Molecular mechanism and preclinical translation of beta2 integrin auto-inhibition on neutrophil arrest and inflammation

摘要中性粒细胞是人类体内含量最丰富的白细胞，对人体的先天免疫和免疫功能至关重要。 炎症，包括心血管炎症性疾病，如缺血再灌注损伤(IRI)， 心肌梗死、炎症和动脉粥样硬化。阻止是中性粒细胞从血液中募集的关键步骤 发炎的组织。使用β2整合素，中性粒细胞在流动状态下抑制激活的内皮细胞。我的 先前的工作证实了已知的Beta2整合素激活途径(扩展E之后是耳机- 打开H；E-H-到E+H-到E+H+)，并发现了一种新的途径，其中头盔在整合素之前打开 在人类主要中性粒细胞(E-H-到E-H+到E+H+)停止期间延伸。新发现的弯开(E- H+)β2整合素结合表达在中性粒细胞上的配体(ICAM)。我证明了这种自动抑制限制了 中性粒细胞在体外和体内的黏附。拟议的工作将在这一领域第一次询问Beta2 用超分辨显微镜观察整合素的激活。我通过分子模拟提炼了初步数据 实现了单分子分辨率。我发现E-H+整合素不是随机定向的，而是显示出一个分子 图案与“面对面”方向一致。在具体目标1中，我将测试这样的假设： Face‘模式是由E-H+整合素与ICAM二聚体结合成对的顺式相互作用引起的。如果是这样，则函数- 阻断ICAM抗体应该会破坏这种“面对面”的模式。将使用非封闭的ICAM抗体 以测试ICAM是否如预期的那样与E-H+Beta2整合素共定位。在《特定目标2》中，我将筛选 小分子变构抑制剂，使β2整合素保持在自动抑制的E-H+构象中。在我的 初步实验，本人已建立了基于流式细胞仪的高通量整合素筛选方法 激活(E+和H+)。我将在三个库中测试化合物以找到候选者。我建议确认一下它的疗效 应用流式细胞术和建立的微流控技术对初级中性粒细胞中成功的候选分子进行检测 粘附性试验。具体目标3是直接测试E-H+整合素的生理意义。我会测试一下 E-H+β2整合素自身抑制保护心肌细胞免受IRI的假说。我会用老鼠 移植了ICAM-1和ICAM-2双敲除骨髓，我之前已经证明消除了 β2整合素对中性粒细胞的自身抑制作用。我预计这些嵌合小鼠会表现出更严重的心肌梗死 IRI和组织丢失。来自AIM 2的成功的抑制剂将在体内的IRI模型中进行测试。在完成后 这些实验，我们将知道在原发人类停搏期间β2整合素激活的分子细节。 中性粒细胞(目标1)，寻找稳定E-H+β2整合素构象的候选小分子抑制剂 (目标2)，并了解E-H+Beta2整合素的体内相关性(目标3)。候选抑制剂代表铅 用于药物开发的化合物，旨在预防和治疗炎症性疾病，如IRI和 慢性血管炎症。

Abstract Neutrophils are the most abundant leukocytes in humans and essential for innate immunity and inflammation, including cardiovascular inflammatory diseases, such as ischemia-reperfusion injury (IRI), post- myocardial infarction inflammation and atherosclerosis. Arrest is a key step in neutrophil recruitment from blood to inflamed tissues. Neutrophils arrest on activated endothelium under flow using the beta2 integrins. My previous work confirmed the known pathway of beta2 integrin activation (extension E followed by headpiece- opening H; E-H- to E+H- to E+H+) and discovered a new pathway where the headpiece opens before the integrin extends during arrest of primary human neutrophils (E-H- to E-H+ to E+H+). The newly identified bent-open (E- H+) beta2 integrin binds ligands (ICAMs) expressed on neutrophils in cis. I showed that this auto-inhibition limits neutrophil adhesion in vitro and in vivo. The proposed work will, for the first time in this field, interrogate beta2 integrin activation by super-resolution microscopy. I refined the preliminary data by molecular modeling and achieved single molecule resolution. I found that E-H+ integrins are not randomly oriented, but show a molecular pattern consistent with a ‘Face-to-Face’ orientation. In specific aim 1, I will test the hypothesis that this ‘Face-to- Face’ pattern is caused by pairwise in-cis interactions of E-H+ integrins binding to ICAM dimers. If so, function- blocking ICAM antibodies should disrupt this ‘Face-to-Face’ pattern. Non-blocking ICAM antibody will be used to test whether ICAMs are co-localized with E-H+ beta2 integrins as expected. In specific aim 2, I will screen small molecule allosteric inhibitors that keep beta2 integrins in the auto-inhibited E-H+ conformation. In my preliminary experiments, I already developed flow-cytometry-based high-throughput screening of integrin activation (E+ and H+). I will test compounds in three libraries to find candidates. I propose to confirm the efficacy of successful candidate molecules in primary neutrophils using flow cytometry and established microfluidic adhesion assays. Specific aim 3 is to directly test the physiologic significance of E-H+ integrins. I will test the hypothesis that auto-inhibition of E-H+ beta2 integrins protects cardiomyocytes from IRI. I will use mice transplanted with ICAM-1 and ICAM-2 double knockout bone marrow, which I have previously shown to eliminate the auto-inhibition of beta2 integrin on neutrophils. I expect these chimeric mice to show more severe myocardial IRI and tissue loss. Successful inhibitors from aim 2 will be tested in this IRI model in vivo. After completion of these experiments, we will know the molecular details of beta2 integrin activation during arrest of primary human neutrophils (aim 1), find candidate small molecule inhibitors that stabilize the E-H+ beta2 integrin conformation (aim 2), and know the in vivo relevance of E-H+ beta2 integrins (aim 3). The candidate inhibitors represent lead compounds for drug development aimed at for preventing and treating inflammatory diseases, such as IRI and chronic vascular inflammation.