Innate Immune Signal Transduction Specificity in Inflammatory Disease

描述(申请人提供)：异常的NOD2信号导致肉芽肿性炎性疾病。功能丧失的NOD2等位基因的患者容易患上克罗恩病，这是一种胃肠道的炎症性疾病。相比之下，具有功能获得NOD2突变的患者会患上早发性结节病(EOS)，这是一种以非干酪性肉芽肿为特征的炎症性疾病，会导致肺、肝脏和眼睛的损害。功能缺失和功能获得突变都会导致炎症性疾病的事实可能是由于NOD2作为变阻器来帮助维持正常的免疫稳态的事实。这种变阻器功能开始于细菌入侵细胞，然后NOD2与细菌肽聚糖的分解产物结合。这激活了NOD2，使其可以调节先天性免疫系统，以帮助定制适应性免疫反应，以根除有害病原体。NOD2激活过多或过少都可能是有害的，这种失衡是炎症性疾病发展的核心。NOD2及其专性激酶RIP2是细胞内细菌识别导致NOD2：RIP2复合体被激活的正向调节电路的一部分。除了刺激自噬、杀菌活性、MHC II类递呈和MAPK激活外，NOD2：RIP2复合体还激活NF-？B。NOD2和RIP2都是受NF-？B调控的基因，因此，它们的激活引起一个正反馈循环，在这个循环中，NOD2：RIP2的激活刺激进一步的激活和进一步的炎症。此外，NOD2和RIP2的表达受到多种炎症介质的刺激，包括肿瘤坏死因子和干扰素。有鉴于此，在之前的授予期间，我们假设抑制这一正向调节回路可能在治疗炎症性疾病方面有效。我们成功地鉴定了抑制RIP2的S激酶活性的纳米分子抑制剂。尽管如此，一个令人不安的问题 事实仍然是：我们仍然不知道RIP2的激酶活性在细胞中起什么作用。一些研究表明，激酶活性对于NOD2的活性是可有可无的，而另一些研究则表明它是必不可少的。我们的工作有助于澄清这一点，因为我们表明RIP2被错误地归类为丝氨酸-苏氨酸激酶。它实际上是一种双重特异性的激酶，这意味着它可以磷酸化丝氨酸、苏氨酸和酪氨酸。我们自己的工作表明，抑制RIP2可以减轻急性NOD2炎症反应。虽然我的实验室已经发现RIP2的S激酶活性有助于调节核因子-βB，但我们不知道它在调节其他NOD2驱动的反应中的作用，如自噬或MAPK信号转导。鉴于制药公司对抑制RIP2治疗炎症性疾病(如结节病、哮喘、IBD和炎症性关节炎)的兴趣，有关RIP2 S激酶活性的不确定性变得更加重要。如果目标是抑制炎症性疾病中的RIP2，了解激酶的活性是必不可少的 确定对这些疾病的疗效和反应。这一知识对于预测炎症性疾病中抑制RIP2的结果也是必不可少的。这项拨款申请旨在回答这些关键问题。

DESCRIPTION (provided by applicant): Aberrant NOD2 signaling causes granulomatous inflammatory disease. Patients with loss-of-function NOD2 alleles are prone to the development of Crohn's disease, an inflammatory disorder of the gastrointestinal tract. In contrast, patients with gain-of-function NOD2 mutations develop Early Onset Sarcoidosis (EOS), an inflammatory disorder characterized by noncaseating granulomas that cause lung, liver and eye damage. The fact that both loss-of-function polymorphisms and gain-of-function mutations both cause inflammatory diseases is likely due to the fact that NOD2 functions as a rheostat to help maintain normal immunologic homeostasis. This rheostat function begins upon bacterial invasion of the cell whereupon NOD2 binds to a breakdown product of bacterial peptidoglycan. This activates NOD2 such that it can modulate the innate immune system to help tailor the adaptive immune response to eradicate the offending pathogen. Either too much or too little NOD2 activation can be deleterious, and this imbalance is central to the development of inflammatory disease. NOD2 and its obligate kinase RIP2 are part of a positive regulatory circuit in which intracellular bacterial recognition causes the NOD2:RIP2 complex to be activated. In addition to stimulating autophagy, bacteriocidal activity, MHC Class II presentation and MAPK activation, the NOD2:RIP2 complex activates NF-?B. Both NOD2 and RIP2 are NF-?B regulated genes, and as such, their activation causes a positive feedback loop in which activation of NOD2:RIP2 stimulates further activation and further inflammation. Additionally, NOD2 and RIP2 expression are stimulated by a variety of mediators of inflammation, including TNF and IFN. Given this, in the prior granting period, we hypothesized that inhibiting this positive regulatory circuit might be efficacious in treating inflammatory disease. We were successful in identifying nanomolar inhibitors of RIP2's kinase activity. Despite this, a troubling fact remains: We still don't know what the kinase activity of RIP2 is doing in the cell. Some studies have shown that the kinase activity is dispensable for NOD2 activity while others have shown that it's essential. Our work has helped clarify this as we showed that RIP2 was misclassified as a serine-threonine kinase. It is actually a dual specificity kinase, meaning that t phosphorylates serines, threonines and tyrosines. Our own work has shown that inhibition of RIP2 attenuates the acute NOD2 inflammatory response. While my lab has found that RIP2's kinase activity helps regulate NF-?B, we don't know its role in regulating other NOD2-driven responses like autophagy or MAPK signaling. The uncertainty regarding RIP2's kinase activity takes on added importance given the interest of pharmaceutical companies in inhibiting RIP2 in inflammatory diseases like sarcoidosis, asthma, IBD and inflammatory arthritis. Understanding the kinase activity is essential if the goal is to inhibit RIP2 in inflammatory disease and to then determine efficacy and response in those diseases. This knowledge is also essential to predict outcomes of RIP2 inhibition in inflammatory disease. This grant application aims to answer these key questions.