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

描述(申请人提供)：哮喘患者的急性支气管收缩是由吸入变应原激活肺中的肥大细胞引发的。肥大细胞对过敏原的反应是通过释放导致呼吸短促、呼吸道炎症的介质，以及来自不变自然杀伤细胞(iNKT细胞)的信号促进肥大细胞的扩张。与正常肺相比，哮喘肺中有更多的肥大细胞，但这方面的哮喘还不可能在小鼠模型中进行研究，因为它们通常不会显示肥大细胞扩张，也不会出现过敏原引发的支气管收缩。然而，在佛蒙特大学，最近开发的使用屋尘螨(HDM)的小鼠模型除了典型的肺部炎症外，还显示了肥大细胞的扩张和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。这个 这一建议的中心假设是，消除iNKT细胞将通过减少肥大细胞的扩张来减少过敏原诱导的支气管收缩和呼吸道高反应性。为这一项目开发了一种新的iNKT抗体(NKT-14)，它可以有效地消除所有iNKT细胞。这一假设将在两个特定的目标中得到解决：SA1：确定消除iNKT细胞是否可以减少过敏性AHR和过敏原诱导的支气管收缩的发生。用NKT-14治疗的过敏小鼠将被用来确定iNKT细胞在肥大细胞扩张和过敏原诱导的支气管收缩中的作用。将测量iNKT细胞在肥大细胞介质释放中的消除效果。INKT细胞的激活会导致AHR，目前尚不清楚这是否依赖于肥大细胞的下游激活。为了解决这个问题，肥大细胞缺陷的小鼠将接受iNKT细胞激活糖脂(GalCer)的挑战，并将测定AHR。SA2：确定IL-33信号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促进激活的肥大细胞介质的释放，促进肥大细胞的成熟。IL-33还能增强iNKT细胞的IL-9信号，从而激活肥大细胞。角色 通过基因沉默siRNA以及在iNKT消除的情况下将重组IL-33给予呼吸道，将在IL-33耗竭的小鼠中建立IL-33的表达。用IL-33扩增肥大细胞，并用48/80触发(诱导肥大细胞脱颗粒)，并测量支气管收缩。这些实验将证明IL-33在HDM过敏中的作用，以及IL-33是否足以促进肥大细胞的增殖。预计这项研究将证实iNKT细胞对肥大细胞增殖至关重要的初步数据，并开始阐明 所涉及的机制。这项申请中提出的研究是创新的，因为它代表着朝着过敏性哮喘的新治疗原则迈出了新的实质性的一步。这项研究的阳性结果将支持对哮喘患者进行人源化iNKT抗体的临床评估；目前已有一种抗人iNKT抗体可用，使这项应用中提出的工作结果迅速转化。

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.