Club cells are the primary source of pIgR in small airways

Background: Loss of secretory immunoglobulin A (SIgA) is common in COPD small airways and likely contributes to disease progression. We hypothesized loss of SIgA results from reduced numbers of cells expressing pIgR, a chaperone protein needed for SIgA transcytosis, in the COPD small airway epithelium. Methods: pIgR-expressing cells were defined and quantified at single-cell resolution in human airways using RNA in-situ hybridization, immunostaining, and single-cell RNA sequencing. Complementary studies in mice utilized immunostaining, primary murine tracheal epithelial cell (MTEC) culture, and transgenic mice with club or ciliated cell-specific knockout of pIgR. SIgA degradation by human neutrophil elastase or secreted bacterial proteases from non-typeable Haemophilus influenzae (NTHi) was evaluated in vitro. Results: Club cells are the predominant cell type responsible for pIgR expression in human and murine airways, but neither loss of pIgR-expressing club cells or reduced pIgR expression in individual cells fully explains loss of SIgA in the airways in patients with advanced COPD. In contrast, we found that neutrophil elastase and NTHi-secreted proteases degrade SIgA in vitro. Interpretation: Club cells are the dominant cell type responsible for pIgR expression and thus play a key role in the maintaining the secretory IgA immunobarrier. However, neither a loss of club cells or reduced pIgR expression within individual cells fully accounts for loss of SIgA in COPD.

A role for club cells in smoking-associated lung adenocarcinoma

The cellular origin of lung adenocarcinoma remains a focus of intense research efforts. The marked cellular heterogeneity and plasticity of the lungs, as well as the vast variety of molecular subtypes of lung adenocarcinomas perplex the field and account for the extensive variability of experimental results. While most experts would agree on the cellular origins of other types of thoracic tumours, great controversy exists on the tumour-initiating cells of lung adenocarcinoma, since this histologic subtype of lung cancer arises in the distal pulmonary regions where airways and alveoli converge, occurs in smokers as well as nonsmokers, is likely caused by various environmental agents, and is marked by vast molecular and pathologic heterogeneity. Alveolar type II, club, and their variant cells have all been implicated in lung adenocarcinoma progeny and the lineage hierarchies in the distal lung remain disputed. Here we review the relevant literature in this rapidly expanding field, including results from mouse models and human studies. In addition, we present a case for club cells as cells of origin of lung adenocarcinomas that arise in smokers.

The Diagnostic Challenges of Ovine Pulmonary Adenocarcinoma

Ovine pulmonary adenocarcinoma (OPA), also known as sheep pulmonary adenomatosis and jaagsiekte, is a contagious pulmonary tumor of sheep, characterized by neoplastic proliferation of type II pneumocyte and club cells. OPA is induced by the oncogenic activity of the envelope glycoprotein (Env) of exogenous jaagsiekte sheep retrovirus (JSRV). This disease is associated with significant economic losses in numerous sheep raising countries. The onset of suggestive clinical signs is often late, making difficult the early diagnosis of the disease and timely implementation of control measures on the affected farms. Further, the lack of diagnostic tests that can be performed routinely by veterinary clinicians to accurately assess infected animals (e.g., serological or others) means that the true prevalence at flock level is not known. Imaging diagnostic methods (e.g., ultrasound, X-ray and computed tomography) can be used to support the clinical diagnosis, even in pre-clinical stages in affected flocks. The diagnosis must be confirmed by PCR of nasal excretions or immunohistochemistry and PCR of tumor lesions. No vaccine for OPA has yet been developed. Thus, in this work, we review the main methods of diagnosis of OPA in order to support the clinician in the identification of the disease, avoid underdiagnosis and allow the implementation of suitable measures to prevent and control its spread.

Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

The club cell, a small airway epithelial (SAE) cell, plays a central role in human lung host defense. We hypothesized that subpopulations of club cells with distinct functions may exist. The SAE of healthy nonsmokers and healthy cigarette smokers were evaluated by single-cell RNA sequencing, and unsupervised clustering revealed subpopulations of SCGCB1A1+KRT5loMUC5AC− club cells. Club cell heterogeneity was supported by evaluations of SAE tissue sections, brushed SAE cells, and in vitro air–liquid interface cultures. Three subpopulations included: (1) progenitor; (2) proliferating; and (3) effector club cells. The progenitor club cell population expressed high levels of mitochondrial, ribosomal proteins, and KRT5 relative to other club cell populations and included a differentiation branch point leading to mucous cell production. The small proliferating population expressed high levels of cyclins and proliferation markers. The effector club cell cluster expressed genes related to host defense, xenobiotic metabolism, and barrier functions associated with club cell function. Comparison of smokers vs. nonsmokers demonstrated that smoking limited the extent of differentiation of all three subclusters and altered SAM pointed domain-containing Ets transcription factor (SPDEF)-regulated transcription in the effector cell population leading to a change in the location of the branch point for mucous cell production, a potential explanation for the concomitant reduction in effector club cells and increase in mucous cells in smokers. These observations provide insights into both the makeup of human SAE club cell subpopulations and the smoking-induced changes in club cell biology.

Cross-talk of inflammatory mediators and airway epithelium reveals CFTR as a major target

Airway inflammation, mucus hyperproduction and epithelial remodelling are hallmarks of many chronic airway diseases, including asthma, Chronic Obstructive Pulmonary Disease and Cystic Fibrosis. While several cytokines are dysregulated in these diseases, most studies focus on the response of airways to IL-4 and IL-13, which were shown to induce mucus hyperproduction and shift the airway epithelium towards a hypersecretory phenotype.We hypothesised that other cytokines might induce the expression of chloride (Cl−) channels/transporters, regulate epithelial differentiation and mucus production. To this end, fully-differentiated human airway basal cells (BCi-NS1.1) were treated with cytokines identified as dysregulated in those diseases, namely interleukins-8, 1β, 4, 17A, 10, 22, and tumour necrosis factor-α (TNF-α).Our results show that CFTR is the main Cl− channel modulated by inflammation, in contrast to TMEM16A, whose levels only changed with IL-4. Furthermore, we identified novel roles for IL-10 and IL-22 by influencing epithelial differentiation towards ciliated cells and away from pulmonary ionocytes. Contrarily, IL-1β and IL-4 reduced the number of ciliated cells while increasing club cells. Interestingly, while IL-1β, IL-4 and IL-10 upregulated CFTR expression, IL-4 was the only cytokine that increased both its function and the number of CFTR-expressing club cells, suggesting that this cell-type may be the main contributor for CFTR function. Additionally, all cytokines assessed increased mucus production through a differential upregulation of MUC5AC and MUC5B transcript levels.Altogether, this study reveals a novel insight into differentiation resulting from the cross-talk of inflammatory mediators and airway epithelial cells, which is particularly relevant for chronic airway diseases.

Inhibition of PDIA3 in club cells attenuates osteopontin production and lung fibrosis

BackgroundThe role of club cells in the pathology of idiopathic pulmonary fibrosis (IPF) is not well understood. Protein disulfide isomerase A3 (PDIA3), an endoplasmic reticulum-based redox chaperone required for the functions of various fibrosis-related proteins; however, the mechanisms of action of PDIA3 in pulmonary fibrosis are not fully elucidated.ObjectivesTo examine the role of club cells and PDIA3 in the pathology of pulmonary fibrosis and the therapeutic potential of inhibition of PDIA3 in lung fibrosis.MethodsRole of PDIA3 and aberrant club cells in lung fibrosis was studied by analyses of human transcriptome dataset from Lung Genomics Research Consortium, other public resources, the specific deletion or inhibition of PDIA3 in club cells and blocking SPP1 downstream of PDIA3 in mice.ResultsPDIA3 and club cell secretory protein (SCGB1A1) signatures are upregulated in IPF compared with control patients. PDIA3 or SCGB1A1 increases also correlate with a decrease in lung function in patients with IPF. The bleomycin (BLM) model of lung fibrosis showed increases in PDIA3 in SCGB1A1 cells in the lung parenchyma. Ablation of Pdia3, specifically in SCGB1A1 cells, decreases parenchymal SCGB1A1 cells along with fibrosis in mice. The administration of a PDI inhibitor LOC14 reversed the BLM-induced parenchymal SCGB1A1 cells and fibrosis in mice. Evaluation of PDIA3 partners revealed that SPP1 is a major interactor in fibrosis. Blocking SPP1 attenuated the development of lung fibrosis in mice.ConclusionsOur study reveals a new relationship with distally localised club cells, PDIA3 and SPP1 in lung fibrosis and inhibition of PDIA3 or SPP1 attenuates lung fibrosis.

Diverse routes of Club cell evolution in lung adenocarcinoma

The high plasticity of lung epithelial cells, has for many years, confounded the correct identification of the cell-of-origin of lung adenocarcinoma (LUAD), one of the deadliest malignancies worldwide. Here, we address the cell-of-origin of LUAD, by employing lineage-tracing mouse models combined with a CRISPR/Cas9 system to induce an oncogenic Eml4-Alk rearrangement in virtually all epithelial cell types of the lung. We find that Club cells give rise to lung tumours with a higher frequency than AT2 cells. Based on whole genome methylome, we identified that tumours retain an epigenetic memory derived from their originating cell type but also develop a tumour-specific pattern regardless of their origin. Single-cell transcriptomic analyses identified two trajectories of Club cell evolution which are similar to the ones used during lung regeneration, providing a link between lung regeneration and cancer initiation. On both routes, tumours lose their Club cell identity and gain an AT2-like phenotype. Together, this study highlights the role of Club cells in LUAD initiation and unveils key mechanisms conferring LUAD heterogeneity.