Human distal airways contain a multipotent secretory cell that can regenerate alveoli.

The human lung differs substantially from its mouse counterpart, resulting in a distinct distal airway architecture affected by disease pathology in chronic obstructive pulmonary disease. In humans, the distal branches of the airway interweave with the alveolar gas-exchange niche, forming an anatomical structure known as the respiratory bronchioles. Owing to the lack of a counterpart in mouse, the cellular and molecular mechanisms that govern respiratory bronchioles in the human lung remain uncharacterized. Here we show that human respiratory bronchioles contain a unique secretory cell population that is distinct from cells in larger proximal airways. Organoid modelling reveals that these respiratory airway secretory (RAS) cells act as unidirectional progenitors for alveolar type 2 cells, which are essential for maintaining and regenerating the alveolar niche. RAS cell lineage differentiation into alveolar type 2 cells is regulated by Notch and Wnt signalling. In chronic obstructive pulmonary disease, RAS cells are altered transcriptionally, corresponding to abnormal alveolar type 2 cell states, which are associated with smoking exposure in both humans and ferrets. These data identify a distinct progenitor in a region of the human lung that is not found in mouse that has a critical role in maintaining the gas-exchange compartment and is altered in chronic lung disease.

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.

Regulation of MHC Class I Expression in Lung Epithelial Cells during Inflammation.

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.

Upregulation of Human Endogenous Retroviruses in Bronchoalveolar Lavage Fluid of COVID-19 Patients.

Severe COVID-19 pneumonia has been associated with the development of intense inflammatory responses during the course of infections with SARS-CoV-2. Given that human endogenous retroviruses (HERVs) are known to be activated during and participate in inflammatory processes, we examined whether HERV dysregulation signatures are present in COVID-19 patients. By comparing transcriptomes of bronchoalveolar lavage fluid (BALF) of COVID-19 patients and healthy controls, and peripheral blood monocytes (PBMCs) from patients and controls, we have shown that HERVs are intensely dysregulated in BALF of COVID-19 patients compared to those in BALF of healthy control patients but not in PBMCs. In particular, upregulation in the expression of specific HERV families was detected in BALF samples of COVID-19 patients, with HERV-FRD being the most highly upregulated family among the families analyzed. In addition, we compared the expression of HERVs in human bronchial epithelial cells (HBECs) without and after senescence induction in an oncogene-induced senescence model in order to quantitatively measure changes in the expression of HERVs in bronchial cells during the process of cellular senescence. This apparent difference of HERV dysregulation between PBMCs and BALF warrants further studies in the involvement of HERVs in inflammatory pathogenetic mechanisms as well as exploration of HERVs as potential biomarkers for disease progression. Furthermore, the increase in the expression of HERVs in senescent HBECs in comparison to that in noninduced HBECs provides a potential link for increased COVID-19 severity and mortality in aged populations. IMPORTANCE SARS-CoV-2 emerged in late 2019 in China, causing a global pandemic. Severe COVID-19 is characterized by intensive inflammatory responses, and older age is an important risk factor for unfavorable outcomes. HERVs are remnants of ancient infections whose expression is upregulated in multiple conditions, including cancer and inflammation, and their expression is increased with increasing age. The significance of this work is that we were able to recognize dysregulated expression of endogenous retroviral elements in BALF samples but not in PBMCs of COVID-19 patients. At the same time, we were able to identify upregulated expression of multiple HERV families in senescence-induced HBECs in comparison to that in noninduced HBECs, a fact that could possibly explain the differences in disease severity among age groups. These results indicate that HERV expression might play a pathophysiological role in local inflammatory pathways in lungs afflicted by SARS-CoV-2 and their expression could be a potential therapeutic target.

Protocol for the generation of murine bronchiolospheres.

Organoid models have been shown to be valuable tools for studying epithelial-mesenchymal crosstalk during biological and pathological settings. Our data identified ACTA2+ PDGFRα+ repair-supportive mesenchymal cells as an important component of the conducting airway niche. Here, we provide a detailed protocol for culturing airway organoids, or bronchiolospheres, which provide an assessment of the ability of mesenchymal cells to support club-cell growth. For complete details on the use and execution of this protocol, please refer to Moiseenko et al. (2020).

SARS-CoV-2 Spike Expression at the Surface of Infected Primary Human Airway Epithelial Cells.

Different serological assays were rapidly generated to study humoral responses against the SARS-CoV-2 Spike glycoprotein. Due to the intrinsic difficulty of working with SARS-CoV-2 authentic virus, most serological assays use recombinant forms of the Spike glycoprotein or its receptor binding domain (RBD). Cell-based assays expressing different forms of the Spike, as well as pseudoviral assays, are also widely used. To evaluate whether these assays recapitulate findings generated when the Spike is expressed in its physiological context (at the surface of the infected primary cells), we developed an intracellular staining against the SARS-CoV-2 nucleocapsid (N) to distinguish infected from uninfected cells. Human airway epithelial cells (pAECs) were infected with authentic SARS-CoV-2 D614G or Alpha variants. We observed robust cell-surface expression of the SARS-CoV-2 Spike at the surface of the infected pAECs using the conformational-independent anti-S2 CV3-25 antibody. The infected cells were also readily recognized by plasma from convalescent and vaccinated individuals and correlated with several serological assays. This suggests that the antigenicity of the Spike present at the surface of the infected primary cells is maintained in serological assays involving expression of the native full-length Spike.

Dysregulation of club cell biology in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive, chronic fibrotic lung disease with an irreversible decline of lung function. "Bronchiolization", characterized by ectopic appearance of airway epithelial cells in the alveolar regions, is one of the characteristic features in the IPF lung. Based on the knowledge that club cells are the major epithelial secretory cells in human small airways, and their major secretory product uteroglobin (SCGB1A1) is significantly increased in both serum and epithelial lining fluid of IPF lung, we hypothesize that human airway club cells contribute to the pathogenesis of IPF. By assessing the transcriptomes of the single cells from human lung of control donors and IPF patients, we identified two SCGB1A1+ club cell subpopulations, highly expressing MUC5B, a significant genetic risk factor strongly associated with IPF, and SCGB3A2, a marker heterogeneously expressed in the club cells, respectively. Interestingly, the cellular proportion of SCGB1A1+MUC5B+ club cells was significantly increased in IPF patients, and this club cell subpopulation highly expressed genes related to mucous production and immune cell chemotaxis. In contrast, though the cellular proportion did not change, the molecular phenotype of the SCGB1A1+SCGB3A2high club cell subpopulation was significantly altered in IPF lung, with increased expression of mucins, cytokine and extracellular matrix genes. The single cell transcriptomic analysis reveals the cellular and molecular heterogeneity of club cells, and provide novel insights into the biological functions of club cells in the pathogenesis of IPF.

Development of a miniaturized 96-Transwell air-liquid interface human small airway epithelial model.

In order to overcome the challenges associated with a limited number of airway epithelial cells that can be obtained from clinical sampling and their restrained capacity to divide ex vivo, miniaturization of respiratory drug discovery assays is of pivotal importance. Thus, a 96-well microplate system was developed where primary human small airway epithelial (hSAE) cells were cultured at an air-liquid interface (ALI). After four weeks of ALI culture, a pseudostratified epithelium containing basal, club, goblet and ciliated cells was produced. The 96-well ALI cultures displayed a cellular composition, ciliary beating frequency, and intercellular tight junctions similar to 24-well conditions. A novel custom-made device for 96-parallelized transepithelial electric resistance (TEER) measurements, together with dextran permeability measurements, confirmed that the 96-well culture developed a tight barrier function during ALI differentiation. 96-well hSAE cultures were responsive to transforming growth factor ß1 (TGF-ß1) and tumor necrosis factor α (TNF-α) in a concentration dependent manner. Thus, the miniaturized cellular model system enables the recapitulation of a physiologically responsive, differentiated small airway epithelium, and a robotic integration provides a medium throughput approach towards pharmaceutical drug discovery, for instance, in respect of fibrotic distal airway/lung diseases.

Intermittent exposure to whole cigarette smoke alters the differentiation of primary small airway epithelial cells in the air-liquid interface culture.

Cigarette smoke (CS) is the leading risk factor to develop COPD. Therefore, the pathologic effects of whole CS on the differentiation of primary small airway epithelial cells (SAEC) were investigated, using cells from three healthy donors and three COPD patients, cultured under ALI (air-liquid interface) conditions. The analysis of the epithelial physiology demonstrated that CS impaired barrier formation and reduced cilia beat activity. Although, COPD-derived ALI cultures preserved some features known from COPD patients, CS-induced effects were similarly pronounced in ALI cultures from patients compared to healthy controls. RNA sequencing analyses revealed the deregulation of marker genes for basal and secretory cells upon CS exposure. The comparison between gene signatures obtained from the in vitro model (CS vs. air) with a published data set from human epithelial brushes (smoker vs. non-smoker) revealed a high degree of similarity between deregulated genes and pathways induced by CS. Taken together, whole cigarette smoke alters the differentiation of small airway basal cells in vitro. The established model showed a good translatability to the situation in vivo. Thus, the model can help to identify and test novel therapeutic approaches to restore the impaired epithelial repair mechanisms in COPD, which is still a high medical need.

Club Cell TRPV4 Serves as a Damage Sensor Driving Lung Allergic Inflammation.

Airway epithelium is the first body surface to contact inhaled irritants and report danger. Here, we report how epithelial cells recognize and respond to aeroallergen alkaline protease 1 (Alp1) of Aspergillus sp., because proteases are critical components of many allergens that provoke asthma. In a murine model, Alp1 elicits helper T (Th) cell-dependent lung eosinophilia that is initiated by the rapid response of bronchiolar club cells to Alp1. Alp1 damages bronchiolar cell junctions, which triggers a calcium flux signaled through calcineurin within club cells of the bronchioles, inciting inflammation. In two human cohorts, we link fungal sensitization and/or asthma with SNP/protein expression of the mechanosensitive calcium channel, TRPV4. TRPV4 is also necessary and sufficient for club cells to sensitize mice to Alp1. Thus, club cells detect junction damage as mechanical stress, which signals danger via TRPV4, calcium, and calcineurin to initiate allergic sensitization.

TiO2 Nanomaterials Non-Controlled Contamination Could Be Hazardous for Normal Cells Located in the Field of Radiotherapy.

Among nanomaterials (NMs), titanium dioxide (TiO2) 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, TiO2 NMs is widely used as a decontaminating agent. Uncontrolled contaminations by TiO2 NMs during their production (professional exposure) or by using products (consumer exposure) are rather frequent. So far, TiO2 NMs cytotoxicity is still a matter of controversy depending on biological models, types of TiO2 NMs, suspension preparation and biological endpoints. TiO2 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, TiO2 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 TiO2 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 TiO2 NMS and radiation.

The role of the non-ciliated bronchiolar cell in tolerance to inhaled vanadium of the bronchiolar epithelium.

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.

An obligatory role for club cells in preventing obliterative bronchiolitis in lung transplants.

Obliterative bronchiolitis (OB) is a poorly understood airway disease characterized by the generation of fibrotic bronchiolar occlusions. In the lung transplant setting, OB is a pathological manifestation of bronchiolitis obliterans syndrome (BOS), which is a major impediment to long-term recipient survival. Club cells play a key role in bronchiolar epithelial repair, but whether they promote lung transplant tolerance through preventing OB remains unclear. We determined if OB occurs in mouse orthotopic lung transplants following conditional transgene-targeted club cell depletion. In syngeneic lung transplants club cell depletion leads to transient epithelial injury followed by rapid club cell-mediated repair. In contrast, allogeneic lung transplants develop severe OB lesions and poorly regenerate club cells despite immunosuppression treatment. Lung allograft club cell ablation also triggers the recognition of alloantigens, and pulmonary restricted self-antigens reported associated with BOS development. However, CD8+ T cell depletion restores club cell reparative responses and prevents OB. In addition, ex-vivo analysis reveals a specific role for alloantigen-primed effector CD8+ T cells in preventing club cell proliferation and maintenance. Taken together, we demonstrate a vital role for club cells in maintaining lung transplant tolerance and propose a new model to identify the underlying mechanisms of OB.

A subpopulation of airway epithelial cells that express hepatocyte nuclear factor 4α - its implication in the development of non-terminal respiratory unit-type lung adenocarcinoma.

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.

Optimal diameter reduction ratio of acinar airways in human lungs.

In the airway network of a human lung, the airway diameter gradually decreases through multiple branching. The diameter reduction ratio of the conducting airways that transport gases without gas exchange is 0.79, but this reduction ratio changes to 0.94 in acinar airways beyond transitional bronchioles. While the reduction in the conducting airways was previously rationalized on the basis of Murray's law, our understanding of the design principle behind the acinar airways has been far from clear. Here we elucidate that the change in gas transfer mode is responsible for the transition in the diameter reduction ratio. The oxygen transfer rate per unit surface area is maximized at the observed geometry of acinar airways, which suggests the minimum cost for the construction and maintenance of the acinar airways. The results revitalize and extend the framework of Murray's law over an entire human lung.

[Chronic diseases, precancer, and cancer of the lung, which are associated with pathology of the club cells of respiratory and terminal bronchioles]. / Khronicheskie zabolevaniia, predrak i rak legkikh, assotsiirovannye s patologiei bulavovidnykh kletok respiratornykh i terminal'nykh bronkhiol.

The review of the literature deals with the participation of Clara cells now called club cells (CCs) of the epithelium in the respiratory and terminal bronchioles in the pathogenesis and morphogenesis of chronic inflammatory diseases, precancer, and cancer of the lung, which develop in the respiratory segments. The review summarizes data on the histophysiology of CCs and their participation in the pathogenesis and morphogenesis of chronic interstitial lung diseases, pneumoconiosis, chronic obstructive diseases, adenomatosis, and adenocarcinoma of the lung. In this area, there is a bronchioloalveolar junction area (BAJA), one of the most important stem cell niches. CCs are located in the BAJA; they are progenitor tissue stem cells and play an important role in the regeneration of the epithelium of the respiratory bronchioles and alveoli. Pathology of CCs in the BAJA leads to the maintenance of chronic inflammation, to the destruction of the lung elastic frame, and to impaired epithelial regeneration, interstitial fibrosis, and adenomatosis. In this case, decompensated inflammation, pathological regeneration, and fibrosis develop, which, along with the action of carcinogenic agents, can contribute to the accumulation of mutations and epigenetic rearrangements in the CCs, which subsequently results in atypical adenomatous hyperplasia and adenocarcinoma of the lung.

Characterization of distal airway stem-like cells expressing N-terminally truncated p63 and thyroid transcription factor-1 in the human lung.

Distal airway stem cells (DASCs) in the mouse lung can differentiate into bronchioles and alveoli. However, it remains unclear whether the same stem cells exist in the human lung. Here, we found that human lung epithelial (HuL) cells, derived from normal, peripheral lung tissue, in monolayer, mostly express both the N-terminally truncated isoform of p63 (∆Np63), a marker for airway basal cells, and thyroid transcription factor-1 (TTF-1), a marker for alveolar epithelial cells, even though these two molecules are usually expressed in a mutually exclusive way. Three-dimensionally cultured HuL cells differentiated to form bronchiole-like and alveolus-like organoids. We also uncovered a few bronchiolar epithelial cells expressing both ∆Np63 and TTF-1 in the human lung, suggesting that these cells are the cells of origin for HuL cells. Taken together, ΔNp63+ TTF-1+ peripheral airway epithelial cells are possibly the human counterpart of mouse DASCs and may offer potential for future regenerative medicine.

Therapeutic targets for inflammation-mediated airway remodeling in chronic lung disease.

INTRODUCTION: Acute exacerbations of chronic lung disease account for substantial morbidity and health costs. Repeated inflammatory episodes and attendant bronchoconstriction cause structural remodeling of the airway. Remodeling is a multicellular response to mucosal injury that results in epithelial cell-state changes, enhanced extracellular deposition, and expansion of pro-fibrotic myofibroblast populations. Areas covered: This manuscript overviews mechanistic studies identifying key sentinel cell populations in the airway and how pattern recognition signaling induces maladaptive mucosal changes and airway remodeling. Studies elucidating how NFκB couples with an atypical histone acetyltransferase, bromodomain-containing protein 4 (BRD4) that reprograms mucosal fibrogenic responses, are described. The approaches to development and characterization of selective inhibitors of epigenetic reprogramming on innate inflammation and structural remodeling in preclinical models are detailed. Expert commentary: Bronchiolar cells derived from Scgb1a1-expressing progenitors function as major sentinel cells of the airway, responsible for initiating antiviral and aeroallergen responses. In these sentinel cells, activation of innate inflammation is coupled to neutrophilic recruitment, mesenchymal transition and myofibroblast expansion. Therapeutics targeting the NFkB-BRD4 may be efficacious in reducing pathological effects of acute exacerbations in chronic lung disease.

Culture and Differentiation of Lung Bronchiolar Epithelial Cells In Vitro.

Club cells are a major bronchiolar epithelial cell type in the lung. Using genetic lineage tracing in mice and in vitro culture of purified cells, we have shown that club cells can differentiate into alveolar type I and II cells. Here we describe the detailed protocol for culturing and differentiating club cells in 3-dimensional culture.

Cell Division Cycle 42 plays a Cell type-Specific role in Lung Tumorigenesis.

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.