Microbial interactions and interferon signaling in cystic fibrosis airway inflammation

Cystic fibrosis (CF) is the most common autosomal recessive disorder among Caucasians. It is caused by mutations in the CFTR gene. These mutations impair chloride transport and increase sodium absorption, resulting in dehydrated airway surfaces and chronic colonization by adaptable motile bacteria. Although Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) are well-characterized pathogens, the CF airway microbiota is highly diverse and reflects the unique immunological landscape of the CF lung. In addition to bacterial infections, respiratory viruses play a significant role in CF pathogenesis. They impair antibacterial defences and fuel inflammation, often exacerbating disease progression. Type I and III interferons (IFNs) are key regulators of innate immunity, with antiviral and immunomodulatory functions. However, CF airway cells exhibit reduced type I IFN levels and impaired dendritic cell activation in response to bacterial stimulation, indicating a diminished antiviral response. Our research group has long been dedicated to studying CF, particularly viral infections and the antiviral role of IFN signalling pathways. Over the years, we have investigated how respiratory viruses interact with the CF airway environment, as well as the contribution of IFNs to protective immunity and chronic inflammation. This background has shaped our current research project, which aims to explore the interplay between rare pathogens and immune regulation in CF. The project will investigate the contribution of understudied viruses and rare bacteria to the inflammatory landscape of CF, focusing particularly on their interaction with IFN signalling. While the immune response to common respiratory pathogens in CF is well documented, the role of less prevalent microbes remains poorly understood. These organisms may persist as colonizers or act as active pathogens, influencing innate immunity and contributing to chronic inflammation. A comprehensive experimental approach was adopted to explore these dynamics. Respiratory samples were collected from patients attending the Regional Reference Centre for CF at the Policlinico Umberto I of Sapienza University in Rome. Clinical data were retrieved from medical records. Although follow-up intervals varied, most patients were evaluated every three months. During these visits, respiratory samples were obtained for routine microbiological analysis and research purposes. Molecular analyses were performed to assess gene expression and viral presence. Total RNA was extracted from the samples and converted to cDNA using standardised kits. Quantitative real-time RT-PCR was used to measure the expression of key immune genes, including TLR9, IFNα, IFNβ, IFNε, IFNλ1, IFNλ2/3, IFNLR1, ISG15, ISG56, IL-8 and IL-1β. DNA was also extracted to detect Merkel cell polyomavirus (MCPyV) using TaqMan-based PCR assays targeting the small T antigen. The role of MCPyV as a respiratory pathogen is controversial and has received little research attention. A large cohort of 539 CF patients was screened for MCPyV, resulting in 1,138 respiratory samples being analysed. MCPyV DNA was detected in 23.5% of the samples, with 25.4% of the patients testing positive at least once. Seasonal variation was noted, with peak prevalence in February. Gene expression analysis revealed age-dependent differences in immune responses. MCPyV-positive children exhibited elevated levels of TLR9 and type I IFNs, whereas adolescents and adults showed reduced expression. These results imply that MCPyV may modulate immune signalling in different ways depending on the stage of infection and the age of the host. One particularly compelling aspect of the PhD project was a family cluster of four cystic fibrosis (CF) siblings who carried the W1282X/3849+10 kb C→T genotype. Three of the siblings were persistently colonised by Pandoraea vervacti, a rare, multidrug-resistant environmental bacterium. Accurate identification was achieved through MALDI-TOF MS and gene sequencing. Gene expression profiling of airway samples from these patients revealed significantly higher levels of IFNβ, IFNε and IFNLR1 than in matched P. vervacti-negative controls, suggesting a distinct mucosal immune response that may be linked to chronic inflammation and disease progression. The final experimental arm focused on the effects of CFTR modulator therapy using elexacaftor/tezacaftor/ivacaftor (ETI). Twenty-six CF patients carrying at least one F508del mutation were enrolled in the study prior to initiating ETI treatment. Respiratory samples were collected at baseline and again at three and six months post-treatment. Clinical outcomes showed significant improvements in lung function (ppFEV1), BMI, and sweat chloride levels, as well as a notable decrease in pulmonary exacerbations. Molecular analysis revealed dynamic changes in immune gene expression following ETI therapy. After six months, IFNβ transcript levels increased significantly while ISG15 and ISG56 levels decreased, indicating a shift in IFN signalling. The pro-inflammatory cytokines IL-8 and IL-1β were dramatically downregulated, with reductions of up to 48-fold, suggesting that ETI therapy promotes a more anti-inflammatory airway environment. In conclusion, this research project highlights the complex interplay between microbial colonization and immune regulation in CF. Emerging viruses and rare bacteria, such as MCPyV and Pandoraea spp., may significantly influence IFN signalling and inflammation. While ETI therapy offers substantial clinical benefits, its modulation of immune gene expression merits further investigation. These findings highlight the importance of understanding how microbial diversity and CFTR modulation influence the evolving immunopathology of CF.