Modeling Long-Term Host Cell-Giardia lamblia Interactions in an In Vitro Co-Culture System

Globally, there are greater than 700,000 deaths per year associated with diarrheal disease. The flagellated intestinal parasite, Giardia lamblia, is one of the most common intestinal pathogens in both humans and animals throughout the world. While attached to the gastrointestinal epithelium, Giardia induces epithelial cell apoptosis, disrupts tight junctions, and increases intestinal permeability. The underlying cellular and molecular mechanisms of giardiasis, including the role lamina propria immune cells, such as macrophages, play in parasite control or clearance are poorly understood. Thus far, one of the major obstacles in ascertaining the mechanisms of Giardia pathology is the lack of a functionally relevant model for the long-term study of the parasite in vitro. Here we report on the development of an in vitro co-culture model which maintains the basolateral-apical architecture of the small intestine and allows for long-term survival of the parasite. Using transwell inserts, Caco-2 intestinal epithelial cells and IC-21 macrophages are co-cultured in the presence of Giardia trophozoites. Using the developed model, we show that Giardia trophozoites survive over 21 days and proliferate in a combination media of Caco-2 cell and Giardia medium. Giardia induces apoptosis of epithelial cells through caspase-3 activation and macrophages do not abrogate this response. Additionally, macrophages induce Caco-2 cells to secrete the pro-inflammatory cytokines, GRO and IL-8, a response abolished by Giardia indicating parasite induced suppression of the host immune response. The co-culture model provides additional complexity and information when compared to a single-cell model. This model will be a valuable tool for answering long-standing questions on host-parasite biology that may lead to discovery of new therapeutic interventions.全球范围内，每年有超过70万例死亡与腹泻疾病相关联。作为鞭毛型肠道寄生虫，蓝氏贾第鞭毛虫（Giardia lamblia）是全球范围内人畜共患的最常见肠道病原体之一。当该寄生虫附着于胃肠道上皮细胞时，可诱导上皮细胞凋亡、破坏上皮紧密连接，并提升肠道通透性。目前学界对贾第虫病（giardiasis）潜在的细胞与分子机制仍缺乏深入认知，其中包括固有层免疫细胞，例如巨噬细胞（macrophages），在寄生虫控制或清除过程中所发挥的具体作用。迄今为止，明确贾第虫致病机制的主要障碍之一，是缺乏可用于该寄生虫体外（in vitro）长期研究的功能相关性模型。本研究报道了一种体外共培养模型的构建方案，该模型可维持小肠的基底外侧-顶端极性结构，并支持该寄生虫的长期存活。研究采用Transwell小室（transwell inserts），将Caco-2肠道上皮细胞与IC-21巨噬细胞在贾第鞭毛虫滋养体（Giardia trophozoites）存在的条件下进行共培养。利用该构建完成的模型，研究证实贾第鞭毛虫滋养体可存活超过21天，并在Caco-2细胞培养基与贾第虫培养基的混合体系中实现增殖。贾第虫可通过激活半胱天冬酶-3（caspase-3）诱导上皮细胞凋亡，而巨噬细胞无法抑制这一凋亡反应。此外，巨噬细胞可诱导Caco-2细胞分泌促炎细胞因子GRO与IL-8，而贾第虫可消除这一促炎应答，表明寄生虫可抑制宿主免疫反应。与单细胞培养模型相比，该共培养模型可提供更为复杂的实验体系与更多研究维度。该模型将成为解答宿主-寄生虫生物学领域长期遗留问题的宝贵工具，有望推动新型治疗干预手段的研发。