PHOSPHOINOSITIDE-3 KINASE IN SEVERE ASTHMA

Abstract

Introduction: A subgroup of severe asthmatics (SA) remain uncontrolled with persistent airway eosinophilia despite high dose glucocorticosteroids (GCS) termed “steroid insensitivity.” A significant proportion of this subgroup get recurrent airway infections. Phosphoinositide-3 kinase (PI3K) pathway may contribute to both GCS insensitivity and further susceptibility to recurrent infections. Objectives: The effect of GCS and PI3K antagonism was evaluated on GCS induced eosinophil apoptosis as a mechanism of GCS insensitivity in healthy, mild to moderate and severe asthmatics. Furthermore, we investigated the relationship between PI3K activation, histone deacetylase (HDAC) and macrophage receptor with collagenous structure (MARCO) expression in SA±recurrent bacterial bronchitis. Methods: Blood eosinophils, isolated from healthy subjects (HS) and asthmatics, were incubated with increasing concentrations of dexamethasone (0.1, 1 and 10 uM) and a pan-PI3K antagonist (LY294002 at 1 and 2 uM). Cell viability was assessed using PrestoBlue cell viability assay, Trypan Blue exclusion test and PE-Annexin-V/7-AAD apoptosis detection (flow cytometry). Furthermore, PI3K activity, MARCO and HDAC levels were measured in macrophages isolated from healthy subjects and SA (±history of recurrent bacterial bronchitis). In a subgroup, sputa were examined for in situ PI3K activity and gene expression of PI3K isoforms by digital PCR. Results: In HS, a time-dependent increase in eosinophil apoptotic% was observed (1.9% at baseline, 25.7% at 16h and 31% at 24h) (p=0.06, p=0.005 respectively). Dexamethasone 10uM increased it to 40.1% at 16h (p=0.03). Dexamethasone induced eosinophil apoptosis was less in severe asthmatics (34.2%) vs. to mild-to-moderate asthmatics (46.7%) (p=0.05) suggesting steroid insensitivity. This was not reversed by co-incubation with LY294002 2uM (39.9% vs. 53.3%) (p=0.04) (Fig. 1B). Asthmatics with recurrent lung infections had higher blood PI3K activity (demonstrated as inverse of biotinylated-PIP3, p=0.02), MARCO expression (p=0.01), and trend for lower HDAC expression (p=0.067) vs. healthy donors. PI3KCD (encoding catalytic 𝛿 isoform) gene expression relative to HPRT1 (housekeeping gene) was increased in SA-infection group (p=0.03). A higher number of asthmatics with recurrent lung infections were on oral corticosteroids (p=0.015) and replacement immunoglobulins (p=0.016). Conclusions: Evaluating dexamethasone-induced apoptosis in blood eosinophil can assess GCS sensitivity ex vivo. Severe asthmatics demonstrate GCS insensitivity, which was not reversed by a pan-PI3K antagonist. PI3K activity is increased in SA with a previous history of recurrent lung infections, which is associated with a decrease in HDAC and MARCO expression. Targeting PI3K pathway, specifically the 𝛿 isoform, may be a potential therapeutic target in SA with mixed-granulocytic bronchitis.