Profound functional and signaling changes in viable inflammatory neutrophils homing to cystic fibrosis airways

Abstract

Blood neutrophils recruited to cystic fibrosis (CF) airways are believed to be rapidly killed by resident bacteria and to passively release elastase and other toxic by-products that promote disease progression. By single-cell analysis, we demonstrate that profound functional and signaling changes readily occur within viable neutrophils recruited to CF airways, compared with their blood counterparts. Airway neutrophils have undergone conventional activation, as shown by decreased intracellular glutathione, increased lipid raft assembly, surface mobilization of CD11b؉ and CD66b؉ granules, and increased levels of the cytoskeleton-associated phospho-Syk kinase. Unexpectedly, they also mobilize to the surface CD63؉ elastase-rich granules, usually confined intracellularly, and lose surface expression of CD16 and CD14, both key receptors in phagocytosis. Furthermore, they express CD80, major histocompatibility complex type II, and the prostaglandin D2 receptor CD294, all normally associated with other lineages, which reflects functional reprogramming. This notion is reinforced by their decreased total phosphotyrosine levels, mirroring a postactivated stage, and increased levels of the phospho-S6 ribosomal protein, a key anabolic switch. Thus, we identified a subset of neutrophils within CF airways with a viable but dysfunctional phenotype. This subset provides a possible therapeutic target and indicates a need to revisit current paradigms of CF airway disease.cystic fibrosis transmembrane conductance regulator ͉ flow cytometry ͉ inflammation ͉ lung disease ͉ phosphoepitope

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

“…In particular, CF airway neutrophils undergo active exocytosis of primary granules, leading to massive release of enzymes (e.g., elastase, myeloperoxidase) that damage the airway tissue and perpetuate inflammation. More intriguingly, we also observed that CF airway neutrophils express high levels of markers conventionally found on long-lived APCs, including class II MHC, the costimulatory molecule CD80, and the chemoattractant receptor of Th2 cells (CD294), all of which suggest profound reprogramming (4).…”

“…Blood was treated with ammonium chloride to lyse RBCs, after which blood leukocytes were spun down and washed with PBS-EDTA. Leukocytes from blood and airway were prestained at 4˚C with Live/Dead (Life Sciences) and anti-CD16 and -CD63 (both from BioLegend) to enable gating of live neutrophils (4). After a wash with PBS-EDTA, leukocytes were equilibrated in serum-free RPMI 1640 (Cellgro) at 4˚C for $45 min.…”

“…In the fatal disease cystic fibrosis (CF), we previously demonstrated that neutrophils recruited from blood into the diseased airway environment undergo marked functional changes (4). In particular, CF airway neutrophils undergo active exocytosis of primary granules, leading to massive release of enzymes (e.g., elastase, myeloperoxidase) that damage the airway tissue and perpetuate inflammation.…”

IL-8 Dictates Glycosaminoglycan Binding and Stability of IL-18 in Cystic Fibrosis

“…In fact, a growing body of data has emerged supporting the belief that inflammatory dyregulation in CF may occur early in life preceding infection and may be prompted by noninfectious stimuli (37). In addition, a fundamental intrinsic abnormality of inflammatory cells, including epithelial cells (1), neutrophils (38), lymphocytes, and granulocytes (39), has been proposed as a leading cause of inflammation. Our results confirm previous observations that CF cells express IL-18 at both the gene and protein level (5).…”

Hyperinflammation in airways of cystic fibrosis patients: what’s new?

“…In the blood of CF patients, the level of apoptosis of neutrophils appears similar to the level of neutrophil apoptosis of healthy subjects, but the interaction of CF airway neutrophils with CF airway epithelial cells reduces their level of apoptosis compared with non-CF neutrophil-airway epithelial cell interaction [50]. Interestingly, Tirouvanziam and colleagues recently established that CF airways contain a significant fraction of nonapoptotic viable neutrophils [51]. They demonstrated that profound functional and signaling changes readily occur within viable neutrophils when recruited to CF airways.…”