Anti-inflammatory activity of hydrogels designed to capture extracellular inflammasomes

项目总结/摘要 称为炎性小体的多蛋白复合物在先天免疫细胞中形成， 检测病原体或组织损伤。异常炎性小体激活导致慢性炎症， 它是许多威胁生命的疾病的罪魁祸首，如癌症，糖尿病，心血管疾病， 以及SARS-CoV-2感染中的细胞因子风暴。炎性小体组装受蛋白质-蛋白质控制 相互作用，因为它需要三种蛋白质的多个拷贝的自缔合和寡聚化：传感器 检测危险信号，激活炎症因子的蛋白酶和连接蛋白ASC 传感器和蛋白酶。炎性小体形成导致质膜破裂和伴随的细胞死亡， 从而导致促炎细胞因子和炎性体颗粒释放到细胞外 环境这些细胞外炎性小体被附近的细胞内化，以延续和放大细胞内的炎症。 炎症反应。去除或隔离细胞外炎性小体可能会抑制或减少 炎症因此，细胞外炎性小体是潜在的治疗靶点。我们的实验室 对适配器ASC的功能和结构及其与其他设备的相互作用有着丰富的经验。 炎症体蛋白，使我们创造了水凝胶，旨在形成特定的蛋白质-蛋白质相互作用， 因此，它们具有通过有效地捕获和释放炎症体来广泛抑制炎症的潜力。 去除细胞外炎性小体。该项目的重点是确定水凝胶化因素导致 炎性小体颗粒在无细胞系统中的最佳结合（Aim 1）; 水凝胶在活化的先天免疫细胞存在下的效率（Aim 2）。我们的实验计划将 结合联合收割机细胞生物学和生物化学方法，包括活/死细胞成像，流式细胞术， 免疫印迹、酶联免疫吸附测定和荧光光谱法。总的来说，我们希望 开发一种广泛适用的水凝胶技术，以在没有药物负载的情况下减少炎症， 靶向与许多炎性疾病有关的炎性体。

PROJECT SUMMARY/ABSTRACT Multiprotein complexes known as inflammasomes form in innate immune cells to trigger inflammation upon detection of pathogens or tissue damage. Abnormal inflammasome activation leads to chronic inflammation, which is the culprit of numerous life-threatening diseases such as cancer, diabetes, cardiovascular disorders, and the cytokine storm in SARS-CoV-2 infection. Inflammasome assembly is controlled by protein-protein interactions as it requires the self-association and oligomerization of multiple copies of three proteins: sensors to detect danger signals, a protease to activate inflammatory factors, and the adaptor protein ASC to connect sensor and protease. Inflammasome formation leads to plasma membrane rupture and concomitant cell death, thus resulting in the release of proinflammatory cytokines and inflammasome particles to the extracellular environment. These extracellular inflammasomes are internalized by nearby cells to perpetuate and amplify the inflammatory response. Removing or sequestering extracellular inflammasomes will likely inhibit or reduce inflammation. Therefore, extracellular inflammasomes are potential therapeutic targets. Our laboratory’s extensive experience on the function and structure of the adaptor ASC, and its interactions with other inflammasome proteins, has led us to create hydrogels designed to form specific protein-protein interactions with inflammasomes; thus, they have the potential to broadly inhibit inflammation by effectively capturing and removing extracellular inflammasomes. This project focuses on identifying the hydrogelation factors leading to optimum biding of inflammasome particles in cell-free systems (Aim 1); and determining the anti-inflammatory efficiency of the hydrogels in the presence of activated innate immune cells (Aim 2). Our experimental plan will combine cell biology and biochemical approaches, including live/dead cell imaging, flow cytometry, immunoblotting, enzyme-linked immunosorbent assays and fluorescence spectroscopy. Overall, we expect to develop a hydrogel technology of broad applicability to reduce inflammation in the absence of drug loading by targeting the inflammasome, which is implicated in many inflammatory diseases.