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, mTOR-inducing 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.

Fig. 1.

Activation of mTOR and CREB pathways in CF airway neutrophils. (A–E) Viable neutrophils from induced sputum (black histograms) are compared to their blood counterparts (gray histograms) for expression of phospho-eIF4E, 4E-BP1, CREB, Stat6, and S6rp, respectively. A fluorescence control (left unstained in the relevant channel) is also shown (open histogram, first line of each panel). Data are from 3 representative patients with low, medium, and high airway neutrophil count, respectively (from Top to Bottom in each panel). Differences in median fluorescence intensities between airway and blood neutrophils were similar across all patients in the study (N.S.: not significant; see Table S1 for detailed statistics).

Fig. 2.

Modulation of mTOR and CREB-associated surface adaptor molecules in CF airway neutrophils. (A–E) Viable neutrophils from induced sputum (black histograms) are compared to their blood counterparts (gray histograms) for expression of CD98, CD114, RAGE, CD39, and CXCR4, respectively. A fluorescence control (left unstained in the relevant channel) is also shown (open histogram, first line of each panel). Data are from 3 representative patients with low, medium, and high airway neutrophil count, respectively (from Top to Bottom in each panel). Differences in median fluorescence intensities between airway and blood neutrophils were similar across all patients (N.S.: not significant; see Table S1 for detailed statistics).

Fig. 3.

Putative model of CF airway inflammation highlighting the role of host-derived metabolic and stress signals. Results presented here and previously (refs. 6, 10, 28), support the existence of 4 discrete states of neutrophils, 1 in CF blood (Neu B) and 3 in the airways (Neu A1–3). We propose that host-derived cues such as oxidants, DAMPs, growth factors, and, in the airways, amino acids (AAs) and other metabolites, drive the transition between these discrete states. Pathogen-derived cues mirroring the categories defined for host-derived cues (i.e., pathogen-associated molecular patterns or PAMPs, pathogen-derived metabolites and virulence factors) also impact the biology of Neu A1, A2, and A3 subsets. Early and sustained activation of mTOR and CREB pathways and associated surface molecules is readily detectable in the Neu A1 subset, while active HNE release defines the Neu A2 subset. Both Neu A1 and A2 subsets are viable and thus may be therapeutically targeted to prevent their transition into the dying Neu A3 subset, which passively releases undigested DNA and actin fibers in the lumen.