Mucosal Immunology and Biology Research Center, Division of Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital, Boston, Massachusetts, USA.
Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA.
Microbiol Spectr. 2021 Sep 3;9(1):e0000321. doi: 10.1128/Spectrum.00003-21. Epub 2021 Jun 9.
Gastrointestinal infections cause significant morbidity and mortality worldwide. The complexity of human biology and limited insights into host-specific infection mechanisms are key barriers to current therapeutic development. Here, we demonstrate that two-dimensional epithelial monolayers derived from human intestinal organoids, combined with like bacterial culturing conditions, provide significant advancements for the study of enteropathogens. Monolayers from the terminal ileum, cecum, and ascending colon recapitulated the composition of the gastrointestinal epithelium, in which several techniques were used to detect the presence of enterocytes, mucus-producing goblet cells, and other cell types following differentiation. Importantly, the addition of receptor activator of nuclear factor kappa-B ligand (RANKL) increased the presence of M cells, critical antigen-sampling cells often exploited by enteric pathogens. For infections, bacteria were grown under like conditions known to induce virulence. Overall, interesting patterns of tissue tropism and clinical manifestations were observed. Shigella flexneri adhered efficiently to the cecum and colon; however, invasion in the colon was best following RANKL treatment. Both Salmonella enterica serovars Typhi and Typhimurium displayed different infection patterns, with . Typhimurium causing more destruction of the terminal ileum and . Typhi infecting the cecum more efficiently than the ileum, particularly with regard to adherence. Finally, various pathovars of Escherichia coli validated the model by confirming only adherence was observed with these strains. This work demonstrates that the combination of human-derived tissue with targeted bacterial growth conditions enables powerful analyses of human-specific infections that could lead to important insights into pathogenesis and accelerate future vaccine development. While traditional laboratory techniques and animal models have provided valuable knowledge in discerning virulence mechanisms of enteric pathogens, the complexity of the human gastrointestinal tract has hindered our understanding of physiologically relevant, human-specific interactions; and thus, has significantly delayed successful vaccine development. The human intestinal organoid-derived epithelial monolayer (HIODEM) model closely recapitulates the diverse cell populations of the intestine, allowing for the study of human-specific infections. Differentiation conditions permit the expansion of various cell populations, including M cells that are vital to immune recognition and the establishment of infection by some bacteria. We provide details of reproducible culture methods and infection conditions for the analyses of , , and pathogenic Escherichia coli in which tissue tropism and pathogen-specific infection patterns were detected. This system will be vital for future studies that explore infection conditions, health status, or epigenetic differences and will serve as a novel screening platform for therapeutic development.
胃肠道感染在全球范围内导致了显著的发病率和死亡率。人类生物学的复杂性以及对宿主特异性感染机制的有限了解是当前治疗开发的主要障碍。在这里，我们证明了源自人类肠道类器官的二维上皮单层，结合类似的细菌培养条件，为肠病原体的研究提供了重大进展。来自回肠末端、盲肠和升结肠的单层再现了胃肠道上皮的组成，其中使用了几种技术来检测分化后肠细胞、产生粘液的杯状细胞和其他细胞类型的存在。重要的是，添加核因子 kappa-B 配体受体激活剂 (RANKL) 增加了 M 细胞的存在，M 细胞是肠道病原体经常利用的关键抗原取样细胞。对于感染，细菌在已知诱导毒力的类似条件下生长。总的来说，观察到了有趣的组织嗜性和临床表现模式。福氏志贺菌有效地黏附在盲肠和结肠上;然而，在用 RANKL 处理后，在结肠中的侵袭效果最佳。伤寒沙门氏菌血清型 Typhi 和 Typhimurium 都表现出不同的感染模式，. Typhimurium 导致回肠末端的破坏更多，. Typhi 比 Typhimurium 更有效地感染盲肠，特别是在黏附方面。最后，验证了各种大肠杆菌的病原菌模式，确认只有这些菌株才会发生黏附。这项工作表明，将人源组织与靶向细菌生长条件相结合，可以对人类特异性感染进行有力分析，从而为发病机制提供重要的见解，并加速未来疫苗的开发。 虽然传统的实验室技术和动物模型在辨别肠道病原体的毒力机制方面提供了有价值的知识，但人类胃肠道的复杂性阻碍了我们对生理相关的人类特异性相互作用的理解;因此，疫苗的开发进展非常缓慢。源自人肠道类器官的上皮单层 (HIODEM) 模型紧密模拟了肠道的多种细胞群体，允许研究人类特异性感染。分化条件允许各种细胞群体的扩增，包括对免疫识别和某些细菌建立感染至关重要的 M 细胞。我们提供了重现性培养方法和感染条件的详细信息，用于分析 、 和致病性大肠杆菌，其中检测到了组织嗜性和病原体特异性感染模式。该系统对于未来探索感染条件、健康状况或表观遗传差异的研究将是至关重要的，并将作为治疗开发的新型筛选平台。
