Introduction Neutrophils are an essential component of the innate immune response. However, their microbicidal mechanisms (generation of reactive oxygen species and release of proteolytic enzymes) may contribute to tissue injury. Neutrophil-mediated tissue damage is a cardinal feature in the pathogenesis/progression of COPD, cystic fibrosis and certain types of asthma. Apoptosis is the key determinant of tissue neutrophil longevity and is critical to the resolution of granulocyte inflammation; pharmacological acceleration of neutrophil apoptosis can promote resolution of inflammation in animal models. The cytokine GM-CSF drives the aberrant neutrophil survival phenotype observed in patients with ARDS, and recent studies suggest the phosphoinositide 3-kinase (PI3K)/AKT pathway is pivotal in signalling GM-CSF-mediated neutrophil survival.

Hypothesis Given the emerging evidence that individual Class I PI3K isoforms (α, β, δ and γ) exert non-redundant signalling roles and represent promising therapeutic targets in inflammation, we hypothesised a distinct contribution of individual Class I PI3K isoforms in mediating constitutive neutrophil apoptosis and the GM-CSF cytoprotective effect.

Methods We established techniques to isolate peripheral blood neutrophils from humans and from knockout/transgenic mice lacking functional PI3K isoforms to 95% purity, and used pan-PI3K inhibitor (LY294002), pan-Class I PI3K inhibitor (PI-103) and novel PI3K isoform-selective small molecule inhibitors (YM-024, TGX-221, IC87114 and AS605240) to determine precise involvement of Class I PI3K isoforms (α, β, δ and γ) in constitutive and GM-CSF-delayed neutrophil apoptosis. Apoptosis was quantified using morphology and annexin V-FITC/propidium iodide staining.

Results GM-CSF-mediated neutrophil survival was reversed by pan-PI3K inhibition but not by individual PI3K isoform inhibition. Combinatorial experiments suggest there is near-complete functional redundancy amongst Class I PI3Ks with regard to GM-CSF-mediated inhibition of neutrophil apoptosis; additionally, neutrophils derived from double knockout PI3K-δ/γ and PI3K-β/δ mice had normal constitutive apoptosis and GM-CSF mediated survival, but were sensitised to inhibition of the remaining isoforms.

Conclusions Thus Class I PI3Ks mediate GM-CSF survival of human and murine neutrophils but there is complete functional redundancy of the PI3K isoforms, necessitating multiple isoform inhibition to reverse GM-CSF-induced neutrophil survival. This finding informs our understanding of the mechanisms regulating neutrophil apoptosis and suggests neutrophil survival would be resilient to individual isoform-selective PI3K inhibitors.