Lipoteichoic Acid Induces Unique Inflammatory Responses when Compared to Other Toll-Like Receptor 2 Ligands

Paul Kubes,Stephen M. Robbins,Elizabeth M. Long,Dominik Hartl,Brandie Millen

doi:10.1371/journal.pone.0005601

Abstract

Toll-like receptors (TLRs) recognize evolutionarily-conserved molecular patterns originating from invading microbes. In this study, we were interested in determining if microbial ligands, which use distinct TLR2-containing receptor complexes, represent unique signals to the cell and can thereby stimulate unique cellular responses. Using the TLR2 ligands, R-FSL1, S-FSL1, Pam2CSK4, Pam3CSK4, and lipoteichoic acid (LTA), we demonstrate that these ligands activate NF-κB and MAP Kinase pathways with ligand-specific differential kinetics in murine macrophages. Most strikingly, LTA stimulation of these pathways was substantially delayed when compared with the other TLR2 ligands. These kinetics differences were associated with a delay in the LTA-induced expression of a subset of genes as compared with another TLR2 ligand, R-FSL1. However, this did not translate to overall differences in gene expression patterns four hours following stimulation with different TLR2 ligands. We extended this study to evaluate the in vivo responses to distinct TLR2 ligands using a murine model of acute inflammation, which employs intravital microscopy to monitor leukocyte recruitment into the cremaster muscle. We found that, although R-FSL1, S-FSL1, Pam2CSK4, and Pam3CSK4 were all able to stimulate robust leukocyte recruitment in vivo, LTA remained functionally inert in this in vivo model. Therefore distinct TLR2 ligands elicit unique cellular responses, as evidenced by differences in the kinetic profiles of signaling and gene expression responses in vitro, as well as the physiologically relevant differences in the in vivo responses to these ligands.

Highlights

Toll-like receptors (TLRs) are key components of the immune system’s capacity to recognize infectious non-self and to mount a rapid and effective immune response [1]
Our studies have revealed that the TLR2 ligands, RFSL1, S-FSL1, Pam3CSK4, Pam2CSK4, and lipoteichoic acid (LTA) each activate typical TLR-dependent pathways in vitro, the kinetics of this activation is substantially delayed in response to LTA as compared with the other TLR2 ligands
When we evaluated the kinetics of the transcriptional activation, we found that LTA and R-FSL1 activated the same transcriptional response, treatment with LTA did not result in the transcription of many inflammation-associated genes nearly as quickly as did R-FSL1

Summary

Introduction

Toll-like receptors (TLRs) are key components of the immune system’s capacity to recognize infectious non-self and to mount a rapid and effective immune response [1] They are type I transmembrane receptors, composed of an extracellular, leucine rich repeat (LRR), ligand-recognition motif, as well as a highly conserved, cytoplasmic, Toll/IL-1R (TIR), signaling-initiating domain [2,3]. The third acyl chain in triacylated lipoproteins is attached via an amide bond to the N-terminal cysteine This reaction depends on the presence of an N-acetyltransferase that is absent in mycoplasma and these organisms produce only diacylated lipopeptides, which are recognized by TLR2/6 heterodimer complexes [8,9]. We were interested in whether the use of these distinct heterodimer complexes would translate to distinct, receptor complex-specific, responses to TLR2 ligands

Methods

Results

Conclusion