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

肺内皮和上皮CXCR2在白细胞转运至感染肺部和肺损伤中的新功能

• 批准号: 491579569
• 负责人: Professor Dr. Alexander Zarbock
• 金额: --
• 依托单位: Department of Systems Immunology
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/491579569?language=en
• 关键词: Novel; functions; pulmonary; endothelial; epithelial

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.

危及生命的细菌性肺病是由过度的中性粒细胞募集和增加的血管通透性引起的，两者都是由促炎介质的释放驱动的。增加的渗透性导致肺泡充满富含蛋白质的水肿液。由此产生的气体交换损失导致急性呼吸衰竭和通常的灾难性疾病，称为急性呼吸窘迫综合征，需要急救和重症监护支持。提高对潜在机制的理解是非常重要的。因此，我们的目标是确定CXCR 2对肺上皮细胞和内皮细胞的确切作用，通过分析CXCR 2的两个假定的功能：影响血管和肺泡通透性和调节趋化因子转运。为了解决这一问题，我们将分别在上皮细胞或内皮细胞中产生CXCR 2缺陷的条件小鼠系，并研究CXCL 1或CXCL 2引入后的白细胞募集。此外，我们将分析这些小鼠的运输的趋化因子的毛细血管腔的生物素标记的趋化因子，这将是可视化的电子显微镜使用链霉亲和素包被的金颗粒intrichelheal注射。将通过使用内皮细胞的体外transwell试验评估血管渗透性，然后在体内分析Evan's Blue渗漏。为了确定囊泡介导的转胞吞作用中可能的功能，我们将使用生物化学方法研究CXCR 2是否参与网格蛋白包被的凹坑或小窝的形成。第二个目标是确定内皮和上皮CXCR 2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，和趋化因子的表达在细菌刺激。在体内肺显微镜检查将使我们能够检测在哪个位置招募的中性粒细胞在其过程中的迁移延迟。作为第三个目标，我们将分析CXCR 2激活后的信号通路，并确定β-arrestin作为多功能衔接分子在这一通路中的作用。使用生物化学方法，我们将确定β-arrestin在CXCR 2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-arrestin在跨上皮迁移过程中的作用。在使用针对β-arrestin的shRNA的体外研究之后，我们将详细分析WT和β-arrestin缺陷小鼠肺部感染期间的白细胞募集、细胞因子表达和细菌负荷。最后，为了估计所观察到的机制是否是肺细胞所独有的，我们将通过活体显微镜研究内皮CXCR 2在提睾肌中白细胞粘附和迁移过程中的作用。总之，这些实验将大大拓宽我们对CXCR 2介导的白细胞募集、细胞因子转胞吞和血管通透性的认识。