Karin Bjorhall’s scientific contributions

Severe eosinophilic asthma remains a clinical burden, potentially due to the multiple mechanisms by which type 2 inflammation can arise. Here we show how epithelial alarmin stimulation of innate lymphoid type 2 cells (ILC2s) can promote Th2 cell activity and subsequent eosinophil influx to the lung via mechanisms dependent upon PI3Kγ and δ isoform activity. A PI3Kgamma and delta (PI3Kγδ) mRNA signature identified a Ubiopred severe asthmatic patient segment with high sputum eosinophil content and a Th2/ILC2/alarmin endotype. Evaluation of potent PI3K inhibitors with γ, δ or dual isoform selectivity supported a role in this endotype as follows: PI3Kγ mediated eosinophil degranulation in an in vitro asthmatic subject blood assay. PI3Kδ-dependent IL-33 signalling/ILC2 activity was observed in an in vitro blood assay. In vivo activity of an inhaled PI3Kγδ inhibitor was demonstrated within a murine mechanistic model of IL-33/ILC2-dependent eosinophil influx, and further confirmed using PI3Kγ knockout and PI3Kδ kinase dead knock-in mice. Finally, we demonstrated therapeutic efficacy of PI3Kγδ pathway inhibition on Th2 mediator release and subsequent eosinophil influx in a rodent in vivo model of pulmonary inflammation. These data indicate a central role for both PI3Kγ and δ isoforms in eosinophilic pulmonary inflammation driven by epithelial damage and innate lymphoid cells, and thereby highlight the therapeutic potential of an inhaled dual PI3Kγδ dual inhibitor in severe eosinophilic asthma.

Severe asthma remains a clinical burden despite the emergence of antibody therapies. Complex disease drivers, which shift between T2, T1 and T17 driven inflammation, termed mixed T cell asthma, likely contribute to ineffective disease control in this population. Here we dissect the mechanisms underlying severe asthma to reveal a potential role (s) for both PI3Kgamma and delta (PI3Kγ and δ) isoform activity in asthmatics with a mixed T cell endotype. A PI3Kγδ gene signature was generated from next generation sequencing of IL-8/GM-CSF-stimulated monocytes where PI3Kγδ signalling was inhibited. This mRNA signature was used to interrogate the Ubiopred database. We observed a strong association between PI3Kγδ pathway activity, severity and mixed eosinophil and neutrophil sputum granulocyte content. To further support a role of PI3Kγδ in a mixed T cell asthma phenotype we demonstrated the following. 1) PI3Kδ-dependent allergen stimulation of mixed T cell activity in grass pollen atopic blood. 2) PI3Kδ-mediated inhibition of IL-5, IFNγ and IL-17 production from asthma patient blood T cells. 3) PI3Kγ mediated signalling was required for activation of asthmatic eosinophil and neutrophil populations. 4) Activity of an inhaled PI3Kγδ dual inhibitor in a rodent in vivo model of steroid resistant allergy/infection on mixed T cell mediator release, granulocyte influx and airways hyperresponsiveness. These data identify a population of mixed T cell asthma patients with increased PI3Kγδ activity and suggest a therapeutic potential of an inhaled PI3K dual inhibitor.

Introduction Severe asthma can be dominated by either Th2/eosinophilic or Th17/neutrophilic pathology yet the specific endotype of a patient may not be clear/stable. We hypothesized that a locally administered PI3kγδ dual isoform inhibitor could provide a more effective and safe treatment paradigm. Methods Nanomolar PI3K inhibitors with >100-fold selectivity for γ, δ or dual γδ isoforms were evaluated following nebulization in the rat OVA allergic model. The effects of these inhibitors on innate and adaptive immune responses were evaluated in human cells isolated from GINA3/4 asthma or healthy subjects. Results Therapeutic treatment (4, 20 & 100μg/kg) within the OVA allergic model showed substantial efficacy of the dual inhibitor on bronchoalveolar lavage (BAL) mediator release (IL-5 and IL-13) and eosinophil/neutrophil influx – reducing all parameters >80% in the absence of measurable systemic exposure. Target engagement in the lung was confirmed using the PI3K pathway marker pS6 in BAL CD4+ T cells. In both healthy and asthmatic subjects, activation, degranulation and chemotaxis of peripheral blood eosinophils or neutrophils was PI3Kγ dependent. However, primed neutrophil respiratory burst exclusively required PI3Kδ. The release of either IL-5, IL-13 or IL-17 from peripheral T cells was PI3Kδ-dependent. CD4+ T cell differentiation toward the Th2 or Th1 phenotype and migration of CD4+ T cells within a 3D collagen matrix, were modestly impaired by mono-selective compounds but effectively inhibited by dual inhibiton. Conclusions Our results provide evidence that an inhaled dual PI3Kγδ isoform inhibitor could be a novel, effective and safer therapy for a broad range of severe asthma patients.