LPS- or Pseudomonas aeruginosa-mediated activation of the macrophage TLR4 signaling cascade depends on membrane lipid composition.

Axel Schoeniger,Herbert Fuhrmann,Julia Schumann

doi:10.7717/peerj.1663

Abstract

It is well known that PUFA impede the LPS-mediated activation of the transcription factor NFkappaB. However, the underlying mode of action has not been clarified yet. To address this issue in a comprehensive approach, we used the monocyte/macrophage cell line RAW264.7 to investigate the consequences of a PUFA supplementation on the TLR4 pathway with a focus on (i) the gene expression of TLR4 itself as well as of its downstream mediators, (ii) the membrane microdomain localization of TLR4 and CD14, (iii) the stimulation-induced interaction of TLR4 and CD14. Our data indicate that the impairment of the TLR4-mediated cell activation by PUFA supplementation is not due to changes in gene expression of mediator proteins of the signaling cascade. Rather, our data provide evidence that the PUFA enrichment of macrophages affects the TLR4 pathway at the membrane level. PUFA incorporation into membrane lipids induces a reordering of membrane microdomains thereby affecting cellular signal transduction. It is important to note that this remodeling of macrophage rafts has no adverse effect on cell viability. Hence, microdomain disruption via macrophage PUFA supplementation has a potential as non-toxic strategy to attenuate inflammatory signaling.

Highlights

The binding of ligands, e.g., lipopolysaccharide (LPS) or the gram-negative bacterium Pseudomonas aeruginosa, to the macrophage Toll-like receptor 4 (TLR4) activates the TLR4 signaling cascade, which eventually results in the activation of the nuclear factor kappa B (NFκB) (McIsaac, Stadnyk & Lin, 2012)
The main steps of the TLR4 pathway include the interaction of TLR4 with its co-receptor CD14, the activation of the adaptor protein MyD88, the recruitment of the IL-1 receptor associated kinase 4 (IRAK-4) to the TLR receptor complex, the phosphorylation-dependent activation of IRAK1, the association of IRAK-1 with the TNF receptor-associated factor 6 (TRAF6) and
On the other hand polyunsaturated fatty acids (PUFA) are reported to disrupt membrane microdomain composition (Parker et al, 2008; Wong et al, 2009; Shaikh, Jolly & Chapkin, 2012), which might hamper the stimulation-induced initiation of the TLR4 signaling cascade via blocking the TLR4-CD14 interaction in lipid rafts. To address this issue in a comprehensive approach we investigated the consequences of a PUFA supplementation on the TLR4 pathway with a focus on (i) the gene expression of TLR4 itself as well as of its downstream mediators, (ii) the membrane microdomain localization of TLR4 and CD14, (iii) the stimulation-induced interaction of TLR4 and CD14 using the monocyte/macrophage cell line RAW264.7

Summary

Introduction

The binding of ligands, e.g., lipopolysaccharide (LPS) or the gram-negative bacterium Pseudomonas aeruginosa, to the macrophage Toll-like receptor 4 (TLR4) activates the TLR4 signaling cascade, which eventually results in the activation of the nuclear factor kappa B (NFκB) (McIsaac, Stadnyk & Lin, 2012). M1 macrophages are characterized by the synthesis and the release of pro-inflammatory cytokines, such as IL-1β, IL-6 or TNF-α, and are able to develop a severe respiratory burst contributing to the defense against microbial pathogens (Sica & Mantovani, 2012). An excessive inflammatory response by M1 macrophages, which is a hallmark of chronic infections with persistent pathogens, such as P. aeruginosa, is reported to cause tissue damage, to lead to functional restrictions and to favor secondary infections (Park et al, 2007; Ambrozova, Pekarova & Lojek, 2010)

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