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

中性粒细胞是人类中最丰富的白细胞群体，在 先天免疫和炎症，包括心血管炎症，如 缺血再灌注损伤(IRI)、心肌梗死后炎症和动脉粥样硬化。 中性粒细胞黏附是中性粒细胞从血管重新聚集到炎症组织的关键步骤。 使用β2整合素使人中性粒细胞在流动状态下被激活的内皮细胞停滞。我的前辈 工作证实了已知的Beta2整合素激活途径(扩展E紧跟高亲和力H； E-H-to E H-to E H)，发现了一种新的途径，即在整合素之前头盔打开 在人类主要中性粒细胞(E-H-到E-H再到E-H)停止期间延伸。新确定的 弯曲开放(E-H)β2整合素结合表达于顺式细胞的中性粒细胞上的配体(ICAM)。我证明了这一点 这种自身抑制作用限制了中性粒细胞在体外和体内的黏附。拟议的工作将是第一次 在这一领域的时间，询问β2整合素激活的超分辨率显微镜。我提炼了 通过分子建模获得初步数据点，实现了单分子分辨。我的数据显示 E-H整合素不是随机定向的，而是显示出与 ‘面对面’的定位。在具体的目标1中，我将测试这样的假设：这种面对面的模式是 由E-H整合素与ICAM二聚体结合的成对顺式相互作用引起。如果是的话， 阻断功能的ICAM抗体应该会破坏这种“面对面”的模式。非阻塞ICAM 抗体将用于测试ICAM是否如预期的那样与E-Hβ2整合素共定位。在……里面 具体目标2，我将筛选使β2整合素保持在细胞内的小分子变构抑制剂 自动抑制E-H构象。在我的初步实验中，我已经开发出一种 基于流式细胞术的整合素活化(E和H)筛选方法我会在两个地方测试化合物 库来查找候选人。我建议确认成功的候选分子在 用流式细胞术和建立的微流控黏附试验检测原代中性粒细胞。特定目标 三是直接检测E-H整合素的生理意义。我将检验这一假设 E-Hβ2整合素的自身抑制保护心肌细胞免受IRI。我会用移植的小鼠 与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。我预计这些嵌合的老鼠会表现出更严重的 心肌缺血再灌注和组织丢失。来自AIM 2的成功的抑制剂将在体内的IRI模型中进行测试。 在完成这些实验后，我们将知道Beta2整合素激活的分子细节。 在抑制原发人类中性粒细胞(目标1)的过程中，找到候选小分子抑制剂 稳定E-Hβ2整合素构象(目标2)，了解E-Hβ2的体内相关性 整合素(目标3)。候选抑制剂代表了药物开发的先导化合物，目的是 用于预防和治疗炎症性疾病，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.