CHARACTERISING ARAP3-MEDIATED INTEGRIN INACTIVATION IN THE NEUTROPHIL

免疫细胞在对抗感染时需要产生炎症来杀死病原体。这是一个严格调控的过程，但偶尔调控出错，即使在没有感染的情况下，免疫细胞也会产生炎症。这种情况发生在慢性炎症条件下，如类风湿关节炎或慢性阻塞性肺病。慢性炎症造成许多痛苦，并对世界各地的卫生服务造成重大消耗。没有已知的药物可以治愈慢性炎症。中性粒细胞是血液中最常见的免疫细胞，在炎症的产生中起着重要作用。中性粒细胞离开血流到达炎症部位。它们只有通过爬过血管壁进入周围组织才能离开血流。这依赖于它们分子上的特殊适配器，即整合素。整合素以“粘性”（活性）和“非粘性”（非活性）状态存在。许多实验室研究了整合素如何变得活跃：这取决于几种细胞内调节因子。没有整合素，或者不能激活它们会阻止中性粒细胞向炎症部位的募集。这可以阻止炎症，但也可以免疫，并导致复发性感染。只有少数研究分析了总是活跃的整合素。事实证明，这也减少了中性粒细胞向炎症部位的募集。我们已经证明，一种叫做“ARAP3”的细胞内调节因子是使中性粒细胞整合素失活所必需的。没有ARAP3，中性粒细胞不能有效地募集到炎症部位，但募集并没有被消除。ARAP3本身受另外两种调节因子（称为pi3激酶和Rap）的调节，并反过来调节另外两种调节因子（称为Arf6和RhoA）。我们将研究Rap、RhoA和Arf6如何在中性粒细胞中与ARAP3一起调节整合素失活。我们将通过在ARAP3中引入小分子变化来实现这一点，这样它就不能再依次与其他调节因子之一对话。我们将使用从没有ARAP3的小鼠骨髓中提取的干细胞，并对它们进行改造，使其具有一种略有变化的ARAP3。改良后的干细胞将被放入培养皿中。当我们改变它们的培养基时，它们会变成中性粒细胞。这些中性粒细胞的整合素将在试管中进行分析。这将使我们了解细胞内调节因子如何与ARAP3一起调节整合素失活。这项工作可能为针对慢性炎症的新治疗方法奠定基础。

Immune cells need to generate inflammation to kill pathogens when they fight infections. This is a tightly regulated process, but occasionally the regulation goes wrong and immune cells generate inflammation even in the absence of infection. This happens in chronic inflammatory conditions, such as rheumatoid arthritis or chronic obstructive pulmonary disease. Chronic inflammation causes much suffering and poses an important drain on health services world-wide. No known drugs can cure chronic inflammation. Neutrophils, the most common immune cells in the blood, are important players in the generation of inflammation. Neutrophils leave the blood stream to travel to sites of inflammation. They can only do this if they can leave the blood stream by crawling through the vessel wall into the surrounding tissue. This relies on specialised adapters on their molecules, the integrins. Integrins exist in a "sticky" (active) and a "non-sticky" (inactive) state. Many labs have studied how integrins become active: this depends on several intracellular regulators. Not having integrins, or not being able to activate them stops neutrophil recruitment to inflammatory sites. This stops inflammation but also immunity and leads to recurrent infections. Only few studies have analysed integrins that are always active. It turns out that this also reduces neutrophil recruitment to inflammatory sites. We have shown that an intracellular regulator called 'ARAP3' is required for inactivating neutrophil integrins. Without ARAP3 neutrophils are not efficiently recruited to inflammatory sites, but recruitment is not abolished. ARAP3 is itself regulated by at two other regulators (called PI3-kinase and Rap), and it in turn regulates two further regulators (called Arf6 and RhoA). We will study how Rap, RhoA and Arf6 are working together with ARAP3 in the neutrophil to regulate integrin inactivation. We will do this by introducing small molecular changes into ARAP3 so that it can no longer talk to one of the other regulators in turn. We will use stem cells from the bone marrow of mice that have no ARAP3, and engineer them to have one of the slightly changed ARAP3s instead. The modified stem cells will then be placed into culture. When we change their culture medium, they will transform into neutrophils. These neutrophils' integrins will be analysed in the test tube. This will allow us to learn how the intracellular regulators work together with ARAP3 to regulate integrin inactivation. This work may build the foundations for new therapeutic approaches that target chronic inflammation.