TLR are primary triggers of the innate immune system by recognizing various microorganisms through conserved pathogen-associated molecular patterns. TLR2 is the receptor for a functional recognition of bacterial lipopeptides (LP) and is up-regulated during various disorders such as chronic obstructive pulmonary disease and sepsis. This receptor is unique in its ability to form heteromers with TLR1 or TLR6 to mediate intracellular signaling. According to the fatty acid pattern as well as the assembling of the polypeptide tail, LP can signal through TLR2 in a TLR1- or TLR6-dependent manner. There are also di- and triacylated LP, which stimulate TLR1-deficient cells and TLR6-deficient cells. In this study, we investigated whether heterodimerization evolutionarily developed to broaden the ligand spectrum or to induce different immune responses. We analyzed the signal transduction pathways activated through the different TLR2 dimers using the three LP, palmitic acid (Pam)octanoic acid (Oct)(2)C-(VPGVG)(4)VPGKG, fibroblast-stimulating LP-1, and Pam(2)C-SK(4). Dominant-negative forms of signaling molecules, immunoblotting of MAPK, as well as microarray analysis indicate that all dimers use the same signaling cascade, leading to an identical pattern of gene activation. We conclude that heterodimerization of TLR2 with TLR1 or TLR6 evolutionarily developed to expand the ligand spectrum to enable the innate immune system to recognize the numerous, different structures of LP present in various pathogens. Thus, although mycoplasma and Gram-positive and Gram-negative bacteria may activate different TLR2 dimers, the development of different signal pathways in response to different LP does not seem to be of vital significance for the innate defense system.

No abstract

Bacterial lipopeptides are strong immune modulators that activate early host responses after infection as well as initiating adjuvant effects on the adaptive immune system. These lipopeptides induce signaling in cells of the immune system through Toll-like receptor 2 (TLR2)-TLR1 or TLR2-TLR6 heteromers. So far it has been thought that triacylated lipopeptides, such as the synthetic N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteine (Pam3)-CSK4, signal through TLR2-TLR1 heteromers, whereas diacylated lipopeptides, like the macrophage-activating lipopeptide from Mycoplasma fermentans (MALP2) or S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteine (Pam2)-CGNNDESNISFKEK, induce signaling through TLR2-TLR6 heteromers. Using new synthetic lipopeptide derivatives we addressed the contribution of the lipid and, in particular, the peptide moieties with respect to TLR2 heteromer usage. In contrast to the current model of receptor usage, not only triacylated lipopeptides, but also diacylated lipopeptides like Pam2CSK4 and the elongated MALP2 analog Pam2CGNNDESNISFKEK-SK4 (MALP2-SK4) induced B lymphocyte proliferation and TNF-a secretion in macrophages in a TLR6-independent manner as determined with cells from TLR6-deficient mice. Our results indicate that both the lipid and the Nterminal peptides of lipoproteins contribute to the specificity of recognition by TLR2 heteromers and are responsible for the ligand-receptor interaction on host cells. IntroductionToll-like receptors (TLR) are prominent pattern-recognition receptors (PRR) of the innate immune system, recognizing various invading microorganisms through conserved molecular structures, so-called pathogenassociated molecular patterns (PAMP) [1]. The receptors differ from each other not only in ligand specificity but also in their expression pattern on different cells of the innate immune system. In addition, distinct TLR are able to address different sets of inflammatory target genes [2]. [3] to attain specificity for a given stimulus [4][5][6][7].LP have been described as strong immunostimulatory compounds initiating early innate host defense reactions in cells such as macrophages and epithelial cells [8,9]. In addition, they are potent polyclonal activators of murine B lymphocytes [10]. Signaling through TLR2-TLRx heteromers by LP leads to the formation of an active signaling complex that includes MyD88 and the IL-1R-associated kinase (IRAK) proteins, finally resulting in the nuclear translocation of NF-jB and subsequent cytokine production [11]. As a consequence TLR2-dependent signaling initiates a variety of host defense responses including the activation of the adaptive immune system [12][13][14].Among the TLR2-dependent PAMP, LP are primary candidates for analyzing the structural requirements for heteromer usage, for several reasons. Firstly, these molecules are available from various different bacterial species expressing different molecular structures. Secondly, the chemical synthesis of LP analogues provides a vast variety of chemically defined m...

No abstract

Bacterial lipoproteins/peptides are composed of di‐O‐acylated‐S‐(2,3‐dihydroxypropyl)‐cysteinyl residues N‐terminally coupled to distinct polypeptides, which can be N‐acylated with a third fatty acid. Using a synthetic lipopeptide library we characterized the contribution of the lipid portion to the TLR2 dependent pattern recognition. We found that the two ester bound fatty acid length threshold is beyond eight C atoms because almost no response was elicited by cellular challenge with analogues carrying shorter acyl chains in HEK293 cells expressing recombinant human TLR2. In contrast, the amide bound fatty acid is of lesser importance. While two ester‐bound palmitic acids mediate a high stimulatory activity of the respective analogue, a lipopeptide carrying one amide‐bound and another ester‐bound palmitic acid molecule was inactive. In addition, species specific LP recognition through murine and human TLR2 depended on the length of the two ester bound fatty acid chains. In conclusion, our results indicate the responsibility of both ester bound acyl chains but not of the amide bound fatty acid molecule for the TLR dependent cellular recognition of canonical triacylated LP, as well as a requirement for a minimal acyl chain length. Thus they might support the explanation of specific immuno‐stimulatory potentials of different microorganisms and provide a basis for rational design of TLR2 specific adjuvants mediating immune activation to distinct levels.