Innate immune signal transduction specificity in inflammatory disease

描述(申请人提供)：先天免疫系统识别病原体并对其作出反应。在这样做的过程中，这个系统负责启动一种细胞因子反应，旨在定制适应性免疫系统，以根除有害生物。这一过程必须受到严格控制，因为太多的活动可能会导致炎症性疾病。由于炎症性疾病的特点是持续的先天免疫激活和细胞因子的释放，因此控制先天免疫反应下调的机制在限制炎症病理中是至关重要的。本次拨款申请旨在通过研究NOD2蛋白(CARD15基因)的信号转导机制以及NOD2的S在启动和维持细胞因子反应中的作用来研究这种下调的机制。NOD2蛋白导致许多炎症性疾病，包括布劳综合征(一种家族性肉芽肿病)，早发性结节病的一个子集，以及15%-20%的遗传性克罗恩病。NOD2在细胞内暴露于革兰氏阳性和革兰氏阴性细菌后被激活，之后它通过IKK支架蛋白Nemo上一个新的位点(K285)的赖氨酸-63(K63)连接的多泛素化来帮助协调NF-？B的激活和细胞因子的释放。我们最近将这一发现扩展到表明主要的细胞外固有免疫信号受体Toll样受体(TLRs)也需要K285 Nemo泛素化才能通过NF-β正确地发出信号。这项工作表明，对IKK支架蛋白Nemo翻译后修饰的调节有助于协调细胞内和细胞外固有免疫途径之间的串扰，也有助于调节释放的细胞因子的身份、数量和持续时间。这些发现还表明，对于NF-？B信号，多个先天免疫信号通路汇聚在NEMO上，NEMO上的翻译后修饰起到控制NF-？B活性的变阻器的作用。因此，这些翻译后修饰也可能是旨在下调由NOD2和其他先天免疫信号通路激活的核因子？B反应的分子的靶标。这项资助的中心假设是，下调NOD2和TLR刺激的核因子-βB激活是避免炎症病理的首要因素。未能适当下调核因子？B的反应和在替代(MAP激酶)信号通路之间协调可能是炎症性疾病的病理生理学基础。对这些下调途径的研究可能会导致对这些疾病的病理生理学的新见解，以及帮助治疗这些疾病的新的药物靶点。为了开始解决这一重要问题，我们已经产生了重要的初步数据。我们已经确定了NEMO上一个新的先天免疫诱导的磷酸化位点，它控制着NEMO的泛素化，从而控制着最终的核因子？B的激活。我们还发现了一条信号通路，它通过一种意想不到的MAP3K来抑制NEMO泛素化，并将天然免疫信号从NF-β活性转移到p38活性。这项拨款申请的具体目的旨在确定天然免疫诱导的核因子-βB活性下调的生化机制，确定MEKK4在天然免疫系统激活下游决定信号特异性的功能，并确定这些信号通路在炎症性疾病病理生理中的作用。公共卫生相关性：作为人类，我们经常暴露在细菌、真菌和病毒中，我们必须对这些病原体做出反应，这样我们才不会被感染。在对这些病原体做出反应后，如果我们的免疫系统没有失活，我们就会发展成炎症性疾病，如哮喘、炎症性肠病、多发性硬化症和动脉粥样硬化(心脏和血管疾病)。这类炎症性疾病是各种人群(从婴儿到老年人)发病率和死亡率的重要原因。由于下调炎症反应的重要性，我们的身体已经发展出复杂的机制来抑制炎症反应。这项拨款申请旨在研究抑制这种炎症反应的机制，以及这种抑制在炎症性疾病中是错误的机制。这项工作旨在帮助确定炎症性疾病的原因，并旨在确定药物干预这些衰弱疾病的新靶点。

DESCRIPTION (provided by applicant): The innate immune system recognizes and responds to pathogenic organisms. In doing so, this system is responsible for initiating a cytokine response designed to tailor the adaptive immune system to eradicate the offending organism. This process must be tightly regulated as too much activity can lead to inflammatory disease. Because inflammatory diseases are characterized by prolonged innate immune activation and cytokine release, the mechanisms controlling downregulation of the innate immune response are paramount in limiting inflammatory pathology. This grant application aims to study the mechanisms of this downregulation by focusing on the signal transduction mechanisms of NOD2 protein (CARD15 gene) and on NOD2's role in initiating and maintaining the cytokine response. The NOD2 protein is responsible for a number of inflammatory disorders including Blau Syndrome (a familial granulomatosis disease), a subset of Early Onset Sarcoidosis and for 15-20% of genetic Crohn's Disease. NOD2 is activated in response to intracellular exposure to both gram-positive and gram-negative bacteria after which it helps to coordinate NF-?B activation and cytokine release through the lysine-63 (K63)-linked polyubiquitination of a novel site (K285) on the IKK scaffolding protein NEMO. We have recently extended this finding to show that the major extracellular innate immune signaling receptors, the Toll-like Receptors (TLRs), also require K285 NEMO ubiquitination to properly signal through NF-?B. This work suggests that regulation of the post-translational modifications on the IKK scaffolding protein, NEMO, helps to coordinate cross-talk between intracellular and extracellular innate immune pathways and also helps to regulate the identity, the amount and the duration of cytokines that are released. These findings also suggest that for NF-?B signaling, multiple innate immune signaling pathways converge on NEMO and that the post-translational modifications on NEMO serve as a rheostat to control NF-?B activity. As such, these post-translational modifications may also be targets for molecules aimed at downregulating the NF-?B response activated by NOD2 and other innate immune signaling pathways. The central hypothesis of this grant is that downregulation of NOD2 and TLR-stimulated NF-?B activation is paramount in avoiding inflammatory pathology. Failure to properly downregulate the NF-?B response and coordinate between alternative (MAP kinase) signaling pathways may underlie the pathophysiology of inflammatory disorders. Study of these pathways of downregulation could lead both to novel insight regarding the pathophysiology of these diseases and to novel druggable target to help treat these diseases. To begin to tackle this important problem, we have generated significant preliminary data. We have identified a novel innate immune-induced phosphorylation site on NEMO that controls NEMO ubiquitination and therefore, controls ultimate NF-?B activation. We have also identified a signaling pathway operating through an unexpected MAP3K which inhibits NEMO ubiquitination and shifts innate immune signaling from NF-?B activity toward p38 activity. The Specific Aims of this grant application aim to determine the biochemical mechanisms by which innate immune-induced NF-?B activity can be downregulated, to determine the function of MEKK4 in dictating signal specificity downstream of innate immune system activation and to determine the role of these signaling pathways in the pathophysiology of inflammatory disease. Public Health Relevance: As humans, we are constantly exposed to bacteria, fungi and viruses, and we must respond to these pathogens so that we do not become infected. After responding to these pathogens, if our immune systems do not deactivate, we develop inflammatory disorders such as asthma, inflammatory bowel disease, multiple sclerosis and atherosclerosis (heart and vascular disease). Inflammatory diseases such as these are a significant cause of morbidity and mortality across a wide range of populations (infants to elderly). Due to the importance of downregulating the inflammatory response, our bodies have developed sophisticated mechanisms to dampen the inflammatory response. This grant application aims to study the mechanisms that dampen this inflammatory response and the mechanisms by which this dampening is faulty in inflammatory disease. This work aims to help determine the causes of inflammatory disease and aims to identify novel targets for pharmaceutical intervention in these debilitating disorders.