Aberrant immune programming in neutrophils in cystic fibrosis.

Cystic fibrosis is a life-shortening genetic disorder, caused by mutations in the gene that encodes cystic fibrosis transmembrane-conductance regulator, a cAMP-activated chloride and bicarbonate channel. Persistent neutrophilic inflammation is a major contributor to cystic fibrosis lung disease. However, how cystic fibrosis transmembrane-conductance regulator loss of function leads to excessive inflammation and its clinical sequela remains incompletely understood. In this study, neutrophils from F508del-CF and healthy control participants were compared for gene transcription. We found that cystic fibrosis circulating neutrophils have a prematurely primed basal state with significantly higher scores for activation, chemotaxis, immune signaling, and pattern recognition. Such an irregular basal state appeared not related to the blood environment and was also observed in neutrophils derived from the F508del-CF HL-60 cell line, indicating an innate characteristic of the phenotype. Lipopolysaccharides (LPS) stimulation drastically shifted the transcriptional landscape of healthy control neutrophils toward a robust immune response; however, cystic fibrosis neutrophils were immune-exhausted, reflected by abnormal cell aging and fate determination in gene programming. Moreover, cystic fibrosis sputum neutrophils differed significantly from cystic fibrosis circulating neutrophils in gene transcription with increased inflammatory response, aging, apoptosis, and necrosis, suggesting additional environmental influences on the neutrophils in cystic fibrosis lungs. Taken together, our data indicate that loss of cystic fibrosis transmembrane-conductance regulator function has intrinsic effects on neutrophil immune programming, leading to premature priming and dysregulated response to challenge.

Neutrophil defect and lung pathogen selection in cystic fibrosis.

Cystic fibrosis is a life-threatening genetic disorder caused by mutations in the CFTR chloride channel. Clinically, over 90% of patients with cystic fibrosis succumb to pulmonary complications precipitated by chronic bacterial infections, predominantly by Pseudomonas aeruginosa and Staphylococcus aureus. Despite the well-characterized gene defect and clearly defined clinical sequelae of cystic fibrosis, the critical link between the chloride channel defect and the host defense failure against these specific pathogens has not been established. Previous research from us and others has uncovered that neutrophils from patients with cystic fibrosis are defective in phagosomal production of hypochlorous acid, a potent microbicidal oxidant. Here we report our studies to investigate if this defect in hypochlorous acid production provides P. aeruginosa and S. aureus with a selective advantage in cystic fibrosis lungs. A polymicrobial mixture of cystic fibrosis pathogens (P. aeruginosa and S. aureus) and non-cystic fibrosis pathogens (Streptococcus pneumoniae, Klebsiella pneumoniae, and Escherichia coli) was exposed to varied concentrations of hypochlorous acid. The cystic fibrosis pathogens withstood higher concentrations of hypochlorous acid than did the non-cystic fibrosis pathogens. Neutrophils derived from F508del-CFTR HL-60 cells killed P. aeruginosa less efficiently than did the wild-type counterparts in the polymicrobial setting. After intratracheal challenge in wild-type and cystic fibrosis mice, the cystic fibrosis pathogens outcompeted the non-cystic fibrosis pathogens and exhibited greater survival in the cystic fibrosis lungs. Taken together, these data indicate that reduced hypochlorous acid production due to the absence of CFTR function creates an environment in cystic fibrosis neutrophils that provides a survival advantage to specific microbes-namely, S. aureus and P. aeruginosa-in the cystic fibrosis lungs.

ABERRANT IMMUNE PROGRAMMING IN NEUTROPHILS IN CYSTIC FIBROSIS.

Cystic fibrosis (CF) is a life-shortening genetic disorder, caused by mutations in the gene that encodes Cystic Fibrosis Transmembrane-conductance Regulator (CFTR), a cAMP-activated chloride and bicarbonate channel. Although multiple organ systems can be affected, CF lung disease claims the most morbidity and mortality due to chronic bacterial infection, persistent neutrophilic inflammation, and mucopurulent airway obstruction. Despite the clear predominance of neutrophils in these pathologies, how CFTR loss-of-function affects these cells per se remains incompletely understood. Here, we report the profiling and comparing of transcriptional signatures of peripheral blood neutrophils from CF participants and healthy human controls (HC) at the single-cell level. Circulating CF neutrophils had an aberrant basal state with significantly higher scores for activation, chemotaxis, immune signaling, and pattern recognition, suggesting that CF neutrophils in blood are prematurely primed. Such an abnormal basal state was also observed in neutrophils derived from an F508del-CF HL-60 cell line, indicating an innate characteristic of the phenotype. LPS stimulation drastically shifted the transcriptional landscape of HC circulating neutrophils towards a robust immune response, however, CF neutrophils were immune-exhausted. Moreover, CF blood neutrophils differed significantly from CF sputum neutrophils in gene programming with respect to neutrophil activation and aging, as well as inflammatory signaling, highlighting additional environmental influences on the neutrophils in CF lungs. Taken together, loss of CFTR function has intrinsic effects on neutrophil immune programming that leads to premature priming and dysregulated response to challenge.

Cftr deletion in mouse epithelial and immune cells differentially influence the intestinal microbiota.

Cystic fibrosis (CF) is a life-threatening genetic disorder, caused by mutations in the CF transmembrane-conductance regulator gene (cftr) that encodes CFTR, a cAMP-activated chloride and bicarbonate channel. Clinically, CF lung disease dominates the adult patient population. However, its gastrointestinal illness claims the early morbidity and mortality, manifesting as intestinal dysbiosis, inflammation and obstruction. As CF is widely accepted as a disease of epithelial dysfunction, it is unknown whether CFTR loss-of-function in immune cells contributes to these clinical outcomes. Using cftr genetic knockout and bone marrow transplantation mouse models, we performed 16S rRNA gene sequencing of the intestinal microbes. Here we show that cftr deletion in both epithelial and immune cells collectively influence the intestinal microbiota. However, the immune defect is a major factor determining the dysbiosis in the small intestine, while the epithelial defect largely influences that in the large intestine. This finding revises the current concept by suggesting that CF epithelial defect and immune defect play differential roles in CF intestinal disease.