Innate Immune Signal Transduction Specificity in Inflammatory Disease

描述（由申请人提供）：异常NOD 2信号传导导致肉芽肿性炎性疾病。NOD 2等位基因功能丧失的患者容易发生克罗恩病，这是一种胃肠道炎性疾病。相反，具有功能获得性NOD 2突变的患者发展为早发性结节病（EOS），这是一种以非干酪化性肉芽肿为特征的炎性疾病，其导致肺、肝和眼损伤。功能丧失型多态性和功能获得型突变均导致炎性疾病的事实可能是由于NOD 2作为变阻器起作用以帮助维持正常的免疫稳态的事实。这种变阻器功能在细菌侵入细胞时开始，此时NOD 2与细菌肽聚糖的分解产物结合。这会激活NOD 2，使其能够调节先天免疫系统，以帮助定制适应性免疫反应，从而根除致病病原体。过多或过少的NOD 2激活都可能是有害的，这种不平衡是炎症性疾病发展的核心。NOD 2及其专性激酶RIP 2是正调控回路的一部分，其中细胞内细菌识别导致NOD 2：RIP 2复合物被激活。除了刺激自噬、杀菌活性、MHC II类呈递和MAPK活化外，NOD 2：RIP 2复合物还活化NF-κB。NOD 2和RIP 2都是NF-κB调节基因，因此，它们的激活引起正反馈环，其中NOD 2：RIP 2的激活刺激进一步的激活和进一步的炎症。此外，NOD 2和RIP 2的表达受到多种炎症介质的刺激，包括TNF和IFN。考虑到这一点，在先前的授权期间，我们假设抑制这种正调控回路可能有效地治疗炎症性疾病。我们成功地鉴定了RIP 2激酶活性的纳摩尔抑制剂。尽管如此，一个令人不安的 事实仍然是：我们仍然不知道RIP 2的激酶活性在细胞中起什么作用。一些研究表明，激酶活性是NOD 2活性的关键，而另一些研究表明它是必需的。我们的工作有助于澄清这一点，因为我们发现RIP 2被错误地归类为丝氨酸-苏氨酸激酶。它实际上是一种双特异性激酶，即磷酸化丝氨酸、苏氨酸和酪氨酸。我们自己的工作已经表明，抑制RIP 2减弱了急性NOD 2炎症反应。虽然我的实验室已经发现RIP 2的激酶活性有助于调节NF-κB，但我们不知道它在调节其他NOD 2驱动的反应（如自噬或MAPK信号）中的作用。鉴于制药公司对抑制结节病、哮喘、IBD和炎症性关节炎等炎症性疾病中的RIP 2的兴趣，RIP 2激酶活性的不确定性变得更加重要。如果目标是在炎症性疾病中抑制RIP 2，然后确定这些疾病的疗效和反应，那么了解激酶活性是必不可少的。这些知识对于预测炎症性疾病中RIP 2抑制的结果也是必不可少的。本研究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-κB. Both NOD2 and RIP2 are NF-κB 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-κB, 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.