iNKT Cells Regulating Lung Mast Cells: New Treatment Opportunity for Asthma

描述（由申请人提供）：哮喘患者的急性支气管收缩由吸入过敏原激活肺中的肥大细胞触发。肥大细胞通过释放引起呼吸短促、气道炎症的介质来响应过敏原，并且来自不变的自然杀伤细胞（iNKT细胞）的信号传导促进肥大细胞扩增。哮喘肺比正常肺有更多的肥大细胞，但哮喘的这一方面还不可能在小鼠模型中进行研究，因为它们通常不显示肥大细胞扩增，也不显示过敏原触发的支气管收缩。然而，在佛蒙特大学，最近开发的使用屋尘螨（HDM）的小鼠模型显示，除了典型的肺部炎症外，肥大细胞扩增以及过敏原诱导的支气管收缩，为研究iNKT细胞在过敏性支气管收缩中的作用奠定了基础。的 该建议的中心假设是消除iNKT细胞将通过减少肥大细胞扩增来减少过敏原诱导的支气管收缩和气道高反应性。为该项目开发了一种新的iNKT抗体（NKT-14），可有效消除所有iNKT细胞。该假设将在两个具体目标中得到解决：SA 1：确定iNKT细胞的消除是否减少过敏性AHR和过敏原诱导的支气管收缩的发展。用NKT-14处理的过敏小鼠将用于确定iNKT细胞对肥大细胞扩增和过敏原诱导的支气管收缩的作用。将测量iNKT细胞消除对肥大细胞介质释放的影响。iNKT细胞的活化引起AHR，并且尚不清楚这是否取决于肥大细胞的下游活化。为了解决这个问题，将用iNKT细胞活化糖脂（β GalCer）攻击肥大细胞缺陷的小鼠，并测定AHR。SA 2：确定IL-33信号传导对于炎症和生理表型是否充分和必要。上皮细胞分泌的细胞因子IL-33增强活化的肥大细胞介质释放并促进肥大细胞成熟。IL-33还增强已知激活肥大细胞的iNKT细胞IL-9信号传导。的作用 使用基因沉默siRNA以及在iNKT消除的情况下将重组IL-33给予气道，将在IL-33耗尽的小鼠中建立IL-33的表达。肥大细胞通过IL-33扩增，并将用48/80触发（诱导肥大细胞脱粒），并将测量支气管收缩。这些实验将证明IL-33在HDM过敏中的作用，以及IL-33是否足以用于肥大细胞扩增。预期本研究将证实初步数据，表明iNKT细胞对肥大细胞扩增至关重要，并开始阐明 所涉及的机制。本申请中提出的研究是创新的，因为它代表了向过敏性哮喘的新治疗原则迈出的新的实质性一步。本研究中的阳性结果将支持哮喘患者中人源化iNKT抗体的临床评价;目前可获得抗人iNKT抗体，使得本申请中提出的工作的发现迅速转化。

DESCRIPTION (provided by applicant): 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.