Development of a human intestinal microphysiological system for the study of immune responses to protozoan parasites

项目摘要 微生理学系统在模拟人类疾病方面具有巨大潜力，但先进的体外微生理学模型 寄生虫感染和免疫力严重不足。寄生虫是发病的主要原因， 死亡率在全球范围内，每年感染数百万人，然而，没有有效的疫苗可用于任何 肠道寄生虫感染通过受污染的食物或水的口服传播是最常见的传播途径。 但是我们对寄生虫/宿主相互作用的知识，包括寄生虫如何相互作用， 与免疫细胞一起引起疾病或引发保护性免疫反应，在肠道内， 非常有限。迫切需要建立改进的人体免疫-寄生虫相互作用的体外模型 以捕获寄生虫感染建立和疾病进展期间存在的关键特征。解决 基于这一需求，我们将开发一种基于LumeNext的微生理肠道脉管系统 微流体装置系统。这种三维细胞培养装置重现了肠道结构， 包括两侧为血液和淋巴管系统的人肠上皮内腔。这些先进的 在体外模型中，我们将寄生虫引入肠上皮，将人类免疫细胞引入肠上皮。 监测寄生虫病和免疫反应。我们的目标是创造一个微生理学的 该系统可用于研究任何原生动物寄生虫。对于这个提议，我们将使用原生动物 寄生虫弓形虫（Toxoplasma gondii（T. gondii）和溶组织内阿米巴（E.因为1）他们是人类 全球重要的病原体，2）它们在原生生物进化树的不同分支上，3）它们有 定义的实验室生长条件和遗传标记菌株，4）我们的实验室最近开发了 具有产生大量高传染性卵囊和T.刚地。我们 数据显示T.在我们的体外模型系统中， 免疫应答和人免疫细胞从脉管系统的迁移。在这个项目中，我们将使用这些 3D仿生肠道血管管腔模型，以解决人类免疫的关键知识缺口 对T. gondii和E.溶组织剂我们会创造一个无氧环境， 可以在缺氧条件下定义反应。这些实验将提供基础 了解人类对肠道T细胞的先天免疫反应。弓形虫感染是必不可少的 疫苗研发。我们也将模型E。使用营养限制和共培养溶组织侵袭性疾病 艰难梭菌感染我们在这项提案中取得的进展将使微生物学和 免疫学领域，以确定对肠道寄生虫的生物学相关阶段的免疫反应 在人体模型中。

PROJECT SUMMARY Microphysiological systems have great potential for modeling human disease but advanced in vitro models of parasitic infection and immunity are severely underrepresented. Parasites are major causes of morbidity and mortality globally, infecting millions of people every year, yet, there are no effective vaccines available for any enteric parasitic infection. Oral transmission via contaminated food or water is the most common route of parasitic infection for humans, but our knowledge of parasite/host interactions, including how parasites interact with immune cells to either cause disease or elicit a protective immune response, within the intestinal tract is very limited. There is a critical need to create improved in vitro models of human immune-parasite interactions to capture key features present during parasitic infection establishment and disease progression. To address this need we will develop a microphysiological gut vasculature lumen system based on the LumeNEXT microfluidic device system. This 3-dimensional cell culture device recapitulates the gut architecture and includes a human intestinal epithelial lumen flanked by blood and lymphatic vasculature. With these advanced in vitro models, we will introduce parasites into the intestinal epithelium and human immune cells into the vasculature to monitor parasitic disease and immune response. Our goal is to create a microphysiological system that can be used for the study of any protozoan parasite. For this proposal, we will use the protozoan parasites Toxoplasma gondii (T. gondii) and Entamoeba histolytica (E. histolytica) because 1) they are human pathogens of global importance, 2) they are on distinct branches of the protist evolutionarily tree, 3) they have defined lab growth conditions and genetically tagged marker strains, 4) our lab has recently developed the unique ability to produce large numbers of the highly infectious oocysts and cysts forms of both T. gondii. Our data shows that T. gondii infection of the intestinal epithelial lumen in our in vitro model system elicits an active immune response and migration of human immune cells from the vasculature. In this project, we will use these 3D biomimetic gut-vasculature lumen models to address critical knowledge gaps of the human immune responses to T. gondii and E. histolytica. We will incorporate an anaerobic environment so that the immune responses can be defined under hypoxic conditions. These experiments will provide the foundational understanding of the human innate immune responses to intestinal T. gondii infection that are essential for vaccine development. We will also model E. histolytica invasive disease using nutrient limitation and co-culture with Clostridiodes difficile. The advances we will achieve in this proposal will allow the microbiology and immunology fields to determine the immune responses to the biologically relevant stages of intestinal parasites in human models.