Chronic Obstructive Pulmonary Disease (COPD) is a significant global health concern, characterized by progressive airflow limitation, heterogeneous endophenotypes, and varied disease trajectories among patients. Current research is hampered by the lack of suitable in vitro models that accurately reflect individual patient variations. While animal models have been used, they fail to fully replicate the genetic and epigenetic complexity of human COPD. The development of readily accessible in vitro models that reproduce the COPD airway microenvironment at the individual level is crucial for understanding molecular mechanisms underlying infection and treatment responses. Stem cell-derived organoids, offering a three-dimensional structure incorporating multiple cell types, represent a promising advancement in this area. Human lung organoids have been successfully generated from various tissues and used to study various diseases and infections, including SARS-CoV-2. Given the clinical association between COPD and severe COVID-19 outcomes, developing a COPD organoid model is critical to investigate the cellular mechanisms underlying this increased risk. This study aimed to establish and characterize COPD lung organoids to study host-pathogen interactions, focusing on SARS-CoV-2 and Pseudomonas aeruginosa infections, to elucidate the underlying mechanisms connecting COPD and severe COVID-19.

The existing literature highlights the significant challenges in modeling COPD in vitro due to its heterogeneity and complexity. While animal models offer some insights, their limitations in replicating human genetics and epigenetics necessitate alternative approaches. The emergence of stem cell-derived organoids has provided a new avenue for studying complex diseases like COPD. Previous studies have successfully generated lung organoids from various tissues, demonstrating their utility in studying lung cancer, cystic fibrosis, and viral infections, including influenza and RSV. However, a dedicated COPD organoid model remained absent, despite the clinical evidence linking COPD to severe COVID-19 outcomes. Existing research on SARS-CoV-2 infection suggests a potential link between the virus's interaction with airway cells and the increased susceptibility of COPD patients. Studies have shown that bacteria play a significant role in COPD exacerbations, with Streptococcus pneumoniae and Pseudomonas aeruginosa being commonly identified.

This study involved the establishment and characterization of COPD lung organoids derived from adult stem cells to investigate host-pathogen interactions. The researchers recruited 28 individuals, including 10 healthy controls and 18 COPD patients, categorized by GOLD stage and exacerbation frequency. Nasopharyngeal and bronchial specimens were obtained as starting materials. Human nasopharyngeal epithelial cells (HNPECs) were isolated from nasopharyngeal swabs, and human bronchial epithelial cells (HBECs) were isolated from bronchial samples using established protocols. Cells were cultured in appropriate media to achieve confluency, then seeded onto transwells for air-liquid interface (ALI) culture. Following differentiation into airway organoids, the researchers performed immunofluorescence staining to identify cell types (basal, club, ciliated, and goblet cells) and quantify their abundance. Quantitative PCR (qPCR) was used to analyze gene expression levels of key markers. Single-cell RNA sequencing (scRNA-seq) was conducted to characterize the cellular composition and transcriptomes of both healthy and COPD organoids. The researchers also assessed ciliary beat frequency (CBF) using micro-optical coherence tomography (µOCT). SARS-CoV-2 infection studies used three distinct SARS-CoV-2 strains (Clade L, O, and G) at a multiplicity of infection (MOI) of 0.1. Viral replication kinetics were assessed using TCID50 assays and qPCR to measure viral load. Cytokine production was analyzed using qPCR and Luminex assays. Bacterial infection studies utilized Pseudomonas aeruginosa and Streptococcus pneumoniae to assess the inflammatory response in COPD organoids. Statistical analyses were performed using appropriate methods for normally and non-normally distributed data. Bulk RNA sequencing was performed on mock-treated and P. aeruginosa-infected NPOs from healthy and COPD subjects to further analyze gene expression changes.

The researchers successfully established and characterized COPD lung organoids that recapitulated key features of the disease. COPD organoids showed goblet cell hyperplasia and significantly reduced ciliary beat frequency compared to healthy organoids. The extent of goblet cell hyperplasia correlated with disease severity and exacerbation frequency in the donors. Single-cell transcriptomics revealed altered cellular differentiation trajectories in COPD organoids, particularly affecting basal cell differentiation into ciliated and goblet cell lineages. SARS-CoV-2 infection experiments demonstrated enhanced viral replication in COPD bronchial organoids (BOs) compared to healthy BOs, especially with the G-614G variant. This increased replication was accompanied by a reduced interferon-β (IFN-β) response in COPD BOs. Interestingly, in nasopharyngeal organoids (NPOs), while the G-614G variant exhibited higher replication, COPD NPOs did not show enhanced replication compared to healthy NPOs. Bacterial infection (P. aeruginosa and S. pneumoniae) induced greater pro-inflammatory responses in COPD NPOs than in healthy NPOs. Bulk transcriptomic analysis of P. aeruginosa-infected COPD NPOs revealed impaired ciliary movement and increased secretory and extracellular matrix remodeling. The study also found a strong inverse relationship between MUC5AC gene expression and FEV1% predicted in COPD organoids.

This study successfully demonstrates a novel in vitro model for COPD using patient-derived organoids that closely reflects the in vivo physiological lung microenvironment. The findings offer crucial insights into the pathogenesis of COPD and its impact on host-pathogen interactions, especially in the context of SARS-CoV-2 infection. The enhanced viral replication observed in COPD bronchi, coupled with impaired interferon responses, provides a potential mechanistic explanation for the increased susceptibility of COPD patients to severe COVID-19. The increased pro-inflammatory response to bacterial infections further highlights the vulnerability of COPD airways to exacerbations. The use of single-cell transcriptomics provided a comprehensive understanding of cellular heterogeneity and the dysregulation of cellular differentiation processes in COPD. This detailed analysis reveals potential therapeutic targets that can be further explored. The model's ability to capture individual variations makes it a powerful tool for personalized medicine approaches to COPD management. The study confirms the significant role of MUC5AC as a potential biomarker for COPD prognosis.

This study successfully generated and characterized COPD organoids, providing a valuable in vitro model for studying host-pathogen interactions in COPD. This model accurately reflects key pathological features of COPD and demonstrates enhanced SARS-CoV-2 replication and impaired antiviral responses in COPD bronchi. The model also shows a heightened pro-inflammatory response to bacterial infections. Future research should expand the sample size and include additional viral and bacterial strains to confirm and strengthen these findings. Further investigations could focus on exploring the impact of different therapeutic interventions on viral and bacterial infections in this model.

The study has several limitations. Nasopharyngeal and bronchial samples were collected from different donors, not paired samples from the same individual. Healthy control donors were relatively younger than the COPD patients. The scRNA-seq analysis was based on a limited number of samples per group. The organoid model lacks the complexity of the in vivo lung environment, such as the immune and vascular systems. Safety and logistical constraints prevented performing scRNA-seq on SARS-CoV-2 infected organoids.