Novel functions of pulmonary endothelial and epithelial CXCR2 in leukocyte trafficking to infected lungs and in lung injury

危及生命的细菌性肺部疾病是由中性粒细胞过度募集和血管通透性增加引起的，两者都是由促炎介质的释放驱动的。通透性增加导致肺泡充盈富含蛋白质的水肿液。由此导致的气体交换损失导致急性呼吸衰竭和典型的灾难性疾病，称为急性呼吸窘迫综合征，需要通气和重症监护支持。提高对潜在机制的理解是非常重要的。因此，我们的目的是通过分析CXCR2的两个可能功能：影响血管和肺泡通透性以及调节趋化因子运输，来确定CXCR2在肺上皮细胞和内皮细胞中的确切作用。为了解决这一问题，我们将分别在上皮细胞或内皮细胞中培养条件性缺乏CXCR2的小鼠系，并研究CXCL1或CXCL2引入后的白细胞募集。此外，我们将通过气管内注射生物素标记的趋化因子来分析这些小鼠向毛细血管腔的趋化因子运输，这些趋化因子将通过链霉亲和素包被金颗粒的电子显微镜观察。血管通透性将通过体外内皮细胞transwell试验进行评估，随后在体内分析Evan’s Blue渗漏。为了确定在囊泡介导的胞吞作用中的可能功能，我们将通过生化方法研究CXCR2是否参与网格蛋白包被的凹坑或小泡的形成。作为第二个目标，我们将确定内皮和上皮CXCR2在细菌性肺部感染中的作用。在这里，我们将详细分析白细胞募集、血管通透性和趋化因子表达对细菌刺激的反应。活体肺显微镜将使我们能够检测到招募的中性粒细胞在迁移过程中延迟的位置。作为第三个目标，我们将分析cxcr2激活后的信号通路，并确定β -阻滞蛋白作为多功能接头分子在该通路中的作用。利用生化方法，我们将确定β -抑制蛋白在cxcr2介导的定位、复合物形成以及细胞因子表达中的作用。此外，我们假设β -阻滞蛋白在跨上皮迁移过程中起作用。在使用shRNA对抗β -抑制素的体外研究之后，我们将详细分析WT和β -抑制素缺乏小鼠肺部感染期间的白细胞募集、细胞因子表达和细菌负担。最后，为了估计所观察到的机制是否为肺细胞所独有，我们将通过活体显微镜研究内皮细胞CXCR2在肌内白细胞粘附和转运中的作用。综上所述，这些实验将大大拓宽我们对cxcr2介导的白细胞募集、细胞因子胞吞作用和血管通透性的认识。

Life threatening bacterial lung diseases are caused by excessive neutrophil recruitment and increased vascular permeability, both driven by the release of pro-inflammatory mediators. Increased permeability leads to alveolar flooding with protein-rich edema fluid. The resulting loss of gas exchange leads to acute respiratory failure and typically catastrophic illness, termed acute respiratory distress syndrome, requiring ventilatory and critical care support. Improved understanding of the underlying mechanism is of keen importance. Therefore, we aim to identify the exact role of CXCR2 on pulmonary epithelial and endothelial cells by analysing two putative functions of CXCR2: Influencing the vascular and alveolar permeability and regulation of chemokine transport. To address this, we will generate conditional mouse lines deficient for CXCR2 in epithelial or in endothelial cells, respectively, and investigate leukocyte recruitment following CXCL1 or CXCL2 introduction. Furthermore, we will analyse these mice for the transport of chemokines to the capillary lumen by intratracheal injection of biotin-labelled chemokines, which will be visualized by electron microscopy using streptavidin coated gold particles. Vascular permeability will be assessed by using in vitro transwell assays with endothelial cells, and later in vivo analysing Evan´s Blue leakage. To identify a possible function in vesicle-mediated transcytosis, we will investigate whether CXCR2 participates in the formation of clathrin-coated pits or caveolae by using biochemical methods. As second aim, we will identify the roles of endothelial and epithelial CXCR2 during bacterial lung infection. Here, we will analyse in detail leukocyte recruitment, vascular permeability, and chemokine expression in response to bacterial stimuli. In vivo lung microscopy will allow us to detect at which location recruited neutrophils are delayed during their course of emigration. As third aim, we will analyse the signalling pathways following CXCR2-activation, and identify the role of beta-arrestin as multifunctional adaptor molecule in this pathway. Using biochemical methods, we will identify the role of beta-arrestin in CXCR2-mediated localization, complex formation, as well as in cytokine expression. Furthermore, we hypothesise a role for beta-arrestin during trans-epithelial migration. Following in vitro-studies using shRNA against beta-arrestin, we will analyse in detail leukocyte recruitment, cytokine expression, and bacterial burden during lung infection in WT and beta-arrestin-deficient mice. Finally, to estimate whether the observed mechanisms are unique to pulmonary cells, we will investigate the role of endothelial CXCR2 during leukocyte adhesion and transmigration in the cremaster muscle by intravital microscopy. Taken together, these experiments will substantially broaden our knowledge concerning CXCR2-mediated leukocyte recruitment, cytokine transcytosis and vascular permeability.