Changes in the Relative Inflammatory Responses in Sheep Cells Overexpressing of Toll-Like Receptor 4 When Stimulated with LPS

Shoulong Deng,Zhuo Deng,Qian Wu,Yuchang Yao,Guoshi Liu,Yunhai Zhang,Wu Li,Kun Yu,Zhengxing Lian,Wenting Li,Zhanjiang Liu

doi:10.1371/journal.pone.0047118

Abstract

BackgroundMany groups of Gram-negative bacteria cause diseases harmful to sheep. TLR4 is an important Toll-like receptor (TLR) which responds to common Gram-negative bacterial infections. Activation of TLR4 leads to the induction of inflammatory responses, which is a linkage between the innate and adaptive immune systems. A vector pTLR4-3S was constructed to overexpress TLR4 gene in sheep. In this study, effects of TLR4 overexpression on inflammation response under LPS stimulated were addressed in vivo and in vitro.Methodology/Principal FindingsSheep fetal fibroblasts were transfected with expression vector pTLR4-3S. Transgenic sheep were produced by microinjection of the constructed plasmids into fertilized eggs. Fetal fibroblasts, monocyte-macrophage and fibroblasts isolated from the transgenic sheep were stimulated by LPS. After that immunoactive factors (TNF-α, IL-10, IL-6, IL-8, IFN-γ), nitric oxide, phagocytize ability and adhesion were detected. Furthermore, transgenic sheep were intradermal injected of LPS in ear and observed pathological changes by HE strain. Overexpression of TLR4 gene was observed on transgenic cells and individuals. In vitro, TLR4 overexpression transgenic cells secreted Th1 and Th2 inducing cytokines with a strong LPS mediated inflammation response and promoting the secretion of nitric oxide, and then recovered to initial level. The phagocytosis index of monocyte/macrophage in transgenic sheep was higher than that of non-transgenic sheep (P<0.05). In vivo, tissue sections showed that transgenic individuals launched inflammation response more quickly.Conclusions/SignificanceOverexpression of TLR4 in transgenic sheep enhanced the clearance of invaded microbe through secretion of cytokines, activation of macrophage, oxidation damage and infiltration of neutrophil.

Highlights

About 90–95% of Gram-negative bacteria are considered to be harmful to their hosts
Real-time quantitative PCR was used. It showed that vectors could strongly drive TLR4 transcription, which peaked at 48 hours (Fig. 1C)
Expression of all three cytokines declined to initial levels within 24 hours of stimulation. All of these findings indicate that transgenic animals can respond rapidly to bacterial infection and that they do so by releasing more cytokines than non-transgenic animals

Summary

Introduction

About 90–95% of Gram-negative bacteria are considered to be harmful to their hosts Many intracellular bacteria, such as, Brucella, Tubercle bacilli, and Salmonella, are pathogenic to both animals and humans and they can be transmitted from animals to humans. These intracellular bacteria are difficult to clear by host immune system and they can cause several syndromes, even death. This is due to inhibit macrophage activity and prevent the formation of phagolysosomes. Interferon-gamma (IFN-c) and CD40L are expressed in Th1 activated macrophages These induce breath-bursts to kill the intracellular bacteria in the macrophages. Effects of TLR4 overexpression on inflammation response under LPS stimulated were addressed in vivo and in vitro

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Results

Conclusion