Changes in the relative inflammatory responses in sheep cells overexpressing of toll-like receptor 4 when stimulated with LPS.
BACKGROUND
Many 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.


RESULTS
Sheep 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
Overexpression of TLR4 in transgenic sheep enhanced the clearance of invaded microbe through secretion of cytokines, activation of macrophage, oxidation damage and infiltration of neutrophil.
Laboratory of Animal Genetics and Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, PR China.
Shoulong
S
Deng
Qian
Q
Wu
Kun
K
Yu
Yunhai
Y
Zhang
Yuchang
Y
Yao
Wenting
W
Li
Zhuo
Z
Deng
Guoshi
G
Liu
Wu
W
Li
Zhengxing
Z
Lian
D000818:Animals; D002478:Cells, Cultured; D007371:Interferon-gamma / Q000378:metabolism; D016753:Interleukin-10 / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects; D020556:Neutrophil Infiltration / Q000187:drug effects; D009569:Nitric Oxide / Q000378:metabolism; D012756:Sheep; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D015850:Interleukin-6; D016209:Interleukin-8; D008070:Lipopolysaccharides; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D009569:Nitric Oxide; D007371:Interferon-gamma