Cellular Interactions between Human Airway Epithelial Cells and Cystic Fibrosis Macrophages result in Elevated Inflammation and Impaired Multiciliogenesis

Abstract

Introduction Patients with chronic lung diseases, such as cystic fibrosis (CF), are often characterized with chronic inflammation and impaired bacterial clearance. This suggests that their respiratory innate immune response is inadequate. Macrophages are the sentinel cells of the respiratory system acting as the first line of defense against invading pathogens, orchestrating wound healing, and maintaining tissue homeostasis. The phenotype and function of macrophages are governed by signals they receive from their microenvironment. Despite this they are often studied in isolation in vitro. We hypothesized that changes in the innate immune response in CF result from pathological signaling between macrophages and airway epithelium. Methods Primary lung macrophages, isolated from lung explants from CF patient and patients with no prior evidence of chronic lung disease, were co-cultured with human bronchial epithelial cell lines with or without mutations in the cystic fibrosis transmembrane regulator (CFTR), NuLi (control) and CuFi (CF). Cell lines were used for the initial experiments to reduce patient to patient variability due to factors such as treatment and bacterial colonization, allowing to focus on the specific role of CFTR mutations. A co-culture model at the air-liquid interface was developed to determine whether epithelial interactions with CFTR-mutant macrophages influenced basal stem cell differentiation. Tumor necrosis factor alpha (TNFα) and interleukins 6 and 8 (IL-6/8) were profiled by stimulating cultures with lipopolysaccharide (LPS) for 24 hours and analyzing the culture supernatants. Results Substantial differences in theinflammatory profile were observed in the presence of CFTR mutations in the co-culture model. A 2-fold increase in TNFα after LPS, was observed comparing CF to WT macrophage monocultures; this suggests they may maintain a hyperinflammatory physiological memory of the CF lung. The presence of epithelial cells suppresses TNFα release when compared to monoculture controls. In contrast, IL-6 is increased in the co-cultures. Additionally, CFTR mutant epithelial cells significantly increase IL-8 (3-fold increase, P<0.05), even in the absence of LPS, this response further exacerbated after LPS stimulation (1.5-fold increase, P<0.05), compared to WT controls. Finally, our data supports a role for macrophages in stimulating ciliogenesis in WT epithelium which is suppressed by the presence of CFTR-mutant macrophages (P<0.05). Conclusion This study highlights the importance of studying macrophages in a physiological microenvironment and supports a pathogenic role for CFTR-mutant epithelium in stimulating lung macrophages to a hyper-inflammatory phenotype. In addition, CF macrophages lose a capacity to promote efficient epithelial regeneration and homeostasis. This highlights the importance of understanding the cellular mechanisms regulated by macrophage-epithelial interactions, regulated by CFTR, to identify new therapeutic targets in CF.