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Biomimetic model of human small airways reveals functional impacts of the cystic fibrosis microenvironment on monocytes and macrophages

ABSTRACT: Several muco-obstructive lung diseases in humans, including cystic fibrosis (CF), feature the chronic recruitment of blood leukocytes and their reprogramming within the lumen of small airways to drive pathological adaptations such as altered immune mediator secretion and defective killing of bacteria. To investigate these mechanisms, we engineered a biomimetic model in which primary human blood monocytes migrate through a human small airway epithelium, andepithelium and differentiate into macrophages upon 4-day exposure to macrophage colony-stimulating factor. We observed that airway-recruited monocytes (ArMos) obtained with the control chemoattractant C-C motif ligand 2 (CCL2) altered their transcriptome and secretome upon transmigration, and further upon differentiation into airway-recruited macrophages (ArMas), as compared to circulating monocytes. We observed specific additional alterations in transcriptomic and secretomic properties of ArMos and ArMas when they were conditioned in CF airway supernatant (derived from cell- and bacteria-free sputum) [CFASN], compared CCL2 conditions. Critically, CF ArMas (but not CF ArMos), showed a much reduced ability to kill P. aeruginosa, a common CF pathogen. These findings extend prior studies on small airway-recruited neutrophils, and identify immune tolerance by macrophages as one underlying cause of bacterial colonization in CF. This biomimetic model support further mechanistic and preclinical investigations of small airway-recruited monocytes and macrophages in CF and other diseases featuring chronic inflammation and infection, like non-CF bronchiectasis, and chronic obstructive pulmonary disease.

ORGANISM(S): Homo sapiens

PROVIDER: GSE311629 | GEO | 2025/12/01

REPOSITORIES: GEO

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Project description:Background: CFTR modulators will decrease some etiologies of inflammation in the CF airway; however, current data indicate that non-resolving airway infection and inflammation will persist in individuals with CF and chronic bacterial infections. Thus, identification of therapies that diminish CF airway inflammation without allowing unrestrained bacterial growth remains a critical research goal. Novel strategies for combatting deleterious airway inflammation require a better understanding of the cellular contributions to chronic CF airway disease, and how cells change after initiation of CFTR modulator therapy. Peripheral blood monocytes, which traffic to the CF airway, can develop both pro-inflammatory and inflammation-resolving phenotypes, and represent intriguing targets for focused therapies to dampen CF airway inflammation. Material and Methods: In order to characterize the inflammatory phenotype of CF blood monocytes, and how these cells change after initiation of CFTR modulator therapy, we studied adults (n=10) with CF, chronic airway infections, and the CFTR-R117H mutations before and 7 days after initiation of ivacaftor. Transcriptomes of freshly isolated blood monocytes were interrogated by RNA-sequencing (RNA-seq). Plasma concentrations of cytokines and chemokines were evaluated by multiplex ELISA. Results: RNAseq identified approximately 50 monocyte genes for which basal expression was significantly changed in all 10 subjects after 7 days of ivacaftor. Of these, the majority were increased in expression post ivacaftor, including many genes traditionally associated with enhanced inflammation and immune responses. Pathway analyses confirmed that transcriptional pathways were overwhelmingly up-regulated in monocytes after 7 days of ivacaftor, including transcriptional modules associated with immunity, cell cycle, oxidative phosphorylation, and the unfolded protein response. Ivacaftor increased plasma concentrations of CXCL2, a neutrophil chemokine secreted by monocytes and macrophages, and CCL2, a monocyte chemokine. These results demonstrate that ivacaftor causes acute changes in blood monocytes and plasma chemokines, and suggest that increased monocyte inflammatory signals and potentially improved trafficking to the lung contribute to changes in airway inflammation in people taking CFTR modulators. To our knowledge, this is the first report investigating the transcriptomic response of isolated blood monocytes in CF subjects.

Project description:Cystic fibrosis (CF) is an inherited, multi-system disease caused by dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a ubiquitous ion channel important for epithelial hydration. A direct consequence of this dysfunction is impaired mucociliary clearance, chronic airway infection and a persistent neutrophilic inflammatory response that results in progressive loss of lung function, development of respiratory failure and premature death. Partial restoration of CFTR function is now possible for most CF patients through mutation specific CFTR modulators. Ivacaftor monotherapy produces significant clinical improvement in CF patients with the G511D mutation. Dual therapy, combining ivacaftor with lumacaftor or tezacaftor, results in modest clinical improvements in patients homozygous for F508del. More recently, triple therapy with elexacaftor/tezacaftor/ivacaftor (ETI) has led to dramatic improvements in lung function and quality of life in patients homozygous and heterozygous for F508del. Sputum proteomics is a powerful research technique capable of identifying important airway disease mechanisms by interrogating the proteome, an entire set of proteins within biological samples. It has confirmed the central role of neutrophilic immune dysregulation in CF and non-CF bronchiectasis, particularly involving the release of antimicrobial proteins and neutrophil-extracellular traps (NETs), and through impaired anti-inflammatory mechanisms. These processes produce distinct molecular signatures within the sputum proteome that become increasingly abnormal with chronic airway infection and progressive lung disease severity. In CF patients, airway and systemic inflammatory cytokines potentially related to these signatures reduce with the various forms of CFTR modulation. To date, no studies of ETI therapy in CF lung disease have assessed large-scale change in protein expression using untargeted proteomics. We hypothesised that ETI therapy would shift the sputum proteome toward health, potentially normalising airway biology in people with CF. The objectives of this study were to investigate changes in the CF sputum proteome with the introduction of ETI, correlate these with changes in clinical markers of disease severity, and make comparisons with the sputum proteome in healthy controls and in repeat samples from CF patients not suitable for ETI therapy. We also explored which molecular pathways associated with CF lung disease did not change with ETI.

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Biomimetic model of human small airways reveals functional impacts of the cystic fibrosis microenvironment on monocytes and macrophages

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