Disentangling the role of shear flow in regulating biomechanical interactions of endothelial cells with intracellular bacterial pathogens

阐明剪切流在调节内皮细胞与细胞内细菌病原体生物力学相互作用中的作用

• 批准号: 532757564
• 负责人: Dr.-Ing. Effie Bastounis, Ph.D.
• 金额: --
• 依托单位: Lehrstuhl für Mikrobiologie / Biotechnologie
• 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/532757564?language=en
• 关键词: Disentangling; role; shear; flow; regulating

Endothelia cells (ECs) lining the inner lumen of blood vessels are constantly exposed to shear stresses (SSs) due to blood flowing over their surfaces. While the effects of fluid SSs on regulating different aspects of EC behavior and contributing to pathologies (e.g., atherosclerosis) have been previously studied, little is known about how fluid SSs modulate the interactions of ECs with intracellular bacterial pathogens like Listeria monocytogenes (LM). The primary site of infection by food-borne LM is the intestinal epithelium. However, in vivo LM can manage to spread to distant tissues by bypassing ECs and/or infecting them, a critical step to breach important barriers, such as the blood-brain, and systemically disseminate causing lethal fatalities. In this proposal, we will investigate how fluid SSs modulate host EC biomechanics, and in turn adhesion, invasion and intercellular spread of LM using an in vitro organotypic model. Taking advantage of this model, which is compatible with time-lapse microscopy, infection assays and parallel conduction of biomechanical measurements like traction force and monolayer stress microscopy, we will first explore how varying levels of fluid SSs modulate the susceptibility of ECs to bacterial infection. We will also investigate the underlying molecular mechanisms taking advantage of a recent transcriptomic screen that revealed key differentially expressed genes between flow exposed ECs as compared to ECs under stationary conditions. Subsequently, we will determine how intracellular bacterial infection alters EC biomechanics, how those alterations are modulated by fluid SSs, and whether SS exposure ultimately acts protectively benefiting the host by obstructing bacterial dissemination, or conversely benefits the bacteria by enhancing intercellular bacterial spread. Through the unique experimental setup in conjunction with biomechanical measurements and precise spatiotemporal quantitation of the infection process, we will be able to uncover both important pathogenesis and host defense mechanisms in a context where the important role of the hemodynamic forces is considered. Moreover, by using the specific pathogen LM as a tool to subtly modulate EC biomechanics (since LM does not destroy host cell integrity because it relies on it), we anticipate revealing novel aspects of vascular EC mechanobiology.

由于血液在其表面流动，血管内腔内衬的内皮细胞 (EC) 不断受到剪切应力 (SS) 的影响。虽然之前已经研究过液体 SS 对调节 EC 行为的不同方面以及导致病理学（例如动脉粥样硬化）的影响，但人们对液体 SS 如何调节 EC 与单核细胞增生李斯特菌 (LM) 等细胞内细菌病原体的相互作用知之甚少。食源性LM 的主要感染部位是肠上皮。然而，体内LM可以通过绕过EC和/或感染EC来设法传播到远处组织，这是突破血脑等重要屏障并系统性传播导致致命死亡的关键一步。在本提案中，我们将利用体外器官模型研究液体 SS 如何调节宿主 EC 生物力学，进而调节 LM 的粘附、侵袭和细胞间扩散。利用该模型，该模型与延时显微镜、感染测定和并行进行生物力学测量（如牵引力和单层应力显微镜）兼容，我们将首先探索不同水平的液体SS如何调节EC对细菌感染的敏感性。我们还将利用最近的转录组筛选来研究潜在的分子机制，该筛选揭示了与静态条件下的 EC 相比，流动暴露的 EC 之间的关键差异表达基因。随后，我们将确定细胞内细菌感染如何改变 EC 生物力学，这些改变如何通过液体 SS 进行调节，以及 SS 暴露最终是否通过阻碍细菌传播来保护性地有益于宿主，或者相反地通过增强细胞间细菌传播来有益于细菌。通过独特的实验设置，结合生物力学测量和感染过程的精确时空定量，我们将能够在考虑血流动力学的重要作用的情况下揭示重要的发病机制和宿主防御机制。此外，通过使用特定病原体 LM 作为巧妙调节 EC 生物力学的工具（因为 LM 不会破坏宿主细胞的完整性，因为它依赖于宿主细胞），我们期望揭示血管 EC 力学生物学的新方面。