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. / Thesis / Master of Science in Medical Sciences (MSMS) / Eosinophils are a type of blood cell that is responsible for causing asthma symptoms and flare-ups. Steroids (in both inhaled and oral forms) decrease eosinophils and so are the main treatment for asthma. But a group of asthmatics continues to have symptoms and eosinophils in their lung secretions even with maximum dose of steroids. Some of these asthmatics also get repeated bacterial lung infections. In blood samples, we studied one of the proteins (phosphoinositide-3 kinase, PI3K), which may be responsible for poor response to steroids and repeated infections. We looked at the effect of steroids on eosinophils extracted from blood samples of healthy people, asthmatics with mild disease and asthmatics with severe disease. We also measured PI3K levels and related proteins in blood and sputum samples from healthy people, asthmatics who get repeated bacterial lung infections (more than two in the previous two years) and asthmatics without recurrent lung infections. We found that eosinophils obtained from asthmatics with severe disease were harder to kill with steroids. But, this was not reversed with a PI3K blocking agent. We also found that asthmatics with a previous history of repeated bacterial lung infections had a higher PI3K level. High PI3K activity was associated with a decrease in two other proteins – histone deacetylase (HDAC) and macrophage receptor with collagenous structure (MARCO). Decrease in HDAC can result in a lower response to steroids, which may result in an increase in eosinophils in the lungs. Lower levels of MARCO can cause a patient to have higher sensitivity to recurrent bacterial infections. Overall, this may lead to a dangerous cycle where repeated lung infection will lead to high PI3K activity, which will cause worse asthma control and more infections. More studies are needed to evaluate drugs that block PI3K, which may be helpful in asthmatics who are less responsive to steroids and also get repeated infections.