INNATE IMMUNE SYSTEM IN THE PATHOGENESIS OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE

Chronic obstructive pulmonary disease (COPD) is a progressive inflammatory disorder of the airways, currently ranked as the fourth leading cause of death worldwide, with rising morbidity and mortality. Traditionally, it is considered a lung-restricted disease; increasing evidence over the past two decades has revealed COPD as a systemic condition, frequently associated with comorbidities and characterized by persistent systemic inflammation. Among the hallmarks of systemic involvement are elevated circulating inflammatory markers, such as C-reactive protein (CRP), and dysregulation of peripheral blood leukocytes, particularly neutrophils. However, the literature on the role and phenotype of circulating neutrophils in COPD remains controversial. Given the limited and conflicting data on circulating neutrophils, this study aims to comprehensively characterise the transcriptional, epigenetic, and functional profiles of circulating neutrophils from COPD patients and to explore their potential contribution to disease pathology. Blood samples have been collected from 39 COPD patients (22 male, 17 female) and 38 age- and sex-matched controls (22 male, 16 female). Human neutrophils (PMNs) were isolated by Ficoll-Paque gradient followed by dextran sedimentation and hypotonic lysis of erythrocytes, under endotoxin-free conditions. Flow cytometric analysis indicated that circulating neutrophils from COPD patients do not display an “activated” phenotype, in terms of modulated expression of CD11b, CD11c, CD35, CD62L, and CD16 activation markers. Transcriptomic analysis identified 718 genes whose expression is upregulated in neutrophils from COPD donors compared to controls (P < 0,05). Gene Ontology (GO) enrichment analysis of upregulated genes identified different pathways upregulated in COPD patients. Among these latter, the interferon (IFN)/viral response pathways were significantly enriched. The presence of this IFN signature was further confirmed by Gene Set Variation Analysis (GSVA) for IFNα and IFNγ response scores. Importantly, the induction of the interferon signature was further supported by the detection of elevated serum levels of IFN-α and IFN-γ in COPD patients. Epigenetic analysis of H3K4me3 of peripheral neutrophils from COPD patients showed an increased H3K4me3 at the promoter level of genes related to cell responsiveness, cytokines, and inflammation. In particular, and consistent with what has been described in “trained” neutrophils, COPD neutrophils exhibited transcriptional reprogramming of metabolically relevant genes. Indeed, gene set variation analysis (GSVA) highlighted a significant (P 4 <0.0001) upregulation of genes previously associated with trained immunity in neutrophils from COPD patients as compared to controls. To verify that the “trained” signature is representative of a true “trained” phenotype, we performed a preliminary study in a pilot cohort of 7 COPD patients and 7 age- and sex-matched controls. Neutrophils from COPD subjects showed increased ROS production in response to PMA and increased C. albicans phagocytosis and an increase in the release of pro-inflammatory cytokines like IL1 and CXCL8 compared to controls. These findings suggest that COPD neutrophils undergo epigenetic, transcriptomic, and metabolic reprogramming, which enhances their responsiveness and aligns with the phenotype of neutrophils previously identified as “trained”, offering mechanistic insight into the functional dysregulation observed in COPD. In addition to the characterisation of neutrophil protein-coding genes transcriptome, this study focused on the role of long non-coding RNAs (lncRNAs) in regulating COPD-related neutrophil dysfunction. COPD aetiology is associated with the complex interaction between environmental and genetic determinants. Numerous genes are involved in the pathogenic process of this disease; among them, long non-coding RNAs have been reported to be involved in COPD molecular mechanisms and classified as potential biomarkers for early diagnosis. This research aims to identify lncRNAs in circulating neutrophils that are reported to play a crucial role in prolonging systemic inflammation. To this aim, we first identified the lncRNAs differentially expressed in COPD neutrophils vs controls; then, a “guilt-by-association” strategy was applied to predict the putative functions of COPD-specific lncRNAs. Transcriptomic analysis of peripheral neutrophils of COPD patients and control donors identified 92 lncRNAs among the 718 genes upregulated in neutrophils from COPD patients vs controls. Weighted gene co-expression network analysis (WGCNA) identified 22 distinct gene modules, one of which was significantly enriched for IFN-responsive genes and included five lncRNAs. Among these, BISPR was the only lncRNA with prior association to the interferon response. Loss-of-function experiments targeting BISPR were conducted to elucidate its regulatory role. These neutrophils, after transfection, are incubated for 4 hours with a recovery medium and then stimulated with IFN-α. BISPR silencing was validated by RT-PCR and RNA-seq. Si-BISPR-silenced neutrophils were subjected to RNA-seq analysis, which identified four clusters. GO enrichment analysis showed that one cluster is associated with IFN response. 5 In this cluster, we found interferon-related genes, whose expression was significantly reduced by BISPR-silencing. Notably, BISPR expression levels were also found to correlate with two clinical parameters, Residual Volume (RV) and Total Lung Capacity (TLC), linked to COPD severity. Collectively, these findings suggest that circulating neutrophils in COPD patients undergo epigenetic and transcriptomic reprogramming, resulting in a trained-like phenotype that may contribute to chronic inflammation. Additionally, BISPR emerges as a potential regulator of the IFN response in neutrophils and a novel contributor to COPD pathogenesis, highlighting its possible relevance as a biomarker or therapeutic target.