A novel 3D culture model of fungal keratitis to explore host-pathogen interactions within the stromal environment.
A novel 3D culture model of fungal keratitis to explore host-pathogen interactions within the stromal environment.
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一种新的真菌性角膜炎3D培养模型，以探索基质环境中宿主与病原体的相互作用。
DOI：
10.1016/j.exer.2021.108581
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发表时间：
2021-06
影响因子：
3.4
通讯作者：
Fuller, Kevin K.
中科院分区：
文献类型：
--
作者：
Brown, Marina E.;Montgomery, Micaela L.;Kamath, Manali M.;Nicholas, Sarah;Liu, Yutao;Karamichos, Dimitrios;Fuller, Kevin K.
关键词：
真菌性角膜炎（FK）的病理是由真菌生长和角膜基质内的炎症驱动的。标准的体外感染模型——包括病原体和角膜细胞在组织培养基中共同培养——足以探测宿主对真菌的反应；然而，他们缺乏基质的生理结构和营养成分来准确地研究真菌的侵袭和代谢过程。因此，我们试图开发一种能够同时评估宿主和真菌细胞生物学的FK培养模型。为此，我们采用了一种先前描述的系统，在该系统中，将原代人角膜成纤维细胞（HCFs）培养在transwell膜上，然后它们分泌类似于人基质的三维（3D）胶原基质。我们证明了两种常见的FK霉菌剂，嗜油镰刀菌和烟曲霉，渗透到这些结构中并引起胶原基质的破坏，这是感染的特征。真菌存在时HCF形态发生改变，电镜显示真菌孢子明显内化到这些细胞中。与这种明显的吞噬细胞样活性相一致，几种促炎细胞因子/趋化因子（包括TNFα， IL-1β， IL-6和IL-8）的mRNA和蛋白水平与未感染的样品相比显着上调。我们同样发现了几种HCF金属蛋白酶（MMPs）的上调，这些酶在伤口愈合过程中分解胶原蛋白，并可能进一步激活促炎信号分子。最后，几个真菌胶原酶基因在构建物的生长过程中相对于单独在组织培养基中生长而上调，这表明真菌代谢向蛋白质分解代谢转变。综上所述，我们的研究结果表明，这种3d基质模型为研究FK期间宿主和真菌细胞病理生物学提供了一个生理学相关的系统。
Fungal keratitis (FK) pathology is driven by both fungal growth and inflammation within the corneal stroma. Standard in vitro infection models–involving co-culture of the pathogen and the corneal cells in tissue culture medium–are sufficient to probe host responses to the fungus; however, they lack the physiological structure and nutrient composition of the stroma to accurately study fungal invasiveness and metabolic processes. We therefore sought to develop a culture model of FK that would allow for both host and fungal cell biology to be evaluated in parallel. Towards this end, we employed a previously described system in which primary human cornea fibroblasts (HCFs) are cultured on transwell membranes, whereupon they secrete a three-dimensional (3D) collagen matrix that resembles the human stroma. We demonstrated that two common mold agents of FK, Fusarium petroliphilum and Aspergillus fumigatus, penetrated into these constructs and caused a disruption of the collagen matrix that is characteristic of infection. HCF morphology appeared altered in the presence of fungus and electron microscopy revealed a clear internalization of fungal spores into these cells. Consistent with this apparent phagocyte-like activity of the HCFs, mRNA and protein levels for several pro-inflammatory cytokines/ chemokines (including TNFα, IL-1β, IL-6, and IL-8) were significantly upregulated compared to uninfected samples. We similarly found an upregulation of several HCF metalloproteases (MMPs), which are enzymes that breakdown collagen during wound healing and may further activate pro-inflammatory signaling molecules. Finally, several fungal collagenase genes were upregulated during growth in the constructs relative to growth in tissue culture media alone, suggesting a fungal metabolic shift towards protein catabolism. Taken together, our results indicate that this 3D-stromal model provides a physiologically relevant system to study host and fungal cell pathobiology during FK.
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