Activation of critical, host-induced, metabolic and stress pathways marks neutrophil entry into cystic fibrosis lungs

Abstract

Cystic fibrosis (CF) patients undergo progressive airway destruction caused in part by chronic neutrophilic inflammation. While opportunistic pathogens infecting CF airways can cause inflammation, we hypothesized that host-derived metabolic and stress signals would also play a role in this process. We show that neutrophils that have entered CF airways have increased phosphorylation of the eukaryotic initiation factor 4E and its partner the 4E-binding protein 1; 2 key effectors in the growth factor-and amino acid-regulated mammalian target of rapamycin (mTOR) pathway. Furthermore CF airway neutrophils display increased phosphorylation of the cAMP response element binding protein (CREB), a major transcriptional coactivator in stress signaling cascades. These active intracellular pathways are associated with increased surface expression of critical adaptor molecules, including the growth factor receptor CD114 and the receptor for advanced glycation end-products (RAGE), a CREB inducer and sensor for host-derived damage-associated molecular patterns (DAMPs). Most CF airway fluids lack any detectable soluble RAGE, an inhibitory decoy receptor for DAMPs. Concomitantly, CF airway fluids displayed high and consequently unopposed levels of S100A12; a potent mucosa-and neutrophil-derived DAMP. CF airway neutrophils also show increased surface levels of 2 critical CREB targets, the purine-recycling enzyme CD39 and the multifunctional, mTORinducing CXCR4 receptor. This coordinated set of events occurs in all patients, even in the context of minimal airway inflammation and well-preserved lung function. Taken together, our data demonstrate an early and sustained activation of host-responsive metabolic and stress pathways upon neutrophil entry into CF airways, suggesting potential targets for therapeutic modulation.CFTR ͉ EN-RAGE ͉ flow cytometry ͉ S6 ribosomal protein ͉ stromal derived factor-1

Metabolic Adaptation of Neutrophils in Cystic Fibrosis Airways Involves Distinct Shifts in Nutrient Transporter Expression

“…Compared with blood neutrophils, the A1 subset of CF airway neutrophils is characterized by a marked exocytosis of secondary granules (high CD66b with low CD63 surface expression), whereas the A2 subset is characterized by a marked exocytosis of both secondary and primary granules (high CD66b and CD63 surface expression) (4,5). Because both subsets showed significant changes in nutrient transporter expression, we tested whether in vitro neutrophil activation and induction of granule exocytosis could alter surface expression of nutrient transporters.…”

“…Cell sorting and quantitative RT-PCR to assess nutrient transporter transcript levels , as described previously (4,5). After a wash with PBS-EDTA, blood and airway neutrophils were sorted with a FACS Jazz Cell Sorter using BD FACS Software (BD Biosciences).…”

“…CF airway neutrophils also undergo strong activation of CREB and mTOR prosurvival pathways (5). The CREB pathway is commonly induced in stress responses, as expected in the inflammatory environment of CF airways (6).…”

In VivoEvaluation of Adeno-Associated Virus Gene Transfer in Airways of Mice with Acute or Chronic Respiratory Infection

“…Analyses showed that day 2 inflammatory responses were dominated by macrophage and neutrophil infiltration, the latter of which is characteristic of the infected CF lung (Makam et al, 2009). However, in the SP-D KO/PAO1 acute infection model, elevated levels of macrophages and neutrophils were present even in uninfected lung samples, suggesting that their presence alone is insufficient to reduce gene transfer.…”

“…Deterioration of the lung in CF patients is caused by a cycle of infection by opportunistic pathogens and neutrophilic inflammation (Makam et al, 2009). By the age of 17, nearly 70% of CF patients have P. aeruginosa in their sputum at levels of 10 6 -10 8 CFU/gr sputum (Davis et al, 1996), and approximately 80% of adult CF patients are chronically infected with P. aeruginosa that cannot be eliminated by antibiotic therapy (Cystic Fibrosis Foundation, 2012).…”