (2) goblet cells can be precursors of multiciliated cells, thus explaining the presence of hybrid cells that co-express markers of goblet and multiciliated cells; and (3) a repertoire of molecules involved in the regeneration process, such as keratins or components of the Notch, Wnt or BMP/TGFß pathways, can be identified. Confirmation of our results on fresh human and pig airway samples, and on mouse tracheal cells, extend and confirm our conclusions regarding the molecular and cellular choreography at work during mucociliary epithelial differentiation.

Evaluation of the human hazard of the liver and lung tumors in mice treated with permethrin based on mode of action.

The non-genotoxic synthetic pyrethroid insecticide permethrin produced hepatocellular adenomas and bronchiolo-alveolar adenomas in female CD-1 mice, but not in male CD-1 mice or in female or male Wistar rats. Studies were performed to evaluate possible modes of action (MOAs) for permethrin-induced female CD-1 mouse liver and lung tumor formation. The MOA for liver tumor formation by permethrin involves activation of the peroxisome proliferator-activated receptor alpha (PPARα), increased hepatocellular proliferation, development of altered hepatic foci, and ultimately liver tumors. This MOA is similar to that established for other PPARα activators and is considered to be qualitatively not plausible for humans. The MOA for lung tumor formation by permethrin involves interaction with Club cells, followed by a mitogenic effect resulting in Club cell proliferation, with prolonged administration producing Club cell hyperplasia and subsequently formation of bronchiolo-alveolar adenomas. Although the possibility that permethrin exposure may potentially result in enhancement of Club cell proliferation in humans cannot be completely excluded, there is sufficient information on differences in basic lung anatomy, physiology, metabolism, and biologic behavior of tumors in the general literature to conclude that humans are quantitatively less sensitive to agents that increase Club cell proliferation and lead to tumor formation in mice. The evidence strongly indicates that Club cell mitogens are not likely to lead to increased susceptibility to lung tumor development in humans. Overall, based on MOA evaluation it is concluded that permethrin does not pose a tumorigenic hazard for humans, this conclusion being supported by negative data from permethrin epidemiological studies.

R-Spondin2, a Positive Canonical WNT Signaling Regulator, Controls the Expansion and Differentiation of Distal Lung Epithelial Stem/Progenitor Cells in Mice.

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/ß-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.

Definitive evidence for Club cells as progenitors for mutant Kras/Trp53-deficient lung cancer.

Accumulating evidence suggests that both the nature of oncogenic lesions and the cell-of-origin can strongly influence cancer histopathology, tumor aggressiveness and response to therapy. Although oncogenic Kras expression and loss of Trp53 tumor suppressor gene function have been demonstrated to initiate murine lung adenocarcinomas (LUADs) in alveolar type II (AT2) cells, clear evidence that Club cells, representing the second major subset of lung epithelial cells, can also act as cells-of-origin for LUAD is lacking. Equally, the exact anatomic location of Club cells that are susceptible to Kras transformation and the resulting tumor histotype remains to be established. Here, we provide definitive evidence for Club cells as progenitors for LUAD. Using in vivo lineage tracing, we find that a subset of Kras12V -expressing and Trp53-deficient Club cells act as precursors for LUAD and we define the stepwise trajectory of Club cell-initiated tumors leading to lineage marker conversion and aggressive LUAD. Our results establish Club cells as cells-of-origin for LUAD and demonstrate that Club cell-initiated tumors have the potential to develop aggressive LUAD.

Imaging lung regeneration by light sheet microscopy.

Optical clearing combined with deep imaging of large biological specimen allows organ-wide visualization of cells in three dimensions (3D) to explore regenerative processes in a spatial context. Here, we investigate the dynamics of airway regeneration following toxin-mediated epithelial injury in cleared whole lung preparations by light sheet microscopy. We use a recently developed knock-in mouse strain labeling bronchiolar Club cells (Scgb1a1-mCherry) to define an optimal clearing procedure that efficiently preserves genetically encoded fluorophores. Dehydration in pH-adjusted tert-butanol followed by clearing in ethyl cinnamate maintained maximum mCherry fluorescence while preventing unfavorable background fluorescence. We apply this technique to depict the course of bronchiolar epithelial renewal from an acute injury phase to early and late recovery stages. 3D reconstructions of whole lungs demonstrate near-complete loss of secretory Club cells throughout the entire respiratory tract 3 days post naphthalene (dpn). Multiple foci of regenerating Club cells emerge at 7 dpn, predominantly at airway bifurcations and in distal terminal bronchioles-anatomical regions assumed to harbor distinct stem/progenitor cells subsets. At 21 dpn, clusters of newly formed Club cells have largely expanded, although the bronchiolar epithelial lining continues to regenerate. This study identifies regional stem cell niches as starting points for epithelial recovery, underscores the enormous regenerative capacity of the respiratory epithelium and demonstrates the power of whole lung 3D imaging for evaluating the extent of pulmonary damage and subsequent repair processes.

The noncanonical BMP signaling pathway plays an important role in club cell regeneration.

The bronchiole is a major site for the development of several life-threatening disorders, including chronic obstructive pulmonary disease and lung adenocarcinomas. The bronchiolar epithelium is composed of club cells and ciliated epithelial cells, with club cells serving as progenitor cells. Presently, the identity of the cells involved in regeneration of bronchiolar epithelium and the underlying mechanisms remain incompletely understood. Here, we show that Prrx1, a homeobox transcription factor, can mark club cells in adult mice during homeostasis and regeneration. We further show that the noncanonical signaling pathway of BMPs, BMPR1A-Tak1-p38MAPK, plays a critical role in club cell regeneration. Ablation of Bmpr1a, Tak1, or Mapk14 (encoding p38α) in Prrx1+ club cells caused minimal effect on bronchiolar epithelium homeostasis, yet it resulted in severe defects in club cell regeneration and bronchiole repair in adult mice. We further show that this pathway supports proliferation and expansion of the regenerating club cells. Our findings thus identify a marker for club cells and reveal a critical role for the BMP noncanonical pathway in club cell regeneration.

Epidermal Club Cells in Fishes: A Case for Ecoimmunological Analysis.

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.

Airway Epithelial Telomere Dysfunction Drives Remodeling Similar to Chronic Lung Allograft Dysfunction.

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 (ß-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 ß-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.

Effect of light on the calretinin and calbindin expression in skin club cells of adult zebrafish.

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.

Localization of Secretory Mucins MUC5AC and MUC5B in Normal/Healthy Human Airways.

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.

Cell proliferation analysis is a reliable predictor of lack of carcinogenicity: Case study using the pyrethroid imiprothrin on lung tumorigenesis in mice.

In the mouse carcinogenicity study, an apparent increase in lung adenocarcinoma was observed in male mice at 7000 ppm. Based on the overall evaluation of toxicology, oncology, pathology and statistics, we concluded that the apparent increase in lung tumors is not relevant for evaluation of carcinogenicity of imiprothrin (Regul Toxicol Pharmacol, 105, 1-14, 2019). To investigate whether imiprothrin has any mitogenic effect on mouse Club cells, the present study examined its effects on replicative DNA synthesis of Club cells and lung histopathology in male mice treated with imiprothrin for 7 days at 3500 and 7000 ppm in the diet. Isoniazid, a known mouse lung mitogen and tumor inducer, was also examined at 1000 ppm in the diet as a positive control of Club cell mitogenesis and morphological changes. Neither imiprothrin nor isoniazid caused any necrotic changes in lung by light or electron microscopy. There were no increases observed in the bromodeoxyuridine (BrdU) labeling index in the imiprothrin groups, while there was a statistically significant increase in the BrdU labeling index in the isoniazid group. These findings demonstrate that imiprothrin does not induce mouse Club cell proliferation or morphologic changes, supporting our previous conclusion described above. Thus, imiprothrin should not be classified as a carcinogen. Furthermore, this study indicates that short-term studies focusing on cell proliferation can be reliable for predicting a lack of carcinogenic potential of test chemicals.

Altered expression of p63 isoforms and expansion of p63- and club cell secretory protein-positive epithelial cells in the lung as novel features of aging.

Aging is a key contributor for subclinical progression of late-onset lung diseases. Basal, club, and type II alveolar epithelial cells (AECs) are lung epithelial progenitors whose capacities of differentiation are extensively studied. The timely transition of these cells in response to environmental changes helps maintain the intricate organization of lung structure. However, it remains unclear how aging affects their behavior. This paper demonstrates that the protein expression profiles of a type II AEC marker, prosurfactant protein C (pro-SPC), and a basal cell marker, p63, are altered in the lungs of 14-mo-old versus 7- to 9-wk-old mice. Expression of NH2-terminal-truncated forms of p63 (ΔNp63), a basal cell marker, and claudin-10, a club cell marker, in cytoplasmic extracts of lungs of 14-mo-old mice was upregulated. In contrast, nuclear expression of full-length forms of p63 (TAp63) decreases with age. These alterations in protein expression profiles coincide with dramatic changes in lung functions including compliance. Whole tissue lysates of middle-aged versus aged rhesus monkey lungs display similar age-associated alterations in pro-SPC expression. An age-associated decrease of TAp63 in nuclear lysates was observed in aged monkey group. Moreover, the lungs of 14-mo-old versus 7- to 9-wk-old mice display a wider spreading of ΔNp63-positive CCSP-positive bronchiolar epithelial cells. This expansion did not involve upregulation of Ki67, a representative proliferation marker. Collectively, it is postulated that 1) this expansion is secondary to a transition of progenitor cells committed to club cells from ΔNp63-negative to ΔNp63-positive status, and 2) high levels of cytoplasmic ΔNp63 expression trigger club cell migration.