Induction of IL-10-balanced immune profiles following exposure to LTA from Staphylococcus epidermidis.

Staphylococcus epidermidis colonises human skin without apparent inflammation, but a dominance of S. epidermidis and S. aureus is characteristic of cutaneous microbial dysbiosis in atopic dermatitis (AD). While S. aureus can trigger AD, the role of S. epidermidis is less understood. We characterised consequences of innate immune sensing of lipoteichoic acid (LTA) preparations derived from S. epidermidis (epi-LTA) or S. aureus (aureus-LTA). Therefore, dendritic cell (DC) activation and consecutive priming of antigen-specific T cells following exposure of DC to epi-LTA or aureus-LTA were investigated. Mimicking acute AD, exposure of DC to IL-4 and LTAs was analysed. Exposure to epi-LTA or aureus-LTA activated human immune cells and murine dendritic cells (DCs) via TLR2/MyD88, however, resulting in divergent immune profiles. Differences between LTAs were significant for IL-6, IL-12p40 and IL-12p70 but not for IL-10, which was best reflected by the IL-12p70-to-IL-10 ratio being IL-10-balanced for epi-LTA but pro-inflammatory for aureus-LTA. LTA-exposed DCs activated CD4+ T cells; however, while T-cell-derived IL-10 was equivalent between LTAs, IFN-γ and IL-17 were significantly higher for aureus-LTA. Mimicking acute AD by exposing DCs to IL-4 and LTAs revealed that IL-4 significantly and uniformly suppressed epi-LTA-induced cytokine production, keeping the IL-12p70-to-IL-10 ratio balanced. In contrast, exposure of DCs to aureus-LTA and IL-4 enhanced IL-12p70 but suppressed IL-10 levels, further unbalancing the IL-12p70-to-IL-10 ratio. These data demonstrate opposing immune consequences following exposure to staphylococcal LTAs. Epi-LTA induced IL-10-balanced, aureus-LTA pro-inflammatory immune profiles.

Priming of alveolar macrophages upon instillation of lipopolysaccharide in the human lung.

The airways are continuously exposed to respiratory pathogens, which may result in bacterial pneumonia, one of the most common infectious diseases and the leading cause of sepsis. Considering that recurrent exposure to microbial products can lead to tolerance of immune cells, and that this might contribute to the susceptibility to nosocomial infection, we investigated the effect of in vivo lipopolysaccharide (LPS) instillation on the responsiveness of alveolar macrophages. In eight healthy humans, sterile saline was instilled into a lung segment by bronchoscope, followed by instillation of LPS into the contralateral lung; 6 hours later, a bilateral bronchoalveolar lavage was performed, and purified alveolar macrophages were ex vivo stimulated with LPS or lipoteichoic acid (LTA), triggering Toll-like receptor (TLR)-4 and -2, respectively. In vivo LPS-exposed alveolar macrophages were primed, as reflected by increased ex vivo LPS- and LTA-induced IL-1 beta and IL-6 gene expression and production compared with in vivo saline-exposed alveolar macrophages. LPS instillation did not influence the surface expression of TLR4 or TLR2. Furthermore, LPS instillation did not impact on the expression of a number of extracellular and intracellular regulators of TLR signaling. However, p38 mitogen-activated protein kinase remained phosphorylated in alveolar macrophages upon LPS instillation. The current data demonstrate that LPS instillation in the human lung primes alveolar macrophages for further stimulation with either LPS or LTA, possibly by sustained p38 mitogen-activated protein kinase activation.

Activation of coagulation and inhibition of fibrinolysis in the human lung on bronchial instillation of lipoteichoic acid and lipopolysaccharide.

OBJECTIVE: Pneumonia is characterized by an acute inflammatory response in the lung, which is frequently associated with changes in coagulation and fibrinolysis in the bronchoalveolar space. Here, we compared the effects of lipoteichoic acid (LTA), a major cell wall component of Gram-positive bacteria, and lipopolysaccharide (LPS), in the human bronchoalveolar space. DESIGN: Controlled in vivo volunteer study. SETTING: Clinical research unit. SUBJECTS: Twenty-three healthy nonsmoking male volunteers. INTERVENTIONS: Sterile saline was instilled into a lung subsegment followed by bronchoscopic instillation of either LTA (Staphylococcus aureus, at a dose of 4, 20, or 100 ng/kg body weight) or LPS (Escherichia coli, 4 ng/kg body weight) into the contralateral lung. Bronchoalveolar lavage fluid was obtained 6 hours thereafter. MEASUREMENTS AND MAIN RESULTS: Bronchial instillation of LTA- or LPS-activated bronchoalveolar coagulation, as reflected by increases in the levels of thrombin-antithrombin complexes, d-dimer, and soluble tissue factor. Concurrently, LTA and LPS inhibited anticoagulant mechanisms, as indicated by reductions in antithrombin, Protein C, and Activated Protein C concentrations together with elevated levels of soluble thrombomodulin. Both LTA and LPS administration was associated with an inhibition of pulmonary fibrinolysis, as measured by a reduction in plasminogen activator activity and elevated levels of plasminogen activator inhibitor type I. CONCLUSIONS: This study is the first to describe the effects of LTA on hemostasis in humans, demonstrating that LTA induces similar changes in the human bronchoalveolar space as LPS, characterized by activation of coagulation with concurrent inhibition of anticoagulant and fibrinolytic pathways.

Lung inflammation induced by lipoteichoic acid or lipopolysaccharide in humans.

RATIONALE: Recognition of pathogen-associated molecular patterns by Toll-like receptors (TLRs) is considered to be important for an appropriate immune response against pathogens that enter the lower airways. OBJECTIVES: We studied the effects of two different TLR agonists relevant for respiratory infections in the human lung: lipoteichoic acid (LTA; TLR2 agonist, component of gram-positive bacteria) and lipopolysaccharide (LPS; TLR4-agonist, component of gram-negative bacteria). METHODS: Fifteen healthy subjects were given LPS or LTA: by bronchoscope, sterile saline was instilled into a lung segment followed by instillation of LTA or LPS into the contralateral lung. After 6 hours, a bronchoalveolar lavage was performed and inflammatory parameters were determined. Isolated RNA from purified alveolar macrophages was analyzed by multiplex ligation-dependent probe amplification. In addition, spontaneous cytokine release by alveolar macrophages was measured. MEASUREMENTS AND MAIN RESULTS: Marked differences were detected between LTA- and LPS-induced lung inflammation. Whereas both elicited neutrophil recruitment, only LPS instillation was associated with activation of neutrophils (CD11b surface expression, degranulation product levels) and consistent rises of chemo-/cytokine levels. Moreover, LPS but not LTA activated alveolar macrophages, as reflected by enhanced expression of 10 different mRNAs encoding proinflammatory mediators and increased spontaneous cytokine release upon incubation ex vivo. Remarkably, only LTA induced C5a release. CONCLUSIONS: This is the first study to report the in vivo effects of LTA in men and to compare inflammation induced by LTA and LPS in the human lung. Our data suggest that stimulation of TLR2 or TLR4 results in differential pulmonary inflammation, which may be of relevance for understanding pathogenic mechanisms at play during gram-positive and gram-negative respiratory tract infection.

The Toll-like receptor 2 R753Q mutation modifies cytokine production and Toll-like receptor expression in atopic dermatitis.

BACKGROUND: Impaired host defense mechanisms may crucially modulate the pathogenesis of atopic dermatitis (AD). More than 10% of patients with AD are heterozygous for the Toll-like receptor 2 (TLR-2) R753Q single nucleotide polymorphism (SNP) and exhibit severe eczema. OBJECTIVE: To elucidate the functional effect of the TLR-2 mutation and its putative relevance for AD. METHODS: Using the human embryonic kidney 293 transfection system, we characterized the properties of the TLR-2 R753Q SNP. Moreover, TLR-2 expression, IL-8 production, and cytokine secretion were analyzed in monocytes and CD4+ T cells of patients with AD with and without the mutant TLR-2 gene. RESULTS: Human embryonic kidney 293 transfectants mimicking this heterozygous mutation produced less IL-8 when stimulated with lipoteichoic acid (LTA), heat-inactivated Staphylococcus aureus or triacylated lipopeptides requiring the TLR-2/1 heterodimer. Suppressed production of IL-8 was confirmed in monocytes from patients with mutant AD after stimulation with peptidoglycan. Cell surface TLR-2 expression was severely impaired in CD3/CD28 activated CD4+ T cells of patients with AD bearing the mutant receptor, which could be restored on LTA stimulation. In contrast, LTA decreased TLR-2 expression among nonatopic individuals and patients with AD with the TLR-2 wild-type gene. T cells from patients with AD exhibited markedly suppressed IL-2 production after macrophage-activating lipopeptide-2 activation. However, no difference was found between mutant and wild-type patients with AD for IL-5, TNF-alpha, IFN-gamma, and IL-2 production. CONCLUSION: Collectively, the outcome of innate and adaptive immune responses in AD is modulated by the TLR-2 R753Q SNP.

Bovine TLR2 and TLR4 properly transduce signals from Staphylococcus aureus and E. coli, but S. aureus fails to both activate NF-kappaB in mammary epithelial cells and to quickly induce TNFalpha and interleukin-8 (CXCL8) expression in the udder.

Staphylococcus aureus, but not E. coli pathogens frequently cause subclinical, chronic infections of the mammary gland. We examined here, if inadequate activation of the bovine TLR2 and TLR4 pathogen receptors by ligands derived from S. aureus pathogens might contribute to molecular mechanisms underpinning the escape strategies from mammary immune defence of this pathogen. We show that infections with live E. coli, but not S. aureus pathogens induce strongly IL-8 and TNFalpha gene expression in the udders. Yet, preparations of heat-killed bacteria from both pathogens activate equally well bovine TLR2 and TLR4 receptors to induce NF-kappaB activation, as shown in the HEK293 reconstitution system of TLR-signal transduction. LTA prepared from the S. aureus strain used to infect the cows activates the bovine TLR2 as strongly as the entire, heat-killed pathogen. Both pathogens induce in primary bovine mammary epithelial cells (pbMEC) IL-8 and TNFalpha gene expression, but S. aureus to less than 5% of the degree caused by E. coli. This impaired proinflammatory activation is paralleled by a complete lack of NF-kappaB activation in pbMEC by S. aureus or LTA. In contrast, E. coli and LPS activate strongly NF-kappaB in these cells. A large proportion of this activation is attributable to TLR-mediated signalling, since a dual transdominant negative DN-MyD88-DN-TRIF factor blocks >80% of the pathogen-related NF-kappaB activation in pbMEC. Our results prove that impaired binding of TLR-ligands from the pathogenic S. aureus strain are not the cause for the inadequate mammary immune response elicited by this pathogen. Rather, the pathogen causing subclinical mastitis impairs NF-kappaB activation in MEC thereby severely weakening the immune response in the udder.

Endogenous beta-adrenergic receptors inhibit lipopolysaccharide-induced pulmonary cytokine release and coagulation.

Beta2-adrenergic receptors are expressed on different cell types in the lung, including respiratory epithelial cells, smooth muscle cells, and macrophages. The aim of the current study was to determine the role of beta-adrenergic receptors in the regulation of lung inflammation induced by instillation via the airways of lipopolysaccharide (LPS) (a constituent of the gram-negative bacterial cell wall) or lipoteichoic acid (LTA) (a component of the gram-positive bacterial cell wall). Mice inhaled the beta-adrenergic antagonist propranolol or saline 30 minutes before and 3 hours after intranasal LPS or LTA administration. LPS and LTA induced a profound inflammatory response in the lungs as reflected by an influx of neutrophils and the release of proinflammatory cytokines and chemokines into bronchoalveolar lavage fluid (BALF). Propranolol inhalation resulted in enhanced LPS-induced lung inflammation, which was reflected by a stronger secretion of TNF-alpha, IL-6, and monocyte chemoattractant protein-1 into BALF and by enhanced coagulation activation (thrombin-antithrombin complexes). In LTA-induced lung inflammation, propranolol did not influence cytokine release but potentiated activation of coagulation. Propranolol did not alter neutrophil recruitment in either model. This study suggests that beta-adrenergic receptors, which are widely expressed in the lungs, serve as negative regulators of pulmonary cytokine release and coagulation induced by LPS and less so during LTA-induced pulmonary inflammation.

D-alanylation of lipoteichoic acid contributes to the virulence of Streptococcus suis.

We generated by allelic replacement a DeltadltA mutant of a virulent Streptococcus suis serotype 2 field strain and evaluated the contribution of lipoteichoic acid (LTA) d-alanylation to the virulence traits of this swine pathogen and zoonotic agent. The absence of LTA D-alanylation resulted in increased susceptibility to the action of cationic antimicrobial peptides. In addition, and in contrast to the wild-type strain, the DeltadltA mutant was efficiently killed by porcine neutrophils and showed diminished adherence to and invasion of porcine brain microvascular endothelial cells. Finally, the DeltadltA mutant was attenuated in both the CD1 mouse and porcine models of infection, probably reflecting a decreased ability to escape immune clearance mechanisms and an impaired capacity to move across host barriers. The results of this study suggest that LTA D-alanylation is an important factor in S. suis virulence.

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.

Intrapulmonary delivery of ethyl pyruvate attenuates lipopolysaccharide- and lipoteichoic acid-induced lung inflammation in vivo.

Ethyl pyruvate (EP) is a stable pyruvate derivative that has been shown to exert anti-inflammatory effects in various models of systemic inflammation including endotoxemia. We here sought to determine the local effects of EP, after intrapulmonary delivery, in models of lung inflammation induced by instillation via the airways of either lipopolysaccharide (LPS, a constituent of the gram-negative bacterial cell wall) or lipoteichoic acid (LTA, a component of the gram-positive bacterial cell wall). For this, we first established that EP dose dependently reduced the responsiveness of mouse MH-S alveolar macrophages and mouse MLE-15 and MLE-12 respiratory epithelial cells to stimulation with LPS or LTA in vitro. We then showed that intranasal administration of EP dose dependently inhibited tumor necrosis factor alpha release in bronchoalveolar lavage fluid of mice challenged with either LPS or LTA via the airways. Moreover, EP reduced the recruitment of neutrophils into the bronchoalveolar space after either LPS or LTA administration. These data suggest that intrapulmonary delivery of EP diminishes lung inflammation induced by LPS or LTA, at least in part by targeting alveolar macrophages and respiratory epithelial cells.

Gender difference in cytokine secretion on immune stimulation with LPS and LTA.

Immune defense capacity differs between men and women. Whereas men are more prone to infection and sepsis, women more commonly develop autoimmune diseases. We investigated the difference in cytokine secretion between males and females in response to different immune stimuli. Whole blood from 154 healthy volunteers (age 24 +/- 5.2; 82 females, 72 males) was collected within 2 h on 2 consecutive days. Blood from males produced significantly more tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, and IL-8 than blood from females in response to a high concentration of either lipopolysaccharide (LPS) or lipoteichoic acid (LTA), whereas IL-10 and interferon-gamma (IFN-gamma) secretion did not differ. Normalization of cytokine measurement to individual monocyte counts cancelled these differences for all parameters except TNF-alpha. Stimulation with a lower concentration of LPS (100 pg/mL) produced even stronger differences in cytokine release, which were not cancelled by normalization to the producing cells. The coefficients of variation (CV) of the LPS-induced and LTA-induced cytokine responses were higher in blood from women than men for all parameters and stimuli measured. Thus, the stronger innate immune response of males in comparison to females appears to stem not only from a difference in monocyte counts but also from the steepness of the response curve.

Endogenous MCP-1 promotes lung inflammation induced by LPS and LTA.

Monocyte chemoattractant protein 1 (MCP-1) plays an important role in leukocyte recruitment to sites of infection and inflammation. In addition, MCP-1 may attenuate inflammation by virtue of its capacity to inhibit the production of proinflammatory cytokines. We here investigated the role of MCP-1 in lung inflammation induced by lipopolysaccharide (LPS) or lipoteichoic acid (LTA), constituents of the gram-negative and gram-positive bacterial cell wall, respectively. Healthy humans demonstrated elevated MCP-1 concentrations in their bronchoalveolar lavage fluid (BALF) 6h after inhalation of LPS. Similarly, intranasal administration of LPS or LTA to mice resulted in a rise in BALF MCP-1 levels. Murine alveolar macrophage-like cells released significant amounts of MCP-1 upon stimulation with LPS or LTA in vitro. Compared to Wt mice, MCP-1(-/-) mice demonstrated lower TNF-α levels and a diminished neutrophil influx into their bronchoalveolar space after either LPS or LTA instillation. After intrapulmonary delivery of LPS MCP-1(-/-) mice had decreased interleukin-6 and KC concentrations and less severe lung inflammation upon histopathological examination. Remarkably, MCP-1 deficiency was associated with an early enhancement of interleukin-10 release in BALF after both LPS and LTA instillation. These data suggest that MCP-1 is a proinflammatory mediator during pulmonary inflammation induced by either LPS or LTA.

Role played by Toll-like receptors 2 and 4 in lipoteichoic acid-induced lung inflammation and coagulation.

BACKGROUND: The cell wall of Streptococcus pneumoniae consists of lipoteichoic acid (LTA), which is released when pneumococci are killed by either the host immune system or antibiotic treatment. Release of excessive amounts of LTA has been implicated in the toxic sequelae of severe gram-positive infection by virtue of its proinflammatory properties. Several in vitro studies have shown that LTA is recognized by Toll-like receptor (TLR) 2 and CD14. Our objective here was to investigate the inflammatory properties of S. pneumoniae LTA in vivo and the role played by TLR2, TLR4, and CD14 therein. METHODS: Wild-type (WT), TLR2 knockout (KO), TLR4 KO, TLR2x4 double-KO, and CD14 KO mice were intranasally inoculated with highly purified pneumococcal LTA. RESULTS: LTA induced a dose-dependent inflammatory response and activation of the coagulation and fibrinolytic pathways in a TLR2-dependent fashion. Surprisingly, TLR4 KO mice also displayed a somewhat diminished pulmonary inflammatory and coagulant response compared with WT mice, possibly as a result of absent TLR4 signaling through LTA-induced release of endogenous mediators. CONCLUSION: Pneumococcal LTA induces a profound inflammatory response and activation of the coagulation pathway in the lungs in vivo through a TLR2-dependent route, which likely is amplified by endogenous TLR4 ligands.

Lipoteichoic acid-induced lung inflammation depends on TLR2 and the concerted action of TLR4 and the platelet-activating factor receptor.

Lipoteichoic acid (LTA) is a major outer cell wall component of Gram-positive bacteria that has been implicated as an important factor in the inflammatory response following bacterial infection. In vitro data indicate roles for TLR2, platelet-activating factor receptor (PAFR), CD14, and LPS-binding protein (LBP) in cellular responsiveness to LTA, whereas the mechanisms contributing to LTA effects in vivo have never been investigated. Using mice deficient for LBP, CD14, TLR2, TLR4, or PAFR, we now examined the role of these molecules in pulmonary inflammation induced by highly purified LTA in vivo. Although pulmonary LBP increased dose-dependently following administration of LTA, the inflammatory response was unaltered in LBP-/- mice. TLR2 proved to be indispensable for the initiation of an inflammatory response, as polymorphonuclear cell influx, TNF-alpha, keratinocyte-derived chemokine, and MIP-2 release were abolished in TLR2-/- mice. Minor effects such as moderately decreased TNF-alpha and MIP-2 levels were observed in the absence of CD14, indicating a role for CD14 as a coreceptor. Quite surprisingly, the absence of TLR4 greatly diminished pulmonary inflammation and the same phenotype was observed in PAFR-/- animals. In contrast to all other mice studied, only TLR4-/- and PAFR-/- mice displayed significantly elevated IL-10 pulmonary concentrations. These data suggest that TLR2 is the single most important receptor signaling the presence of LTA within the lungs in vivo, whereas TLR4 and PAFR may influence lung inflammation induced by LTA either by sensing LTA directly or through recognition and signaling of endogenous mediators induced by the interaction between LTA and TLR2.

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-1beta, IL-6, IL-8, IL-10 and TGF-beta 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 (1)H-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.

Molecular interaction between lipoteichoic acids and Lactobacillus delbrueckii phages depends on D-alanyl and alpha-glucose substitution of poly(glycerophosphate) backbones.

Lipoteichoic acids (LTAs) have been shown to act as bacterial counterparts to the receptor binding proteins of LL-H, LL-H host range mutant LL-H-a21, and JCL1032. Here we have used LTAs purified by hydrophobic interaction chromatography from different phage-resistant and -sensitive strains of Lactobacillus delbrueckii subsp. lactis. Nuclear magnetic resonance analyses revealed variation in the degree of alpha-glucosyl and D-alanyl substitution of the 1,3-linked poly(glycerophosphate) LTAs between the phage-sensitive and phage-resistant strains. Inactivation of phages was less effective if there was a high level of D-alanine residues in the LTA backbones. Prior incubation of the LTAs with alpha-glucose-specific lectin inhibited the LL-H phage inactivation. The overall level of decoration or the specific spatial combination of alpha-glucosyl-substituted, D-alanyl-substituted, and nonsubstituted glycerol residues may also affect phage adsorption.

Functional analysis of D-alanylation of lipoteichoic acid in the probiotic strain Lactobacillus rhamnosus GG.

Lipoteichoic acid (LTA) is a macroamphiphile molecule which performs several functions in gram-positive bacteria, such as maintenance of cell wall homeostasis. D-alanylation of LTA requires the proteins encoded by the dlt operon, and this process is directly related to the charge properties of this polymer strongly contributing to its function. The insertional inactivation of dltD of the probiotic strain Lactobacillus rhamnosus GG (ATCC 53103) resulted in the complete absence of D-alanyl esters in the LTA as confirmed by nuclear magnetic resonance analysis. This was reflected in modifications of the bacterial cell surface properties. The dltD strain showed 2.4-fold-increased cell length, a low survival capacity in response to gastric juice challenge, an increased sensitivity to human beta-defensin-2, an increased rate of autolysis, an increased capacity to initiate growth in the presence of an anionic detergent, and a decreased capacity to initiate growth in the presence of cationic peptides compared to wild-type results. However, in vitro experiments revealed no major differences for adhesion to human intestinal epithelial cells, biofilm formation, and immunomodulation. These properties are considered to be important for probiotics. The role of the dlt operon in lactobacilli is discussed in view of these results.

Cell activation of human macrophages by lipoteichoic acid is strongly attenuated by lipopolysaccharide-binding protein.

Lipoteichoic acid (LTA) represents immunostimulatory molecules expressed by Gram-positive bacteria. They activate the innate immune system via Toll-like receptors. We have investigated the role of serum proteins in activation of human macrophages by LTA from Staphylococcus aureus and found it to be strongly attenuated by serum. In contrast, the same cells showed a sensitive response to LTA and a significantly enhanced production of tumor necrosis factor alpha under serum-free conditions. We show that LTA interacts with the serum protein lipopolysaccharide-binding protein (LBP) and inhibits the integration of LBP into phospholipid membranes, indicating the formation of complexes of LTA and soluble LBP. The addition of recombinant human LBP to serum-free medium inhibited the production of tumor necrosis factor alpha and interleukins 6 and 8 after stimulation of human macrophages with LTA in a dose-dependent manner. Using anti-LBP antibodies, this inhibitory effect could be attributed to soluble LBP, whereas LBP in its recently described transmembrane configuration did not modulate cell activation. Also, using primary alveolar macrophages from rats, we show a sensitive cytokine response to LTA under serum-free culture conditions that was strongly attenuated in the presence of serum. In summary, our data suggest that innate immune recognition of LTA is organ-specific with negative regulation by LBP in serum-containing compartments and sensitive recognition in serum-free compartments like the lung.

Comparison of lipoteichoic acid from different serotypes of Streptococcus pneumoniae.

Pneumococcal lipoteichoic acid (LTA) is known to have a completely different chemical structure compared with that of Staphylococcus aureus: the polyglycerophosphate in the backbone is replaced in the pneumococcal LTA by a pentamer repeating unit consisting of one ribitol and a tetrasaccharide carrying the unusual substituents phosphocholine and N-acetyl-D-galactosamine. Neither D-alanine nor N-acetyl-D-glucosamine, which play central roles in the biological activity of the staphylococcal LTA, has been reported. The extraction using butanol is more gentle compared with the previously reported chloroform-methanol extraction and results in a higher yield of LTA. We characterized the LTA of two different strains of Streptococcus pneumoniae:R6 (serotype 2) and Fp23 (serotype 4). NMR analysis confirmed the structure of LTA from R6 but showed that its ribitol carries an N-acetyl-D-galactosamine substituent. The NMR data for the LTA from Fp23 indicate that this LTA additionally contains ribitol-bound D-alanine. Dose-response curves of the two pneumococcal LTAs in human whole blood revealed that LTA from Fp23 was significantly more potent than LTA from R6 with regard to the induction of all cytokines measured (tumor necrosis factor, interleukin-1 (IL-1), IL-8, IL-10, granulocyte colony-stimulating factor, and interferon gamma). However, other characteristics, such as lack of inhibition by endotoxin-specific LAL-F, Toll-like receptor 2 and not 4 dependence, and lack of stimulation of neutrophilic granulocytes, were shared by both LTAs. This is the first report of a difference in the structure of LTA between two pneumococcal serotypes resulting in different immunostimulatory potencies.

Heterozygous Arg753Gln polymorphism of human TLR-2 impairs immune activation by Borrelia burgdorferi and protects from late stage Lyme disease.

Lyme disease (LD) is caused by Borrelia burgdorferi and displays different stages, including localized, early disseminated, and persistent infection, all of which are associated with profound inflammatory reactions in the host. Induction of proinflammatory cytokines by B. burgdorferi is mainly mediated by outer surface proteins interacting with TLR-2/TLR-1 heterodimers. In this study, we show that TNF-alpha induction by Borrelia lysate was impaired in heterozygous TLR-2 knockout mice, while reactivity to lipoteichoic acid, another TLR-2 ligand signaling via TLR-2/TLR-6 heterodimers, was unaffected. Blood from individuals heterozygous for the TLR-2 polymorphism Arg753Gln was tested for cytokine release upon stimulation with Borrelia lysate, and induction of TNF-alpha and IFN-gamma was significantly lower as compared with individuals not exhibiting this variation. Overexpression of TLR-2 carrying the Arg753Gln polymorphism in HEK 293 cells led to a significantly stronger impairment of activation by TLR-2/TLR-1 ligands as compared with TLR-2/TLR-6 ligands. To study whether heterozygosity for the Arg753Gln variant of TLR-2 influenced susceptibility for LD, we analyzed 155 patients for this polymorphism. The Arg753Gln variant occurs at a significantly lower frequency in LD patients as compared with matched controls (5.8 vs 13.5%, odds ratio 0.393, 95% confidence interval 0.17-0.89, p = 0.033), with an even more pronounced difference when late stage disease was observed (2.3 vs 12.5%, odds ratio 0.163, 95% confidence interval 0.04-0.76, p = 0.018). These data suggest that Arg753Gln may protect from the development of late stage LD due to a reduced signaling via TLR-2/TLR-1.