Susanne Deininger

Cellular trafficking of lipoteichoic acid and Toll-like receptor 2 in relation to signaling; role of CD14 and CD36

Lipoteichoic acid (LTA) is a central inducer of inflammatory responses caused by Gram‐positive bacteria, such as Staphylococcus aureus, via activation of TLR2. Localization of TLR2 in relation to its coreceptors may be important for function. This study explores the signaling, uptake, and trafficking pattern of LTA in relation to expression of TLR2 and its coreceptors CD36 and CD14 in human monocytes. We found TLR2 expressed in early endosomes, late endosomes/lysosomes, and in Rab‐11‐positive compartments but not in the Golgi apparatus or endoplasmic reticulum (ER). Rapid internalization of fluorescently labeled LTA was observed in human monocytes, colocalizing with markers for early and late endosomes, lysosomes, ER, and Golgi network. Blocking CD14 and CD36 with antibodies inhibited LTA binding and LTA‐induced TNF release from monocytes, emphasizing an important role for both molecules as coreceptors for TLR2. Importantly, blocking CD36 did not affect TNF release induced by N‐palmitoyl‐S‐[2,3‐bis(palmitoyloxy)‐(2R,S)‐propyl]‐(R)‐cysteinyl‐seryl‐(lysyl)3‐lysine or LPS. Expression of CD14 markedly enhanced LTA binding to the plasma membrane and also enhanced NF‐κB activation. LTA internalization, but not NF‐κB activation, was inhibited in Dynamin‐I K44A dominant‐negative transfectants, suggesting that LTA is internalized by receptor‐mediated endocytosis but that internalization is not required for signaling. In fact, immobilizing LTA and thereby inhibiting internalization resulted in enhanced TNF release from monocytes. Our results suggest that LTA signaling preferentially occurs at the plasma membrane, is independent of internalization, and is facilitated by CD36 and CD14 as coreceptors for TLR2.

Definition of Structural Prerequisites for Lipoteichoic Acid-Inducible Cytokine Induction by Synthetic Derivatives

The controversy about the immune stimulatory properties of lipoteichoic acid (LTA) from Staphylococcus aureus was solved recently by showing decomposition and inactivation of LTA obtained by conventional purification strategies, as well as pronounced LPS contamination of commercial preparations. By introducing a novel preparation method, the structure of bioactive LTA was elucidated. This structure was confirmed by chemical synthesis. In this work, synthetic LTA derivatives were employed to study the structure-function relationship of cytokine induction in human monocytes. Synthetic LTA induced the same cytokine pattern as highly purified natural LTA. The gentiobiose core could be omitted without affecting bioactivity. The polyglycerophosphate backbone amplified the response to the lipid anchor (∼100-fold) only when substituted with d-alanine, whereas α-d-N-acetylglucosamine substituents could be omitted. Replacing d-alanine substituents with l-alanine reduced the activity of the molecule at least 10-fold, indicating stereoselectivity. These results define for the first time the crucial patterns required for the immune recognition of LTA.

Inflammatory neurodegeneration induced by lipoteichoic acid from Staphylococcus aureus is mediated by glia activation, nitrosative and oxidative stress, and caspase activation.

In this study we investigated the mechanisms of neuronal cell death induced by lipoteichoic acid (LTA) and muramyl dipeptide (MDP) from Gram‐positive bacterial cell walls using primary cultures of rat cerebellum granule cells (CGCs) and rat cortical glial cells (astrocytes and microglia). LTA (± MDP) from Staphylococcus aureus induced a strong inflammatory response of both types of glial cells (release of interleukin‐1β, tumour necrosis factor‐α and nitric oxide). The death of CGCs was caused by activated glia because in the absence of glia (treatment with 7.5 µm cytosine‐d‐arabinoside to inhibit non‐neuronal cell proliferation) LTA + MDP did not cause significant cell death (less than 20%). In addition, staining with rhodamine‐labelled LTA confirmed that LTA was bound only to microglia and astrocytes (not neurones). Neuronal cell death induced by LTA (± MDP)‐activated glia was partially blocked by an inducible nitric oxide synthase inhibitor (1400 W; 100 µm), and completely blocked by a superoxide dismutase mimetic [manganese (III) tetrakis (4‐benzoic acid)porphyrin chloride; 50 µm] and a peroxynitrite scavenger [5,10,15,20‐tetrakis (4‐sulfonatophenyl) porphyrinato iron (III); 100 µm] suggesting that nitric oxide and peroxynitrite contributed to LTA‐induced cell death. Moreover, neuronal cell death was inhibited by selective inhibitors of caspase‐3 (z‐DEVD‐fmk; 50 µm) and caspase‐8 (z‐Ile‐Glu(O‐Me)‐Thr‐Asp(O‐Me) fluoromethyl ketone; 50 µm) indicating that they were involved in LTA‐induced neuronal cell death.

Cytokine induction by Gram-positive bacteria

Despite similar clinical relevance of Gram-positive and Gram-negative infections, immune activation by Gram-positive bacteria is by far less well understood than immune activation by Gram-negative bacteria. Our group has made available highly purified lipoteichoic acids (LTA) as a key Gram-positive immunostimulatory component. We have characterized the reasons for lower potency of LTA compared to Gram-negative lipopolysaccharide (LPS), identifying lack of IL-12/IFNgamma induction as a general characteristic of TLR2 agonists, and need for presentation of LTA on surfaces for enhanced immunostimulatory potency, as major aspects. Aspects of chemokine induction, where LTA is more potent than LPS, have been addressed. Furthermore, novel complement and plant defence activation, as well as CD36 as a new LTA receptor, were identified. The bacterial costimuli and modulators of LTA inducible responses are being investigated: LTA isolated from so far 16 bacterial species, although different in structure, behave remarkably similar while whole live and killed bacteria differ with regard to the pattern of induced responses. The purification and characterization of the respective components of the bacterial cell wall has begun.

Presentation of lipoteichoic acid potentiates its inflammatory activity

Lipoteichoic acid (LTA) is a major immunostimulatory molecule in the cell wall of Gram-positive bacteria. Adhesion of LTA to a polystyrene surface drastically increased its immunostimulatory potency in human whole blood in comparison to soluble LTA, although only 1% of the LTA had bound, as determined using rhodamine-labelled LTA. The release of the proinflammatory cytokines IL-1beta, TNF and IL-6 and the chemokines IL-8 and G-CSF was increased 2- to 10-fold, but IL-10 release was unaltered. This presentation effect was not shared by lipopolysaccharide (LPS) or other toll-like receptor 2 agonists and was less pronounced in polypropylene vessels. LTA did not induce cytokine release in silicone-coated borosilicate vessels, but covalent coupling of LTA to polystyrene beads restored cytokine induction in these vessels, indicating that presentation of LTA on a surface is in fact essential for its immunostimulatory potency. This novel aspect of presentation as a factor in the recognition of LTA may reflect the physiological situation in the bacterial cell wall, where LTA is anchored in the bacterial membrane and projects through the peptidoglycan. In practical terms, contamination of medical devices with components of Gram-positive bacteria may pose an underestimated inflammatory risk.

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 gram-negative 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 μM. 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.

Polypropylene glycol is a selective binding inhibitor for LTA and other structurally related TLR2 agonists

Polypropylene glycol (PPG) is commonly added to bacterial cultures to avoid foaming. However, lipoteichoic acid (LTA) from bacteria grown with PPG lacked cytokine‐inducing potency in human blood. We tested the blocking efficacy of several glycols on the cytokine response to staphylococcal LTA in human blood. PPG 1200 was the most potent inhibitor tested, shown for TNF, IL‐1β, IL‐6, IL‐8, IL‐10 and TGF‐β induction, and displayed no cytotoxic effects. TNF induction by Staphylococcus aureus or by Toll‐like receptor (TLR)2 agonists (di‐ and triacylated lipopeptides and LTA) was also inhibited by PPG 1200, but not that induced by Escherichia coli or TLR4 agonists. In flow cytometric studies, PPG‐carrying nanobeads bound more rhodamine‐labeled LTA than those with glycerol. Additionally, the methyl group peak in the 1H‐NMR of LTA shifted after incubation with increasing PPG 1200 concentrations. Sequential incubation of polystyrene plates with LTA, then PPG 1200 and then blood, with washing steps in between, showed that LTA‐induced TNF release was inhibited. But when PPG 1200 was pre‐incubated with blood that was washed before LTA was added, TNF induction was not repressed, demonstrating that PPG binds LTA and not cellular structures. In summary, PPG 1200 is a novel inhibitor of cytokine induction by TLR2 agonists, which interferes directly with the ligands.

Apolipoprotein B100 is a suppressor of Staphylococcus aureus-induced innate immune responses in humans and mice.

Plasma lipoproteins such as LDL (low‐density lipoprotein) are important therapeutic targets as they play a crucial role in macrophage biology and metabolic disorders. The impact of lipoprotein profiles on host defense pathways against Gram‐positive bacteria is poorly understood. In this report, we discovered that human serum lipoproteins bind to lipoteichoic acid (LTA) from Staphylococcus aureus and thereby alter the immune response to these bacteria. Size‐exclusion chromatography and solid‐phase‐binding analysis of serum revealed the direct interaction of LTA with apolipoproteins (Apo) B100, ApoA1, and ApoA2. Only ApoB100 and the corresponding LDL exerted biological effects as this binding significantly inhibited LTA‐induced cytokine releases from human and murine immune cells. Serum from hypercholesterolemic mice or humans significantly diminished cytokine induction in response to S. aureus or its LTA. Sera taken from the patients with familial hypercholesterolemia before and after ApoB100‐directed immuno‐apheresis confirmed that ApoB100 inhibited LTA‐induced inflammation in humans. In addition, mice in which LDL secretion was pharmacologically inhibited, displayed significantly increased serum cytokine levels upon infection with S. aureus in vivo. The present study identifies ApoB100 as an important suppressor of innate immune activation in response to S. aureus and its LTA.