Titolo della tesi: Microbial Interactions and Interferon Signaling in Cystic Fibrosis Airway Inflammation
Cystic fibrosis (CF) is the most common autosomal recessive disorder among Caucasians and is characterized by a broad spectrum of pulmonary and extrapulmonary manifestations resulting from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. These mutations impair chloride transport and enhance sodium absorption, leading to dehydrated airway surfaces and chronic colonization by motile and adaptable bacteria. Although Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) are extensively studied, the airway microbiota in CF is highly heterogeneous reflecting the unique environment of the CF lung. Less-characterized viruses and uncommon bacterial species can persist as long-term colonizers or behave as active pathogens, shaping innate immune responses and potentially driving the progression of disease.
Type I and III interferons (IFNs) have emerged as key players in the innate immune landscape of CF, balancing antiviral defense with the modulation of airway inflammation. Their activation is often sustained by continuous microbial exposure, and when dysregulated, can contribute to the persistent inflammatory state that characterizes CF lung disease. Unraveling how these IFN pathways respond to specific pathogens and how they are reshaped by CFTR modulator therapies is essential for improving long-term patient outcomes and advancing personalized treatment strategies.
The study is structured into three interconnected sections, each exploring complementary aspects of airway type I/III IFN regulation in CF through an integrated virological, bacteriological, and modulator therapy perspective, with the aim of deepening our understanding of airway immunology.
The role of Merkel cell polyomavirus (MCPyV) as a respiratory pathogen is controversial and has received little research attention. MCPyV and other oncogenic dsDNA viruses have been shown to evade immune recognition by altering Toll-like receptor 9 (TLR9) expression and downstream signaling. Interestingly, CF patients with rhinosinusitis exhibit altered TLR9 expression in the upper respiratory tract compared to healthy individuals. MCPyV DNA was detected in 268 of 1,138 respiratory samples from 539 CF patients, with 25.4% testing positive at least once, supporting the hypothesis that the conditions of the respiratory tract of CF patients may favour MCPyV persistence. Gene expression analysis of TLR9, IFNβ, IFNε, and IFNα was performed on cells from respiratory samples using RT/Real Time-PCR in 95 MCPyV-positive and 147 negative patients, stratified by age. When comparing MCPyV-positive and negative patients, MCPyV-positive children displayed higher expression levels of TLR9, IFNβ, IFNε, and IFNα (p = 0.02, p < 0.001, p = 0.002, p = 0.27), whereas MCPyV-positive adolescents and adults showed reduced expression (p < 0.001, p = 0.01, p = 0.02, p = 0.26; p = 0.04, p = 0.001, p < 0.001, p = 0.12, respectively). These findings suggest a primary immune activation in children, in contrast to possible viral reactivation and TLR9 modulation in older patients. In line with this, TLR9 levels inversely correlated with age in MCPyV-positive patients (r = –0.34, p = 0.001). Microbiological status influenced TLR9 expression: lower in MCPyV-positive patients with P. aeruginosa (p = 0.009), higher with S. aureus (p = 0.006), and reduced in co-infected individuals (p = 0.003).
The Lazio Regional Reference Center for Cystic Fibrosis, based at Policlinico Umberto I – Sapienza University of Rome, provides care to approximately 600 patients annually. Among them, a family cluster of four siblings with CF was analyzed, three of whom were chronically colonized by Pandoraea spp., an uncommon and multidrug-resistant environmental bacterium. The clinical history of these patients was retrospectively reviewed from 2008 onward, tracing the progression of their respiratory condition and associated microbiological findings. Initial cultures revealed a non-fermenting Gram-negative bacillus, initially misidentified as Brevundimonas vesicularis and Cupriavidus pauculus, and subsequently reclassified as Pandoraea vervacti through MALDI-TOF mass spectrometry, with confirmation obtained via 16S rRNA and gyrB gene sequencing. Among 15 respiratory samples compared with those from 31 Pandoraea-negative CF patients, the colonized individuals showed significantly higher transcript levels of IFNβ, IFNε, and the type III IFN receptor IL28R1 (p = 0.002, p = 0.033, p < 0.01), indicating a distinct and potentially pro-inflammatory mucosal immune response. By contrast no difference was recorded for IFNα, IFNλ1 and IFNλ2/3 (p > 0.05). The absence of colonization in the sibling who had undergone lung transplantation suggests that a pre-existing inflammatory airway environment may be necessary for the pathogen’s persistence.
The advent of CFTR modulators has revolutionized CF management. In particular, the triple combination therapy elexacaftor/tezacaftor/ivacaftor (ETI) has led to substantial improvements in lung function, nutritional status, and overall quality of life. The clinical and immunological effects of CFTR modulator therapy with ETI were evaluated in 26 CF patients carrying at least one F508del mutation. After 6 months, significant improvements were observed in lung function (+17.6% ppFEV1, p < 0.001), body mass index (+1.4 kg/m², p < 0.001), and sweat chloride concentration (–44.4 mEq/L, p = 0.001). No hospitalizations for respiratory exacerbations occurred during the observation period. A total of 74 respiratory samples were collected before ETI treatment and at 3 and 6 months. Expression levels of type I/III IFNs, IL28R1, IFN-stimulated genes ISG15 and ISG56, and inflammatory cytokines IL-8 and IL-1β were analyzed in airway samples to clarify ETI’s role in modulating innate immune responses. Increased levels of IFNβ (p < 0.001) were found after 6 months of ETI therapy, while ISG15 and ISG56 decreased significantly (p = 0.022, p = 0.004). After 3 months, CF patients produced reduced levels of IL-8 and IL-1β (p = 0.001, p = 0.011) and even significantly lower levels after 6 months (p < 0.001, p < 0.001).