Gram-negative bacterial infections have a major economic impact on both the livestock industry and public health. Toll-like receptor 4 (TLR4) plays a crucial role in host defence against Gram-negative bacteria. Exploring the defence mechanism regulated by TLR4 may provide new targets for treatment of inflammation and control of bacterial infections. In a previous study, we generated transgenic sheep overexpressing TLR4 by microinjection to improve disease resistance. The defence mechanism through which TLR4 overexpression protected these sheep against pathogens is still not fully understood. In the present study, we used Escherichia coli to infect monocytes isolated from peripheral blood of the animal model. The overexpression of TLR4 strongly enhanced the percentage of endocytosis and capacity of elimination in monocytes during the early stages of infection. This phenomenon was mainly due to overexpression of TLR4 promoting caveolae-mediated endocytosis. Pretreatment of the transgenic sheep monocytes with inhibitors of TLR4, Src signalling, or the caveolae-mediated endocytosis pathway reduced the internalization of bacteria, weakened the ability of the monocytes to eliminate the bacteria, and increased the pH of the endosomes. Together, our results reveal the effects of TLR4 on the control of E. coli infection in the innate immunity of sheep and provide crucial evidence of the caveolae-mediated endocytosis pathway required for host resistance to invading bacteria in a large animal model, providing theoretical support for breeding disease resistance in the future. Furthermore, Src and caveolin 1 (CAV1) could be potentially valuable targets for the control of infectious diseases.

中文翻译：

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Toll样受体4的过表达通过增强小窝依赖性细胞内吞作用而促进绵羊内在大肠杆菌的内在化和消除

革兰氏阴性细菌感染对畜牧业和公共卫生都具有重大的经济影响。Toll样受体4（TLR4）在宿主抵抗革兰氏阴性细菌的防御中起着至关重要的作用。探索由TLR4调节的防御机制可能为治疗炎症和控制细菌感染提供新的靶标。在先前的研究中，我们通过显微注射产生了过表达TLR4的转基因绵羊，以提高抗病能力。TLR4过表达保护这些绵羊免受病原体侵害的防御机制仍不完全清楚。在本研究中，我们使用大肠杆菌感染从动物模型外周血中分离的单核细胞。在感染的早期，TLR4的过表达极大地增强了单核细胞内吞作用的百分率和消除能力。这种现象主要是由于TLR4的过表达促进了小窝介导的胞吞作用。用TLR4，Src信号转导或小窝介导的胞吞途径抑制剂预处理转基因绵羊单核细胞会减少细菌的内在化，削弱单核细胞消除细菌的能力，并增加内体的pH。总之，我们的研究结果揭示了TLR4对绵羊先天免疫的大肠杆菌感染控制的影响，并提供了在大型动物模型中宿主抵抗入侵细菌所需的小窝介导的内吞途径的关键证据，为今后的抗病育种提供理论支持。此外，Src和小窝蛋白1（CAV1）可能是控制传染病的潜在有价值目标。