Toll-Like Receptor 4 Promotes NO Synthesis by Upregulating GCHI Expression under Oxidative Stress Conditions in Sheep Monocytes/Macrophages.

Shoulong Deng,Yuchang Yao,Baolu Zhang,Ning Li,Jinlong Zhang,Zhengxing Lian,Guoshi Liu,Yixun Liu,Kun Yu,Xiaosheng Zhang,Zhixian Wang

doi:10.1155/2015/359315

Abstract

Many groups of Gram-negative bacteria cause diseases that are harmful to sheep. Toll-like receptor 4 (TLR4), which is critical for detecting Gram-negative bacteria by the innate immune system, is activated by lipopolysaccharide (LPS) to initiate inflammatory responses and oxidative stress. Oxidation intermediates are essential activators of oxidative stress, as low levels of free radicals form a stressful oxidative environment that can clear invading pathogens. NO is an oxidation intermediate and its generation is regulated by nitric oxide synthase (iNOS). Guanosine triphosphate cyclohydrolase (GCHI) is the rate-limiting enzyme for tetrahydrobiopterin (BH4) synthesis, which is essential for the production of inducible iNOS. Previously, we made vectors to overexpress the sheep TLR4 gene. Herein, first generation (G1) of transgenic sheep was stimulated with LPS in vivo and in vitro, and oxidative stress and GCHI expression were investigated. Oxidative injury caused by TLR4 overexpression was tightly regulated in tissues. However, the transgenic (Tg) group still secreted nitric oxide (NO) when an iNOS inhibitor was added. Furthermore, GCHI expression remained upregulated in both serum and monocytes/macrophages. Thus, overexpression of TLR4 in transgenic sheep might accelerate the clearance of invading microbes through NO generation following LPS stimulation. Additionally, TLR4 overexpression also enhances GCHI activation.

Highlights

In both host defense and inflammation, monocytes/ macrophages are the most prominent immune cell type activated by lipopolysaccharide (LPS) to release various pro- and anti-inflammatory mediators
Significantly higher at 1 and 8 h (P < 0.05) and returned to a normal level at 48 h, similar to iNOS (Figure 5(c)). These findings indicated that Toll-like receptor 4 (TLR4) enhances the activity of iNOS by upregulating GCHI to promote synthesis and secretion
We have shown that TLR4 was overexpressed at the RNA level and protein levels

Summary

Introduction

In both host defense and inflammation, monocytes/ macrophages are the most prominent immune cell type activated by lipopolysaccharide (LPS) to release various pro- and anti-inflammatory mediators. TLR4 recruits downstream adaptors to activate both the myeloid differentiation primary response gene 88 (MyD88) and TIR-domain-containing adaptorinducing interferon-β- (TRIF-) dependent pathways, which function through nuclear factor-κB- (NF-κB-) associated signaling events [2]. The actions of these proteins trigger the release of oxidation intermediates, including reactive oxygen species (ROS), reactive nitrogen species (RNS), and cytokines [3]. Apart from the inflammatory reaction, oxidation intermediates are involved in many pathological processes, such as insulin resistance and type 2 diabetes [4].

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Results

Conclusion