Dataset Information

Rhodobacter azotoformans LPS (RAP99-LPS) Is a TLR4 Agonist That Inhibits Lung Metastasis and Enhances TLR3-Mediated Chemokine Expression.

ABSTRACT: The lipopolysaccharides (LPSs) of Rhodobacter are reported to be TLR4 antagonists. Accordingly, the extract of Rhodobacter azotoformans (RAP99) is used as a health supplement for humans and animals in Japan to regulate immune responses in vivo. We previously analyzed the LPS structure of RAP99 (RAP99-LPS) and found it is different from that of E. coli-LPS but similar to lipid A from Rhodobacter sphaeroides (RSLA), a known antagonist of TLR4, with both having three C14 fatty acyl groups, two C10 fatty acyl groups, and two phosphates. Here we show that RAP99-LPS has an immune stimulatory activity and acts as a TLR4 agonist. Pretreatment of RAP99-LPS suppressed E. coli-LPS-mediated weight loss, suggesting it is an antagonist against E. coli-LPS like other LPS isolated from Rhodobacter. However, injections of RAP99-LPS caused splenomegaly and increased immune cell numbers in C57BL/6 mice but not in C3H/HeJ mice, suggesting that RAP99-LPS stimulates immune cells via TLR4. Consistently, RAP99-LPS suppressed the lung metastasis of B16F1 tumor cells and enhanced the expression of TLR3-mediated chemokines. These results suggest that RAP99-LPS is a TLR4 agonist that enhances the activation status of the immune system to promote anti-viral and anti-tumor activity in vivo.

SUBMITTER: Murakami K

PROVIDER: S-EPMC8185171 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:The molecular determinants underpinning how hexaacylated lipid A and tetraacylated precursor lipid IVa activate Toll-like receptor 4 (TLR4) are well understood, but how activation is induced by other lipid A species is less clear. Species specificity studies have clarified how TLR4/MD-2 recognises different lipid A structures, for example tetraacylated lipid IVa requires direct electrostatic interactions for agonism. In this study, we examine how pentaacylated lipopolysaccharide from Rhodobacter sphaeroides (RSLPS) antagonises human TLR4/MD-2 and activates the horse receptor complex using a computational approach and cross-species mutagenesis. At a functional level, we show that RSLPS is a partial agonist at horse TLR4/MD-2 with greater efficacy than lipid IVa. These data suggest the importance of the additional acyl chain in RSLPS signalling. Based on docking analysis, we propose a model for positioning of the RSLPS lipid A moiety (RSLA) within the MD-2 cavity at the TLR4 dimer interface, which allows activity at the horse receptor complex. As for lipid IVa, RSLPS agonism requires species-specific contacts with MD-2 and TLR4, but the R2 chain of RSLA protrudes from the MD-2 pocket to contact the TLR4 dimer in the vicinity of proline 442. Our model explains why RSLPS is only partially dependent on horse TLR4 residue R385, unlike lipid IVa. Mutagenesis of proline 442 into a serine residue, as found in human TLR4, uncovers the importance of this site in RSLPS signalling; horse TLR4 R385G/P442S double mutation completely abolishes RSLPS activity without its counterpart, human TLR4 G384R/S441P, being able to restore it. Our data highlight the importance of subtle changes in ligand positioning, and suggest that TLR4 and MD-2 residues that may not participate directly in ligand binding can determine the signalling outcome of a given ligand. This indicates a cooperative binding mechanism within the receptor complex, which is becoming increasingly important in TLR signalling.

Project description:Binge/moderate alcohol suppresses TLR4-MyD88 proinflammatory cytokines; however, alcohol's effects on TLR-TRIF signaling, especially after in vivo exposure in humans, are unclear. We performed a comparative analysis of the TLR4-MyD88, TLR4-TRIF, and TLR3-TRIF pathways in human monocytes following binge alcohol exposure. Mechanistic regulation of TLR-TRIF signaling by binge alcohol was evaluated by analyzing IRF3 and TBK1, upstream regulator protein phosphatase 1 (PP1), and immunoregulatory stress proteins HspA1A and XBP-1 in alcohol-treated human and mouse monocytes/macrophages. Two approaches for alcohol exposure were used: in vivo exposure of primary monocytes in binge alcohol-consuming human volunteers or in vitro exposure of human monocytes/murine macrophages to physiological alcohol concentrations (25-50 mM ethanol), followed by LPS (TLR4) or polyinosinic-polycytidylic acid (TLR3) stimulation ex vivo. In vivo and in vitro binge alcohol exposure significantly inhibited the TLR4-MyD88 cytokines TNF-? and IL-6, as well as the TLR4-TRIF cytokines/chemokines IFN-?, IP-10, and RANTES, in human monocytes, but not TLR3-TRIF-induced cytokines/chemokines, as detected by quantitative PCR and ELISA. Mechanistic analyses revealed TBK-1-independent inhibition of the TLR4-TRIF effector IRF3 in alcohol-treated macrophages. Although stress protein XBP-1, which is known to regulate IRF3-mediated IFN-? induction, was not affected by alcohol, HspA1A was induced by in vivo alcohol in human monocytes. Alcohol-induced HspA1A was required for inhibition of TLR4-MyD88 signaling but not TLR4-TRIF cytokines in macrophages. In contrast, inhibition of PP1 prevented alcohol-mediated TLR4-TRIF tolerance in macrophages. Collectively, our results demonstrate that in vivo and in vitro binge alcohol exposure in humans suppresses TLR4-MyD88 and TLR4-TRIF, but not TLR3-TRIF, responses. Whereas alcohol-mediated effects on the PP1-IRF3 axis inhibit the TLR4-TRIF pathway, HspA1A selectively suppresses the TLR4-MyD88 pathway in monocytes/macrophages.

Dataset Information

Rhodobacter azotoformans LPS (RAP99-LPS) Is a TLR4 Agonist That Inhibits Lung Metastasis and Enhances TLR3-Mediated Chemokine Expression.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets