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

嗜中性粒细胞是人类中最丰富的白细胞群体，并且在免疫调节中发挥重要作用。 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）、心肌梗死后炎症和动脉粥样硬化。 中性粒细胞粘附是中性粒细胞从血管募集到炎症组织的关键步骤。 使用β 2整联蛋白在流动下在活化的内皮上的人中性粒细胞停滞。我以前的 工作证实了已知的β 2整联蛋白活化途径（延伸E后接高亲和力H; E-H-到E + H-到E + H+），并发现了一个新的途径，其中头片段在整合素之前打开 在原代人嗜中性粒细胞停滞期间延长（E-H-至E-H+至E + H+）。新识别的 β-开放（E-H+）β 2整联蛋白结合在中性粒细胞上顺式表达的配体（ICAM）。我证明了 这种自身抑制在体外和体内限制了嗜中性粒细胞的粘附。拟议的工作将首先 时间在这个领域，询问β 2整合素激活的超分辨率显微镜。我提炼了 初步数据点的分子建模和实现单分子分辨率。我的数据显示 E-H+整联蛋白不是随机取向的，而是显示出与 "面对面"取向。在具体目标1中，我将检验这种"面对面"模式是 由E-H+整联蛋白与ICAM二聚体结合的成对顺式相互作用引起。如果是这样的话， 功能阻断ICAM抗体应该破坏这种"面对面"模式。非阻塞性ICAM 抗体将用于测试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，我将筛选小分子变构抑制剂，保持β 2整合素在 自抑制E-H+构象。在我的初步实验中，我已经开发了一种 基于流式细胞术的整联蛋白活化（E+和H+）的筛选测定。我将测试两种化合物 图书馆寻找候选人我建议确认成功的候选分子的功效， 使用流式细胞术和已建立的微流体粘附测定法检测原代中性粒细胞。具体目标 3是直接检测E-H+整合素的生理意义。我将检验这个假设， E-H + β 2整联蛋白的自身抑制保护心肌细胞免受IRI。我会用移植了 ICAM-1和ICAM-2双敲除骨髓，我以前已经证明， β 2整联蛋白对中性粒细胞的自身抑制。我预计这些嵌合体小鼠会表现出更严重的 心肌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。 完成这些实验后，我们将了解β 2整合素激活的分子细节 在原代人嗜中性粒细胞停滞期间（aim 1），寻找候选的小分子抑制剂， 稳定E-H + β 2整联蛋白构象（目的2），并了解E-H + β 2的体内相关性 整合素（aim 3）。候选抑制剂代表药物开发的先导化合物， 用于预防和治疗炎性疾病，例如IRI和慢性血管炎症。

Neutrophils are the most abundant population of leukocytes in humans and play essential roles in innate immunity and inflammation, including cardiovascular inflammatory diseases, such as ischemia-reperfusion injury (IRI), post-myocardial infarction inflammation and atherosclerosis. Neutrophil adhesion is a key step in neutrophil recruitment from blood vessels to inflamed tissues. Human 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 high-affinity 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 points by molecular modeling and achieved single molecule resolution. My data show 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 a flow-cytometry-based screening assay of integrin activation (E+ and H+). I will test compounds in two libraries to find candidates. I propose to confirm the efficacy of successful candidate molecules in primary neutrophils using flow cytometry and in 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.