Acute bronchoconstriction in asthmatics is triggered by inhaled allergen activating mast cells in the lung. Mast cells respond to allergen by releasing mediators that cause shortness of breath, inflammation of the airways, and signaling from invariant natural killer cells (iNKT cells) promotes mast cell expansion. Asthmatic lungs have more mast cells than normal lungs but this aspect of asthma has not been possible to investigate in mouse models because they typically do not show mast cell expansion nor allergen triggered bronchoconstriction. However, at the University of Vermont, a recently developed mouse model using house dust mite (HDM) demonstrates both mast cell expansion as well as allergen induced bronchoconstriction in addition to typical lung inflammation, setting the stage to investigate the role of iNKT cells in the context of allergic bronchoconstriction. The central hypothesis of this proposal is that eliminating iNKT cells will reduce allergen induced bronchoconstriction and airways hyperresponsiveness via a reduction of mast cell expansion. A new iNKT antibody (NKT-14) that efficiently eliminates all iNKT cells was developed for this project. The hypothesis will be addressed in two specific aims: SA1: To determine if elimination of iNKT cells reduces the development of allergic AHR and allergen induced bronchoconstriction. Allergic mice treated with NKT-14 will be used to determine the role of iNKT cells on mast cell expansion and allergen-induced bronchoconstriction. The effect of iNKT cell elimination in mast cell mediator release will be measured. Activation of iNKT cells cause AHR and it is unknown if this depends on downstream activation of mast cells. To address this issue, mice deficient in mast cells will be challenged with an iNKT cell activating glycolipid (?GalCer) and AHR will be determined. SA2: To determine if IL-33 signaling is sufficient and necessary for the inflammatory and physiological phenotype. The epithelial cell-secreted cytokine IL-33 potentiates activated mast cell mediator release and promotes mast cell maturation. IL-33 also potentiates iNKT cell IL-9 signalling known to activate mast cells. The role of IL-33 will be established in IL-33 depleted mice using gene silencing siRNA as well as giving recombinant IL-33 to the airways in the context of iNKT elimination. Mast cells expanded by IL-33 and will be triggered with 48/80 (induces mast cell degranulation) and bronchoconstriction will be measured. These experiments will demonstrate the role of IL-33 during HDM allergy and also if IL-33 is sufficient for mast cell expansion. It is expected that this study will confirm preliminary data showing that iNKT cells are critical for mast cell expansion and start elucidating the mechanisms involved. The research proposed in this application is innovative, because it represents a new and substantial step towards a novel treatment principle of allergic asthma. Positive results in this study will support clinical evaluation of a humanized iNKT antibody in asthmatics;an anti-human iNKT antibody is currently available making the findings of the work proposed in this application rapidly translational.
The proposed research is relevant to public health because it addresses one of the core mechanisms in asthma;the expansion of mast cells in the allergic lung. Using a new mouse model of allergic asthma we will be able to elucidate the role of a particular inflammatory cell, the invariant natural killer cell (iNKT cell), and how it participate in the mast cell maturation and expansion. We have access to a unique antibody that will eliminate the iNKT cells allowing us to address this important inflammatory condition and it is expected that successful completion of this research will be quickly translated to studies in patients.