BACKGROUND Severe, steroid-insensitive asthma is a substantial clinical problem accounting for >50% of asthma-associated health-care-costs. Effective treatments are urgently required, however, their development is hampered by a lack of understanding of the mechanisms that promote the pathogenesis of disease. Steroid-insensitive asthma is associated with respiratory infections, and non-eosinophilic endotypes of disease, including neutrophilic asthma. The mechanisms that underpin infection-induced, severe, neutrophilic, steroid-insensitive asthma may be elucidated using mouse models of disease. OBJECTIVES To develop representative mouse models of this endotype of asthma. To use these models to identify novel pathogenic mechanisms involving microRNA (miR)-21, phosphoinositide-3-kinase (PI3K) pathways and histone deacetylase (HDAC)2, and to investigate new treatment approaches. METHODS Novel mouse models of Chlamydia, and Haemophilus, respiratory infection and ovalbumin-induced, severe, neutrophilic, steroid-insensitive allergic airway disease (SSIAAD) in BALB/c mice were developed. The roles and potential for targeting of infection-induced miR-21 expression and PI3K-dependent signalling in the lung were examined using therapeutic treatments with a specific miR-21 inhibitor (antagomir, Ant-21) and the pan-PI3K inhibitor LY294002. RESULTS Respiratory infection induced a miR-21-dependent, PI3K-mediated cell-signalling pathway that promoted steroid-insensitive, neutrophilic inflammation and airway hyper-responsiveness (AHR) in AAD. This involved the suppression of nuclear HDAC2 activity. Inhibition of miR-21 or PI3K suppressed nuclear pAkt levels and restored HDAC2 levels. Treatment also restored sensitivity to steroid administration. CONCLUSIONS We have identified a previously unrecognised pathogenic role for a miR-21/PI3K/pAkt/HDAC2 signalling axis in severe, neutrophilic, steroid-insensitive AAD. Our data highlights miR-21 as a novel therapeutic target for the treatment of this form of asthma.