Regulation of ITK in Lung Allergy

描述(申请人提供)：肺部过敏，特别是支气管哮喘，是美国最常见的慢性疾病之一，影响到大约1500万美国人，并导致严重疾病和数千人死亡。这种疾病造成的直接和间接经济负担高达数十亿美元。这种疾病的基本症状包括IgE抗体，淋巴细胞和嗜酸性粒细胞渗入肺，粘液分泌，以及支气管平滑肌过度活跃。Th2淋巴细胞分泌的细胞因子调节过敏性哮喘的炎症反应。特别是，IL-4、IL-5和IL-13等细胞因子对IgE抗体的产生、嗜酸性粒细胞的募集以及随后炎性血管活性胺和脂质介质的释放至关重要。可诱导T细胞激酶ITK的激活已被证明可以调节Th2细胞因子的产生。有趣的是，ITK基因突变的小鼠(ITK-KO小鼠)表现出明显较轻的肺部炎症症状，这是在哮喘的实验模型中进行的评估。ITK的激活需要接头蛋白SLP76的存在和Src激酶LCK的转磷酸化。在没有SLP 76的情况下，ITK不会发生转磷酸化，因此在酶的作用下仍然是无效的。体外研究揭示了ITK的SH3结构域与SLP 76的多聚脯氨酸区(PR)之间的相互作用。然而，这种相互作用对活细胞中ITK激活和随后的细胞因子产生的生物学意义尚未确定。在目前的研究中，我们将使用竞争性多肽抑制剂，试图破坏ITK和SLP 76之间的联系，并抑制活细胞中ITK的激活。这可能会创造一种类似于ITK-KO小鼠的情况。为了实现这一点，我们将评估一个新的研究领域的可行性，以设计和测试代表SLP 76的PR区域中与ITK的SH3结构域相互作用的特定位置的细胞渗透性合成肽。代表这个位置的合成肽将通过添加精氨酸残基而变成阳离子，并测试它们移位到细胞质环境的能力。这些多肽对ITK-SLP 76结合、ITK磷酸化和酶活性以及细胞因子产生(Th1和Th2)的影响将在Jurkat细胞系统和原代小鼠脾T细胞上进行测试。此外，我们将把多聚Arg-SLP 76肽注射到小鼠体内，并测试它们对脾T细胞的作用，如上所述。最后，我们将测试这些多肽对小鼠哮喘模型的影响。这一建议是新颖的，尽管存在一些风险，但它有可能加强与健康相关的研究，并为治疗和管理肺部过敏的药物的合理设计开辟道路。公共卫生相关性：声明过敏性哮喘是一种基于免疫的疾病，涉及呼吸道炎症。过敏性哮喘被认为是一个严重的全球公共卫生问题，具有重大的经济和社会影响。在这里，我们提出的研究可能会发现控制过敏性哮喘发病机制的新方法。

DESCRIPTION (provided by applicant): Lung allergies, in particular Bronchial Asthma, rank among the most common chronic conditions in the United States, affecting approximately 15 million Americans and causing serious illness and several thousands of deaths. The direct and indirect financial burden created by this disease runs in the billions of dollars. The symptoms underlying this disease involve IgE antibodies, infiltration of lungs by lymphocytes and eosinophils, secretion of mucous, and hyperactivity of the bronchial smooth muscle. Cytokines secreted by Th2 lymphocytes regulate the inflammatory response seen in allergic asthma. In particular, cytokines such as IIL-4, IL-5, and IL-13 are critical for the production of IgE antibodies, recruitment of eosinophils, and the subsequent release of inflammatory vasoactive amines and lipid mediators. The activation of the Inducible T cell Kinase, ITK, has been shown to regulate the production of Th2 cytokines. Interestingly, mice with disrupted ITK genes (ITK-KO mice) display significantly milder symptoms of lung inflammation, as assessed in an experimental model of bronchial asthma. The activation of ITK requires the presence of the adaptor protein SLP 76 and transphosphorylation by the Src kinase LCK. In the absence of SLP 76 ITK does not become transphosphorylated and thus remains enzymatically inactive. In vitro studies have uncovered an interaction between ITK's SH3 domain and the polyproline region (PR) of SLP 76. However, the biological significance of this interaction on ITK activation and subsequent cytokine production in live cells has not been determined. In the present investigation we will use competitive peptide inhibitors in an attempt to disrupt the association between ITK and SLP 76 and inhibit the activation of ITK in live cells. This presumably should create a condition that would mimic the situation with ITK-KO mice. To accomplish this we will assess the feasibility of a novel area of investigation for designing and testing cell-permeable synthetic peptides that represent the specific site in the PR region of SLP 76 with which ITK's SH3 domain interacts. Synthetic peptides representing this site will be rendered cationic by the addition of Arginine residues and tested for their ability to translocate into the cytoplasmic milieu. The effects of such peptides on ITK-SLP 76 association, ITK phosphorylation and enzymatic activity, as well as cytokine production (both Th1 and Th2) will be tested using both the Jurkat cell system and primary murine splenic T cells. In addition, we will inject the polyArg-SLP 76 peptides into mice and test their effects, as above, on splenic T cells ex vivo. Finally, we will test the effects of these peptides on a murine model of Bronchial Asthma. This proposal is novel and in spite of some risk, it has the potential to enhance health-related research and open avenues for the rationale design of drugs for the treatment and management of lung allergies. PUBLIC HEALTH RELEVANCE: Statement Allergic asthma is an immunologically based disease that involves inflammation of the airway. Allergic asthma is considered a serious public health problem worldwide with a significant economic and social impact. Here, we propose studies that may uncover new ways of controlling the mechanisms underlying the pathogenesis of allergic asthma.