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

摘要中性粒细胞是人体内最丰富的白细胞，对先天免疫和 炎症，包括心血管炎性疾病，如缺血-再灌注损伤（IRI），缺血后再灌注损伤（IRI）， 心肌梗死炎症和动脉粥样硬化。阻止是从血液中募集中性粒细胞的关键步骤 到发炎的组织使用β 2整联蛋白在流动下活化的内皮上的中性粒细胞停滞。我 先前的工作证实了已知的β 2整联蛋白活化途径（延伸E，随后是头片段， 打开H; E-H-到E + H-到E + H+），并发现了一个新的途径，其中头部在整合素之前打开 在原代人嗜中性粒细胞停滞期间延长（E-H-至E-H+至E + H+）。新鉴定的E-open（E- H+）β 2整联蛋白以顺式结合在嗜中性粒细胞上表达的配体（ICAM）。我证明了这种自我抑制限制了 体外和体内中性粒细胞粘附。拟议的工作将在该领域首次询问beta2 通过超分辨率显微镜观察整合素活化。我通过分子建模完善了初步数据， 实现了单分子分辨率。我发现E-H+整合素并不是随机取向的，而是显示出分子的 与“面对面”方向一致的图案。在具体目标1中，我将检验这种"面对面"的假设， Face '模式由E-H+整合素与ICAM二聚体结合的成对顺式相互作用引起。如果是，函数- 阻断ICAM抗体会破坏这种"面对面"模式。将使用非封闭性ICAM抗体 以测试ICAM是否如预期的那样与E-H + β 2整联蛋白共定位。在具体目标2中，我将筛选 小分子变构抑制剂，其保持β 2整联蛋白处于自抑制E-H+构象。在我 在初步的实验中，我已经开发了基于流式细胞术的整合素高通量筛选 活化（E+和H+）。我将在三个库中测试化合物以找到候选化合物。我建议确认 成功的候选分子在初级中性粒细胞使用流式细胞术和建立微流控 粘附测定。具体目标3是直接测试E-H+整联蛋白的生理学意义。我将测试 E-H + β 2整联蛋白的自身抑制保护心肌细胞免受IRI的假说。我会用老鼠 移植ICAM-1和ICAM-2双敲除骨髓，我以前已经证明，消除 β 2整联蛋白对中性粒细胞的自身抑制。我希望这些嵌合体小鼠能表现出更严重的心肌梗死， IRI和组织损失。来自目标2的成功抑制剂将在该IRI模型中进行体内测试。完成后 通过这些实验，我们将了解β 2整合素在原发性人肝癌细胞凋亡停滞期间活化的分子细节。 中性粒细胞（目的1），寻找稳定E-H + β 2整合素构象的候选小分子抑制剂 (aim 2），并了解E-H + β 2整联蛋白的体内相关性（目的3）。候选抑制剂代表铅 用于药物开发的化合物，其目的在于预防和治疗炎性疾病，例如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.