A Novel 3D Skin Explant Model to Study Anaerobic Bacterial Infection.
A Novel 3D Skin Explant Model to Study Anaerobic Bacterial Infection.
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DOI：
10.3389/fcimb.2017.00404
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发表时间：
2017
影响因子：
5.7
通讯作者：
Tötemeyer S
中科院分区：
文献类型：
--
作者：
Maboni G;Davenport R;Sessford K;Baiker K;Jensen TK;Blanchard AM;Wattegedera S;Entrican G;Tötemeyer S
皮肤感染研究经常受到财政和伦理约束的限制，而替代方法，如单层细胞培养，不反映许多细胞过程，限制了它们的应用。对于更多功能的替代，3D皮肤培养模型提供了许多优点，例如维持组织结构和存在于宿主环境中的细胞类型。可以使用外科手术或屠宰后获得的组织建立3D皮肤培养模型，使其成为动物实验的成本效益和有吸引力的替代方案。大多数3D培养模型都是针对需氧病原体建立的，但目前还没有针对无氧皮肤感染的模型。足癣是一种影响绵羊指间皮肤的厌氧细菌感染，在世界范围内造成重大的动物福利和经济影响。结节双杆菌是一种革兰氏阴性厌氧菌，是引起手足口病的病原体。结核分枝杆菌感染和宿主免疫反应的机制尚不清楚。在这里，我们提出了一个新的3D皮肤离体模型来研究厌氧菌感染的绵羊皮肤外植体感染结节棘球绦虫。我们的研究结果表明，结节棘球蚴可以侵入皮肤外植体，并且在培养基中可以量化关键炎症标志物的表达改变。通过76 h的组织完整性（组织病理学特征）和细胞死亡（DNA断裂）来评估外植体的活力，结果表明模型在28 h内是稳定的。通过qPCR对所有感染的皮肤外植体中的结节病菌进行量化，并通过荧光原位杂交（fluorescence in situ Hybridization）观察到细菌侵入表皮。培养基中促炎细胞因子/趋化因子的测定显示，外植体释放il - 1β以响应细菌。相比之下，CXCL8的产生水平与模拟感染的外植体没有什么不同。3D皮肤模型真实地模拟了指间皮肤，并证明结节芽胞杆菌侵入皮肤并引发了对这种细菌的早期细胞炎症反应。这种新型模型是研究厌氧细菌感染的第一种。
Skin infection studies are often limited by financial and ethical constraints, and alternatives, such as monolayer cell culture, do not reflect many cellular processes limiting their application. For a more functional replacement, 3D skin culture models offer many advantages such as the maintenance of the tissue structure and the cell types present in the host environment. A 3D skin culture model can be set up using tissues acquired from surgical procedures or post slaughter, making it a cost effective and attractive alternative to animal experimentation. The majority of 3D culture models have been established for aerobic pathogens, but currently there are no models for anaerobic skin infections. Footrot is an anaerobic bacterial infection which affects the ovine interdigital skin causing a substantial animal welfare and financial impact worldwide. Dichelobacter nodosus is a Gram-negative anaerobic bacterium and the causative agent of footrot. The mechanism of infection and host immune response to D. nodosus is poorly understood. Here we present a novel 3D skin ex vivo model to study anaerobic bacterial infections using ovine skin explants infected with D. nodosus. Our results demonstrate that D. nodosus can invade the skin explant, and that altered expression of key inflammatory markers could be quantified in the culture media. The viability of explants was assessed by tissue integrity (histopathological features) and cell death (DNA fragmentation) over 76 h showing the model was stable for 28 h. D. nodosus was quantified in all infected skin explants by qPCR and the bacterium was visualized invading the epidermis by Fluorescent in situ Hybridization. Measurement of pro-inflammatory cytokines/chemokines in the culture media revealed that the explants released IL1β in response to bacteria. In contrast, levels of CXCL8 production were no different to mock-infected explants. The 3D skin model realistically simulates the interdigital skin and has demonstrated that D. nodosus invades the skin and triggered an early cellular inflammatory response to this bacterium. This novel model is the first of its kind for investigating an anaerobic bacterial infection.