Kukoamine B promotes TLR4-independent lipopolysaccharide uptake in murine hepatocytes.

Xinchuan Zheng,Dong Yang,Qian Chen,Yongling Lu,Ning Wang,Jiang Zheng,Shijun Fan,Yongjun Yang,Xin Liu

doi:10.18632/oncotarget.11292

Abstract

Free bacterial lipopolysaccharide (LPS) is generally removed from the bloodstream through hepatic uptake via TLR4, the LPS pattern recognition receptor, but mechanisms for internalization and clearance of conjugated LPS are less clear. Kukoamine B (KB) is a novel cationic alkaloid that interferes with LPS binding to TLR4. In this study, KB accelerated blood clearance of LPS. KB also enhanced LPS distribution in the hepatic tissues of C57 BL/6 mice, along with LPS uptake in primary hepatocytes and HepG2 cells. By contrast, KB inhibited LPS internalization in Kupffer and RAW 264.7 cells. Loss of TLR4 did not affect LPS uptake into KB-treated hepatocytes. We also detected selective upregulation of the asialoglycoprotein receptor (ASGPR) upon KB treatment, and ASGPR colocalized with KB in cultured hepatocytes. Molecular docking showed that KB bound to ASGPR in a manner similar to GalNAc, a known ASGPR agonist. GalNAc dose-dependently reduced KB internalization, suggesting it competes with KB for ASGPR binding, and ASGPR knockdown also impaired LPS uptake into hepatocytes. Finally, while KB enhanced LPS uptake, it was protective against LPS-induced inflammation and hepatocyte injury. Our study provides a new mechanism for conjugated LPS hepatic uptake induced by the LPS neutralizer KB and mediated by membrane ASGPR binding.

Highlights

Recognition of lipopolysaccharide (LPS), a glycolipid compound that constitutes much of the outer membrane in Gram-negative bacteria, is essential for triggering the host immune response [1,2,3]
In direct fluorescence imaging detection, we observed increased Fluorescein isothiocyanate (FITC)-LPS distribution in liver sections of mice co-injected with Kukoamine B (KB) (Figure 1C)
Free LPS is metabolized in the liver via TLR4-dependent mechanisms, the pathways that mediate hepatic uptake of conjugated LPS remain unclear

Summary

Introduction

Recognition of lipopolysaccharide (LPS), a glycolipid compound that constitutes much of the outer membrane in Gram-negative bacteria, is essential for triggering the host immune response [1,2,3]. LPS is generally recognized via its pattern recognition receptor, TLR4, and induces pro-inflammatory MyD88-dependent or -independent signaling pathways [4,5,6]. Whereas essential TLR4 activation is required to facilitate infection control, excessive TLR4 stimulation by LPS may result in serious consequences, such as sepsis, multiple organ dysfunction (MODS) and shock [7, 8]. Lipoprotein-bound LPS exhibits much weaker activity than free LPS in stimulating macrophages to release pro-inflammatory cytokines like TNF-α and IL-6. This is presumably due to blockage of the LPS lipid A moiety-TLR4 interaction by lipoproteins [13, 14]. There are few reports describing the possible roles of such drugs in accelerating LPS uptake and removal

Methods

Results

Conclusion