Abstract
BackgroundGram-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.ResultsIn 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.ConclusionTogether, 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.
Highlights
Gram-negative bacterial infections have a major economic impact on both the livestock industry and public health
When bacteria bind to Toll-like receptor 4 (TLR4)-bound myeloid differentiation protein 2 (MD2) on the plasma membrane, Toll-interleukin receptor domain containing adaptor protein (TIRAP) and myeloid differentiation factor 88 (MyD88) participate in the rapid activation of the TIRAP-MyD88-dependent pathway, which results in the production of proinflammatory cytokines [8]
The monocyte markers cluster of differentiation 14 (CD14) and CD11b were detected on the cell membranes by antibody staining (Fig. 1c), and TLR4 expression in the transgenic and wild-type sheep was compared at the transcription and protein levels (Fig. 1d–f)
Summary
Gram-negative bacterial infections have a major economic impact on both the livestock industry and public health. Toll-like receptor 4 (TLR4) is known to be a patternrecognition receptor that triggers innate immunity [1,2,3] This molecule is expressed mainly in the immunocytes of mammals, including macrophages, monocytes, neutrophils, and dendritic cells [4]. TLR4 recognizes Gram-negative bacteria via their signature molecules to activate the host’s innate immune response, which is tightly regulated via distinct factors and pathways. The localization of TLR4 has emerged as a key determinant of TLR4 function These factors include subcellular TLR4 localization and ligand sensing, which occurs on the cell surface through activation of the Toll-interleukin 1 receptor (TIR) domain containing adaptor protein (TIRAP)-myeloid differentiation factor 88 (MyD88)-dependent pathway and through endocytosis that activates the TRIF-related adaptor molecule (TRAM)-TIR domain containing adaptor-inducing interferon-β (TRIF)-dependent pathway [6]. With the help of the bridging factor TRAM, TLR4 recruits the TRIF receptor that drives the TRAM-TRIF-dependent pathway and leads to the production of type I interferons [10]
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