The epithelium of the human sinonasal cavities is colonized by a diverse microbial community, modulating epithelial development and immune priming and playing a role in respiratory disease. Here, we present a novel in vitro approach enabling a 3-day coculture of differentiated Calu-3 respiratory epithelial cells with a donor-derived bacterial community, a commensal species (Lactobacillus sakei), or a pathobiont (Staphylococcus aureus). We also assessed how the incorporation of macrophage-like cells could have a steering effect on both epithelial cells and the microbial community. Inoculation of donor-derived microbiota in our experimental setup did not pose cytotoxic stress on the epithelial cell layers, as demonstrated by unaltered cytokine and lactate dehydrogenase release compared to a sterile control. Epithelial integrity of the differentiated Calu-3 cells was maintained as well, with no differences in transepithelial electrical resistance observed between coculture with donor-derived microbiota and a sterile control. Transition of nasal microbiota from in vivo to in vitro conditions maintained phylogenetic richness, and yet a decrease in phylogenetic and phenotypic diversity was noted. Additional inclusion and coculture of THP-1-derived macrophages did not alter phylogenetic diversity, and yet donor-independent shifts toward higher Moraxella and Mycoplasma abundance were observed, while phenotypic diversity was also increased. Our results demonstrate that coculture of differentiated airway epithelial cells with a healthy donor-derived nasal community is a viable strategy to mimic host-microbe interactions in the human upper respiratory tract. Importantly, including an immune component allowed us to study host-microbe interactions in the upper respiratory tract more in depth.IMPORTANCE Despite the relevance of the resident microbiota in sinonasal health and disease and the need for cross talk between immune and epithelial cells in the upper respiratory tract, these parameters have not been combined in a single in vitro model system. We have developed a coculture system of differentiated respiratory epithelium and natural nasal microbiota and incorporated an immune component. As indicated by absence of cytotoxicity and stable cytokine profiles and epithelial integrity, nasal microbiota from human origin appeared to be well tolerated by host cells, while microbial community composition remained representative for that of the human (sino)nasal cavity. Importantly, the introduction of macrophage-like cells enabled us to obtain a differential readout from the epithelial cells dependent on the donor microbial background to which the cells were exposed. We conclude that both model systems offer the means to investigate host-microbe interactions in the upper respiratory tract in a more representative way.

中文翻译：

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具有供体来源的微生物群和THP-1巨噬细胞的双重和三重上皮共培养模型系统，以模拟人类鼻腔中的宿主-微生物相互作用。

人鼻窦腔的上皮细胞被多种微生物群落定殖，调节上皮细胞的发育和免疫引发，并在呼吸系统疾病中起作用。在这里，我们提出了一种新颖的体外方法，该方法可以使分化的Calu-3呼吸道上皮细胞与供体来源的细菌群落，共生物种（日本乳酸杆菌）或病原菌（金黄色葡萄球菌）共培养3天。我们还评估了巨噬细胞样细胞的掺入如何对上皮细胞和微生物群落具有指导作用。在我们的实验装置中接种供体来源的微生物群不会对上皮细胞层造成细胞毒性应激，与无菌对照组相比，细胞因子和乳酸脱氢酶的释放没有改变。分化的Calu-3细胞的上皮完整性也得以维持，在与供体来源的菌群共培养与无菌对照之间观察到的跨上皮电阻没有差异。鼻微生物群从体内到体外的转变保持了系统发育丰富性，但注意到系统发育和表型多样性降低。THP-1衍生的巨噬细胞的其他包涵和共培养没有改变系统发育多样性，但是观察到供体独立的向较高的莫拉氏菌和支原体丰度的转变，而表型多样性也增加了。我们的研究结果表明，将分化的气道上皮细胞与健康的供体来源的鼻腔共同培养是在人类上呼吸道中模拟宿主-微生物相互作用的可行策略。重要的是，包括免疫成分使我们能够更深入地研究上呼吸道中的宿主-微生物相互作用。重要提示尽管常驻菌群与鼻窦健康和疾病相关，并且需要上层免疫细胞与上皮细胞之间的相互干扰呼吸道，这些参数尚未在单个体外模型系统中组合。我们已经开发了分化呼吸上皮和天然鼻微生物群的共培养系统，并整合了免疫成分。如无细胞毒性和稳定的细胞因子谱以及上皮完整性所表明的那样，宿主细胞对人类来源的鼻微生物群似乎具有良好的耐受性，而微生物群落组成仍然代表了人（鼻）鼻腔。重要的，巨噬细胞样细胞的引入使我们能够从上皮细胞中获得不同的读数，这取决于细胞所接触的供体微生物背景。我们得出结论，两个模型系统都提供了一种以更具代表性的方式研究上呼吸道中宿主与微生物相互作用的方法。