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.

Fig. 1.

Activation pattern of viable CF airway neutrophils. Viable neutrophils from induced sputum are compared with their blood counterparts (three representative CF patients) for levels of activation markers. Intracellular GSH levels, as measured by GSH-bimane adducts (see SI Methods), are decreased in sputum neutrophils (Upper Left). CD16 and CD14 are also decreased in sputum neutrophils (Upper Middle and Right, respectively). By contrast, surface lipid rafts (labeled by cholera toxin B, or CTB), as well as CD11b, CD66b, and CD63 (specific for NE-rich granules) are all increased in sputum neutrophils (Lower, from left to right). Differences in median fluorescence intensities between sputum and blood neutrophils were highly significant among the 33 patients included in the study (P < 10⁻³ for all).

Fig. 2.

Unconventional surface markers found on viable CF airway neutrophils. Viable neutrophils from induced sputum are compared with their blood counterparts (three representative CF patients) for levels of CD80, major histocompatibility class II (MHCII), and the prostaglandin D2 receptor CD294 (from left to right). All three markers appear significantly up-regulated on sputum versus blood neutrophils (P < 10⁻³ for all).

Fig. 3.

Methanol-compatible method for phosphoprofiling of viable airway neutrophils. From a complex mixture of viable, apoptotic, and necrotic cells (Upper Left), viable neutrophils are gated (Upper Middle) by using a combination of a fixable viability dye and a methanol-resistant CD66b-quantum dot conjugate (see SI Methods), yielding a clean subset as visualized by forward scatter and side scatter (Upper Right). This subset is then compared with the equivalent subset in blood for phosphoepitope expression (Lower, showing ph-S6rp expression, as an example, in three representative CF patients).