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

根据世界卫生组织(WHO)的数据，据估计，全球90%的人口呼吸着受污染的空气，这可能导致2016年全球420万人过早死亡(世卫组织，2018年)。肺上皮是抵御外界侮辱的第一道防线，受到机械力的作用，与吸入的物质接触，因此经常会受到损伤(Whitsett&Alenghat，2015)。众所周知，肺上皮通过将它们捕获到粘液细胞(主要是杯状细胞)产生的粘液中，并通过一组纤毛细胞将粘液协调地移出肺外，从而参与一些外来颗粒的清除(Davies&Moore，2010；Knight&Holgate，2003)。在基底细胞的帮助下，肺上皮细胞也能够参与免疫监视，启动先天免疫反应，并调节肺液和近端结缔组织(Deckers等人，2017；Knight&Holgate，2003；Loxham，Davies&Blume，2014)。当受到损伤或刺激时，支气管上皮可发生重塑，如杯状细胞增生、粘液高分泌、呼吸道增厚。此外，炎症反应可由促炎细胞因子、先天免疫细胞特异性趋化因子和蛋白水解酶的分泌启动(Knight&Holgate，2003)。在慢性呼吸系统疾病中，慢性或反复炎症和慢性呼吸道重塑与各种呼吸系统疾病的进展有关，如哮喘和COPD。然而，由于现有实验模型的局限性，这些环境刺激在人体气流剪切力作用下的接触所引起的确切的生理和病理机制还不够深入，因此需要一个稳健的模型来模拟气道腔内的气流。此前，Benam和其他人(2016)开发了一种芯片上的小气道，它由暴露在气流中的分化的肺泡上皮以及一层内皮和一层代表血流的介质组成。与其他小组发表的其他论文(未在此列出)一样，他们的主要研究集中在研究上皮基底部(例如，与内皮细胞相互作用以促进中性粒细胞黏附)。但对上皮顶端的研究在很大程度上被忽视了。在本项目中，我们将建立一种基于可控气流剪应力的肺上皮芯片模型，以帮助在体外组织水平上研究环境刺激对肺上皮细胞的影响。微流体芯片将由分化的支气管上皮细胞组成，支撑在膜上，顶端有‘呼吸’或振荡的气流，底部有中介流(Benam等人，2016)。一旦成功建立，这个模型可以可靠地检测不同水平的切应力在呼吸道中的影响，以及个别或吸入环境刺激组合的影响。这将为呼吸道细胞对污染物等刺激的生理和病理生理学反应提供新的机械性见解。这将从上皮屏障通透性、粘液产生、纤毛功能和免疫反应(如受体表达以及细胞因子和趋化因子的产生)方面进行评估。微流控芯片的设计还允许直接成像，以可视化上皮的任何结构变化，以及实时细胞因子测量。通过密切模拟上皮细胞在体内暴露的环境。这种微工程模型有望减少这一领域中动物模型的使用，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道疾病的发展。

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.