Search

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. [ABSTRACT FROM AUTHOR]

Primary bronchial epithelial cells (pBECs) obtained from donors have limited proliferation capacity. Recently, conditional reprogramming (CR) technique has overcome this and has provided the potential for extended passaging and subsequent differentiation of cells at air-liquid interface (ALI). However, there has been no donor-specific comparison of cell morphology, baseline gene expression, barrier function, and antiviral responses compared with their "parent" pBECs, especially cells obtained from donors with asthma. We, therefore, collected and differentiated pBECs at ALI from mild donors with asthma (n = 6) for the parent group. The same cells were conditionally reprogrammed and later differentiated at ALI. Barrier function was measured during the differentiation phase. Morphology and baseline gene expression were compared at terminal differentiation. Viral replication kinetics and antiviral responses were assessed following rhinovirus (RV) infection over 96 h. Barrier function during the differentiation phase and cell structural morphology at terminal differentiation appear similar in both parent and CR groups, however, there were elongated cell structures superficial to basal cells and significantly lower FOXJ1 expression in CR group. IFN gene expression was also significantly lower in CR group compared with parent asthma group following RV infection. The CR technique is a beneficial tool to proliferate pBECs over extended passages. Considering lower FOXJ1 expression, viral replication kinetics and antiviral responses, a cautious approach should be taken while choosing CR cells for experiments. In addition, as lab-to-lab cell culture techniques vary, the most appropriate technique must be utilized to best match individual cell functions and morphologies to address specific research questions and experimental reproducibility across the labs. [ABSTRACT FROM AUTHOR]

Alveolar epithelial cell (AEC) senescence is implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Mitochondrial dysfunction including release of mitochondrial DNA (mtDNA) is a feature of senescence, which led us to investigate the role of the DNA-sensing guanine monophosphate-adenine monophosphate (GMP-AMP) synthase (cGAS) in IPF, with a focus on AEC senescence. cGAS expression in fibrotic tissue from lungs of patients with IPF was detected within cells immunoreactive for epithelial cell adhesion molecule (EpCAM) and p21, epithelial and senescence markers, respectively. Submerged primary cultures of AECs isolated from lung tissue of patients with IPF (IPF-AECs, n = 5) exhibited higher baseline senescence than AECs from control donors (Ctrl-AECs, n = 5–7), as assessed by increased nuclear histone 2AXγ phosphorylation, p21 mRNA, and expression of senescence-associated secretory phenotype (SASP) cytokines. Pharmacological cGAS inhibition using RU.521 diminished IPF-AEC senescence in culture and attenuated induction of Ctrl-AEC senescence following etoposide-induced DNA damage. Short interfering RNA (siRNA) knockdown of cGAS also attenuated etoposide-induced senescence of the AEC line, A549. Higher levels of mtDNA were detected in the cytosol and culture supernatants of primary IPF- and etoposide-treated Ctrl-AECs when compared with Ctrl-AECs at baseline. Furthermore, ectopic mtDNA augmented cGAS-dependent senescence of Ctrl-AECs, whereas DNAse I treatment diminished IPF-AEC senescence. This study provides evidence that a self-DNA-driven, cGAS-dependent response augments AEC senescence, identifying cGAS as a potential therapeutic target for IPF. [ABSTRACT FROM AUTHOR]

The recurrent emergence of novel, pathogenic coronaviruses (CoVs) severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1; 2002), Middle East respiratory syndrome (MERS)-CoV (2012), and most recently SARS-CoV-2 (2019) has highlighted the need for physiologically informative airway epithelial cell infection models for studying immunity to CoVs and development of antiviral therapies. To address this, we developed an in vitro infection model for two human coronaviruses; alphacoronavirus 229E-CoV (229E) and betacoronavirus OC43-CoV (OC43) in differentiated primary human bronchial epithelial cells (pBECs). Primary BECs from healthy subjects were grown at air-liquid interface (ALI) and infected with 229E or OC43, and replication kinetics and timecourse expression of innate immune mediators were assessed. OC43 and 229E-CoVs replicated in differentiated pBECs but displayed distinct replication kinetics: 229E replicated rapidly with viral load peaking at 24 h postinfection, while OC43 replication was slower peaking at 96 h after infection. This was associated with diverse antiviral response profiles defined by increased expression of type I/III interferons and interferon-stimulated genes (ISGs) by 229E compared with no innate immune activation with OC43 infection. Understanding the host-virus interaction for previously established coronaviruses will give insight into pathogenic mechanisms underpinning SARS-CoV-2-induced respiratory disease and other future coronaviruses that may arise from zoonotic sources. [ABSTRACT FROM AUTHOR]

Asthma is a common chronic inflammatory disease associated with intermittent airflow obstruction caused by airway inflammation, mucus overproduction, and bronchial hyperresponsiveness. Despite current treatment and management options, a large number of patients with asthma still have poorly controlled disease and are susceptible to acute exacerbations, usually caused by a respiratory virus infection. As a result, there remains a need for novel therapies to achieve better control and prevent/treat exacerbations. Nanoparticles (NPs), including extracellular vesicles (EV) and their synthetic counterparts, have been developed for drug delivery in respiratory diseases. In the case of asthma, where airway epithelium dysfunction, including dysregulated differentiation of epithelial cells, impaired barrier, and immune response, is a driver of disease, targeting airway epithelial cells with NPs may offer opportunities to repair or reverse these dysfunctions with therapeutic interventions. EVs possess multiple advantages for airway epithelial targeting, such as their natural intrinsic cell-targeting properties and low immunogenicity. Synthetic NPs can be coated with muco-inert polymers to overcome biological barriers such as mucus and the phagocytic response of immune cells. Targeting ligands could be also added to enhance targeting specificity to epithelial cells. The review presents current understanding and advances in NP-mediated drug delivery to airway epithelium for asthma therapy. Future perspectives in this therapeutic strategy will also be discussed, including the development of novel formulations and physiologically relevant preclinical models. [ABSTRACT FROM AUTHOR]

Patients with frequent exacerbations represent a chronic obstructive pulmonary disease (COPD) subgroup requiring better treatment options. The aim of this study was to determine the innate immune mechanisms that underlie susceptibility to frequent exacerbations in COPD. We measured sputum expression of immune mediators and bacterial loads in samples from patients with COPD at stable state and during virus-associated exacerbations. In vitro immune responses to rhinovirus infection in differentiated primary bronchial epithelial cells (BECs) sampled from patients with COPD were additionally evaluated. Patients were stratified as frequent exacerbators (≥2 exacerbations in the preceding year) or infrequent exacerbators (<2 exacerbations in the preceding year) with comparisons made between these groups. Frequent exacerbators had reduced sputum cell mRNA expression of the antiviral immune mediators type I and III interferons and reduced interferon-stimulated gene (ISG) expression when clinically stable and during virus-associated exacerbation. A role for epithelial cell-intrinsic innate immune dysregulation was identified: induction of interferons and ISGs during in vitro rhinovirus (RV) infection was also impaired in differentiated BECs from frequent exacerbators. Frequent exacerbators additionally had increased sputum bacterial loads at 2 wk following virus-associated exacerbation onset. These data implicate deficient airway innate immunity involving epithelial cells in the increased propensity to exacerbations observed in some patients with COPD. Therapeutic approaches to boost innate antimicrobial immunity in the lung could be a viable strategy for prevention and treatment of frequent exacerbations. [ABSTRACT FROM AUTHOR]