Anti-inflammatory activity of hydrogels designed to capture extracellular inflammasomes

项目摘要/摘要 被称为炎症体的多蛋白复合体在先天免疫细胞中形成，以触发炎症 检测病原体或组织损伤。炎症小体异常激活会导致慢性炎症， 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、 SARS-CoV-2感染中的细胞因子风暴。炎症体组装受蛋白质-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关联和寡聚：传感器 检测危险信号，一种激活炎症因子的蛋白酶，以及连接的接头蛋白ASC 传感器和蛋白酶。炎性小体的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症体颗粒释放到细胞外 环境。这些细胞外的炎性小体被附近的细胞内化，以持久和放大 炎症反应。去除或隔离细胞外炎性小体可能会抑制或减少 发炎。因此，细胞外炎性小体是潜在的治疗靶点。我们实验室的 对适配器ASC的功能和结构及其与其他设备的交互具有丰富的经验 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 炎症小体；因此，它们有可能通过有效地捕获和抑制炎症而广泛抑制炎症。 清除细胞外炎性小体。该项目的重点是确定导致水凝胶的因素 无细胞系统中炎性小体颗粒的最佳结合(目标1)；以及抗炎作用的测定 水凝胶在激活的天然免疫细胞存在下的效率(目标2)。我们的实验计划将 结合细胞生物学和生化方法，包括活/死细胞成像，流式细胞仪， 免疫印迹、酶联免疫吸附试验和荧光光谱分析。总体而言，我们预计 开发一种具有广泛适用性的水凝胶技术，通过以下方式在没有载药的情况下减少炎症 针对与许多炎症性疾病有关的炎症性小体。

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.