Engineering the pulmonary epithelium "on a chip" to investigate immune responses to the inhaled external environmental stimuli

据世界卫生组织（世卫组织）估计，2016年，全球90%的人口呼吸被污染的空气，可能导致420万人过早死亡（世卫组织，2018年）。肺上皮作为抵御外界伤害的第一道防线，受到机械力的作用，并与吸入剂接触，因此经常受伤（Whitsett & Alenghat, 2015）。众所周知，肺上皮通过将一些外来颗粒捕获在黏液细胞（主要是杯状细胞）产生的黏液中，并通过一组纤毛细胞协同将黏液移出肺部，从而参与对它们的清除（Davies & Moores, 2010; Knight & Holgate, 2003）。在基底细胞的帮助下，肺上皮还能够参与免疫监视，启动先天免疫反应，调节肺液和邻近结缔组织（Deckers等人，2017;Knight & Holgate, 2003; Loxham, Davies & Blume, 2014）。受到损伤或刺激后，支气管上皮可发生重塑，如杯状细胞增生、粘液分泌增多、气道增厚。此外，炎性反应可由促炎细胞因子、先天免疫细胞和蛋白酶特异性趋化因子的分泌引发（Knight & Holgate, 2003）。在慢性呼吸系统疾病中，慢性或反复炎症和慢性气道重塑与各种呼吸系统疾病（如哮喘和COPD）的进展有关。然而，在气流剪切应力的背景下，这些环境刺激接触人体所引起的确切生理和病理机制在很大程度上尚未得到充分的研究。由于现有实验模型的局限性，需要一个具有模拟气道腔内气流特征的鲁棒模型。此前，Benam等人（2016）开发了一种小型芯片气道，该芯片由暴露于空气流动的分化肺泡上皮、内皮层和代表血液流动的介质电流组成。与其他小组的其他出版物（此处未列出）一样，他们的主要研究重点是研究上皮的基底侧（例如与内皮相互作用以促进中性粒细胞粘附）。但对上皮顶端侧的研究在很大程度上被忽视。本项目将开发一种基于可控气流剪切应力的肺支气管上皮芯片模型，以帮助研究体外组织水平环境刺激对肺上皮的影响。微流体芯片将由支撑在膜上的分化支气管上皮组成，其顶端有“呼吸”或振荡气流，底部有介质流（Benam et al., 2016）。一旦成功建立，该模型可以可靠地检查气道通道中不同水平剪切应力的影响以及单个或组合吸入环境刺激的影响。这将为气道细胞对污染物等刺激的生理和病理生理反应提供新的机制见解。这将根据上皮屏障渗透性、粘液产生、纤毛功能和免疫反应（如受体表达、细胞因子和趋化因子产生）进行评估。微流控芯片的设计还允许直接成像可视化上皮的任何结构变化以及实时细胞因子测量。通过密切模仿上皮细胞在体内暴露的环境。这种微工程模型有望减少动物模型在该领域的使用，揭示器官水平对环境刺激的反应，并有助于我们理解慢性呼吸系统疾病的发展。

According to the World Health Organisation (WHO), it was estimated that 90% of the worldwide population breathes polluted air and can contributed to 4.2 million premature deaths worldwide in 2016 (WHO, 2018). The pulmonary epithelium, being the first line of defence against the external insults, are subjected to mechanical forces and contact with the inhaled agents thus can be frequently injured (Whitsett & Alenghat, 2015). The pulmonary epithelium is well known to participate in the clearance of some foreign particles by trapping them in the mucus produced by mucous cells (mainly goblet cells) and concerted movement of mucus out of the lungs by a group of ciliated cells (Davies & Moores, 2010; Knight & Holgate, 2003). Together with the help of basal cells, the pulmonary epithelium is also able to engage in immune surveillance, initiate an innate immune response and regulate lung fluids and the proximate connective tissue (Deckers et al., 2017; Knight & Holgate, 2003; Loxham, Davies & Blume, 2014). Upon damage or stimulation, the bronchial epithelium can undergo remodelling such as goblet cell hyperplasia, mucus hypersecretion, airway thickening. In addition, an inflammatory response can be initiated by the secretion of proinflammatory cytokines, chemokines specific for innate immune cells and proteases (Knight & Holgate, 2003). In chronic respiratory diseases, chronic or repeated inflammation and chronic airway remodelling are associated with the progression of various respiratory diseases such as asthma and COPD. However, the exact physiological and pathological mechanisms induced by contact of these environmental stimuli in the context of airflow shear stress in human are largely underexplored.Due to the limitations of the available experimental models, a robust model with features mimicking the airflow in the airway lumen is required. Previously, Benam and others (2016) have developed a small airway-on-a-chip which consists of a differentiated alveoli epithelium exposed to flow of air as well as a layer of endothelium and a current of media representing the blood flow. Along with other publications by other groups (not listed here), their predominant research focuses on studying the basal side of the epithelium (e.g. interactions with endothelium to promote neutrophil adherence). But the research on the apical side of the epithelium has been largely ignored. In this project, a pulmonary bronchial epithelium-on-chip model with controlled airflow-based shear stress will be developed to assist in studying the effect of environmental stimuli on the pulmonary epithelium at the tissue level in vitro. The microfluid chip will consist of a differentiated bronchial epithelium supported on a membrane with a 'breathing' or oscillating airflow on its apical side and a media flow on the basal side (Benam et al., 2016). Once successfully established, this model can reliably examine the effect of different levels of shear stress in the airway channel and the effects of individual or combinations of inhaled environmental stimuli. This will provide novel mechanistic insights in the physiological and pathophysiological responses of the airway cells to stimuli such as pollutants. This will be assessed in terms of epithelial barrier permeability, mucus production, cilia function and immunological responses, such as receptor expression as well as cytokine and chemokine production. The design of the microfluidic chips also allows direct imaging to visualise any structural changes of the epithelium as well as real-time cytokine measurements. By closely mimicking the environment that the epithelium cells are exposed to in vivo. This micro-engineered model will hopefully reduce the use of animal models in this field, shed light into organ-level responses to environmental stimuli and contribute to our understanding of the development of chronic respiratory diseases.