Use of Synthetic Derivatives To Determine the Minimal Active Structure of Cytokine-Inducing Lipoteichoic Acid

Abstract

Lipoteichoic acid (LTA) from gram-positive bacteria is the counterpart to lipopolysaccharide from gramnegative bacteria. LTA, which activates Toll-like receptor 2 (TLR2), induces a unique cytokine and chemokine pattern. The chemical synthesis of LTA proved its immunostimulatory properties. To determine the minimal active structure of LTA, we reduced synthetic LTA in a number of steps down to the synthetic anchor and employed these molecules to stimulate interleukin-8 (IL-8) release in human whole blood. Ten times more of the synthetic structures with four to six D-alanine-substituted polyglycerophosphate units (50 nM) than of the native LTA preparation was required to induce IL-8 release. A further reduction to three backbone units with two or no D-alanine residues resulted in cytokine induction only from 500 nM. The synthetic anchor was not able to induce IL-8 release even at 5 M. When the LTA derivatives were used at 500 nM, they induced increasing levels of IL-8 and tumor necrosis factor alpha with increasing elongation of the backbone. Peritoneal macrophages were less responsive than human blood to the synthetic structures. Therefore, TLR2 dependency could be shown only with cells from TLR2-deficient mice for the two largest synthetic structures. This was confirmed by using TLR2-transfected HEK 293 cells. Taken together, these data indicate that although the synthetic anchor (which, unlike the native anchor, contains only myristic acid) cannot induce cytokine release, the addition of three backbone units, even without D-alanine substituents, confers this ability. Lengthening of the chain with D-alanine-substituted backbone units results in increased cytokine-inducing potency and a more sensitive response.Recognition of conserved bacterial structures called pathogen-associated molecular patterns occurs via pattern recognition receptors on immune cells and leads to activation of the innate immune system and the induction of a variety of cytokines. Lipopolysaccharide (LPS) has been known as the most important pathogen-associated molecular pattern of gramnegative bacteria for more than 50 years (16) and has been well examined in detail over the decades. Immune recognition takes place by the binding of LPS to Toll-like receptor 4 (TLR4) and also involves the cofactors CD14 (17) and 14).The immunostimulatory component of gram-positive bacteria was not clear for a long time, although a structural counterpart to LPS, called lipoteichoic acid (LTA), was found in the bacterial membrane. Like LPS, LTA is an amphiphilic molecule with a lipid anchor and a negatively charged backbone. Inefficient preparation methods on the basis of hot phenol, which resulted in the decomposition and the subsequent loss of activity, or LPS contamination during preparation led to inconsistent findings (9). Meanwhile, an improved preparation method based on n-butanol extraction at an ambient temperature was developed to purify the biologically active LTA of Staphylococcus aureus (8) and other organisms (3, 4). Structural analysis by nuclear ...

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