Vimentin-mediated regulation of the inflammasome in acute lung injury

 描述(申请人提供)：炎症小体被激活，以响应已知的引起急性肺损伤(ALI)的广泛的感染性因素，但它也必须受到严格的调控，以避免来自广泛的病原体、病原体相关的分子模式和危险相关的分子模式的有害影响，这些已知触发其激活。炎症小体的激活需要两种不同的促炎刺激。在第一步中，Toll样受体信号激活了核因子-κB，上调了原IL-1β。第二步涉及多蛋白复合体的组装，它诱导caspase-1的自我切割，然后介导前-IL-1β的切割成为其生物活性形式。越来越多的证据表明，IL-1β是急性炎症和急性肺损伤的重要介质。我们将使用两个成熟的NLRP3和NLRC4炎症小体激活的小鼠模型来验证我们的假设，分别是内毒素和铜绿假单胞菌。我们将通过实验证明NLR蛋白NLRP3和NLRC4通过其富含亮氨酸的重复序列与波形蛋白相互作用，这种蛋白质-蛋白质相互作用是将前IL-1β加工和成熟为具有生物活性的IL-1β所必需的。我们将进一步研究NLR蛋白在线粒体膜外膜的定位是否需要在炎性肌小体激活过程中与波形蛋白相互作用。我们的数据表明，波形蛋白IF网络作为一个额外的检查点来控制炎症小体的激活，这一假说我们将在体外和体内使用波形蛋白/NLR蛋白相互作用的药物抑制剂进行验证。这些发现可能具有重要的意义 针对NLRs和Vimentin之间相互作用的生物学和临床意义可以为ALI/ARDS患者的治疗提供一套新的治疗方法。我们制定了三个相互关联的特异性目标，以研究在体内和体外急性肺损伤模型中波形蛋白中间丝的调节：特异性目标1：确定III型中间纤维蛋白激活炎症小体的机制，这是炎症和急性肺损伤所必需的。特定目标2：确定蛋白质：波形蛋白和NLRs之间的蛋白质相互作用，NLRP3和NLRC4，激活炎症体所需的。具体目的3：确定NLR蛋白移位到线粒体膜上是否需要波形蛋白介导的炎性小体的组装和激活。

 DESCRIPTION (provided by applicant): The inflammasome is activated in response to a broad spectrum of infectious agents known to cause acute lung injury (ALI), but it must also be tightly regulated to avoid deleterious effects from the wide array of pathogens, pathogen-associated molecular patterns and danger-associated molecular patterns, which are known to trigger its activation. Activation of the inflammasome requires two distinct pro-inflammatory stimuli. In the first step, Toll-like receptor (TLR) signaling activates NF-κB and upregulates pro IL-1β. The second step involves the assembly of the multiprotein complex, which induces self-cleavage of caspase-1, and then mediates the cleavage of pro- IL-1β into its biologically active form. Growing evidence indicates that IL-1β is a critical mediator of acute inflammation and acute lung injury. We will test our hypothesis using two well- established murine models of NLRP3 and NLRC4 inflammasome activation, LPS and Pseudomonas aeruginosa, respectively. We will conduct experiments to demonstrate that NLR proteins, NLRP3 and NLRC4, via their leucine-rich-repeat (LRR) interact with vimentin and that this protein-protein interaction is required for the processing and maturation of pro-IL-1β into biologically active IL-1β. We will further examine whether the localization of the NLR proteins at the outer mitochondrial membrane is required for the interaction with vimentin during inflammasome activation. Our data suggest that the vimentin IF network acts as an additional checkpoint to control the activation of the inflammasome, a hypothesis we will test using a pharmacologic inhibitor of vimentin/NLR protein interactions in vitro and in vivo. These findings are likely to have important biological and clinical significance as targeting the interaction between NLRs and vimentin could provide a suite of novel therapeutics to treat patients with ALI/ARDS. We have formulated three interrelated specific aims to study the regulation of vimentin intermediate filaments in both in vivo and in vitro models of acute lung injury: Specific Aim 1: To determine the mechanism by which vimentin, a type III intermediate filament protein, activates the inflammasome, which is required for inflammation and acute lung injury. Specific Aim 2: To identify the protein:protein interactions between vimentin and NLRs, NLRP3 and NLRC4, required for the activation of the inflammasome. Specific Aim 3: To determine whether translocation of NLR proteins to the mitochondrial membrane is required for the vimentin-mediated assembly and activation of the inflammasome.