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.

Internalization and coreceptor expression are critical for TLR2-mediated recognition of lipoteichoic acid in human peripheral blood.

Lipoteichoic acid (LTA), a ubiquitous cell wall component of Gram-positive bacteria, represents a potent immunostimulatory molecule. Because LTA of a mutant Staphylococcus aureus strain lacking lipoproteins (Deltalgt-LTA) has been described to be immunobiologically inactive despite a lack of ascertained structural differences to wild-type LTA (wt-LTA), we investigated the functional requirements for the recognition of Deltalgt-LTA by human peripheral blood cells. In this study, we demonstrate that Deltalgt-LTA-induced immune activation critically depends on the immobilization of LTA and the presence of human serum components, which, to a lesser degree, was also observed for wt-LTA. Under experimental conditions allowing LTA-mediated stimulation, we found no differences between the immunostimulatory capacity of Deltalgt-LTA and wt-LTA in human blood cells, arguing for a limited contribution of possible lipoprotein contaminants to wt-LTA-mediated immune activation. In contrast to human blood cells, TLR2-transfected human embryonic kidney 293 cells could be activated only by wt-LTA, whereas activation of these cells by Deltalgt-LTA required the additional expression of TLR6 and CD14, suggesting that activation of human embryonic kidney 293 cells expressing solely TLR2 is probably mediated by residual lipoproteins in wt-LTA. Notably, in human peripheral blood, LTA-specific IgG Abs are essential for Deltalgt-LTA-mediated immune activation and appear to induce the phagocytic uptake of Deltalgt-LTA via engagement of FcgammaRII. In this study, we have elucidated a novel mechanism of LTA-induced cytokine induction in human peripheral blood cells that involves uptake of LTA and subsequent intracellular recognition driven by TLR2, TLR6, and CD14.

Engagement of scientists with the public and policymakers to promote alternative methods.

Scientists are usually good at teaching, sometimes even to lay audiences. But communicating with journalists, activists, or policymakers can be a different story - hesitancy to make mistakes as well as the temptation to disproportionally promote one's own case come into play. The multitude of social media and other web-based outlets has diversified and accelerated the communication of science. Real-time reactions, sharing of data, tools and results, increasing invitation of personal opinion, demand for transparency, political correctness, and loss of trust in experts are challenges to researchers in general. The field of alternatives to animal testing is more political and important to lay audiences than many others, so its scientists must be especially aware of these challenges. Public engagement offers the opportunity to form community and create wide support for non-animal research and its implementation. This requires scientists to step out of the ivory tower of higher education and engage with diverse interest groups by outreach activities, interviews, and press releases, etc. by employing tailored communication.

IL-33 enhances lipopolysaccharide-induced inflammatory cytokine production from mouse macrophages by regulating lipopolysaccharide receptor complex.

Bacterial LPS triggers monocytes and macrophages to produce several inflammatory cytokines and mediators. However, once exposed to LPS, they become hyporesponsive to a subsequent endotoxin challenge. This phenomenon is defined as LPS desensitization or tolerance. Previous studies have identified some components of the biochemical pathways involved in negative modulation of LPS responses. In particular, it has been shown that the IL-1R-related protein ST2 could be implicated in LPS tolerance. The natural ligand of ST2 was recently identified as IL-33, a new member of the IL-1 family. In this study, we investigated whether IL-33 triggering of ST2 was able to induce LPS desensitization of mouse macrophages. We found that IL-33 actually enhances the LPS response of macrophages and does not induce LPS desensitization. We demonstrate that this IL-33 enhancing effect of LPS response is mediated by the ST2 receptor because it is not found in ST2 knockout mice. The biochemical consequences of IL-33 pretreatment of mouse macrophages were investigated. Our results show that IL-33 increases the expression of the LPS receptor components MD2 (myeloid differentiation protein 2) and TLR-4, the soluble form of CD14 and the MyD88 adaptor molecule. In addition, IL-33 pretreatment of macrophages enhances the cytokine response to TLR-2 but not to TLR-3 ligands. Thus, IL-33 treatment preferentially affects the MyD88-dependent pathway activated by the TLR.

2-APB and capsazepine-induced Ca2+ influx stimulates clathrin-dependent endocytosis in alveolar epithelial cells.

Calcium as a second messenger influences many cellular and physiological processes. In lung, alveolar type II (ATII) cells sense mechanical stress and respond by Ca(2+) dependent release of surfactant, which is essential for respiratory function. Nevertheless, Ca(2+) signaling mechanisms in these cells--in particular Ca(2+) entry pathways are still poorly understood. Herein, we investigated pharmacological properties of non-voltage-gated Ca(2+) channel modulators in ATII and NCI-H441 cells and demonstrate that 2-Aminoethoxydiphenyl-borinate (2-APB) and capsazepine (CPZ) activate Ca(2+) entry with pharmacologically distinguishable components. Surprisingly, 2-APB and CPZ activated clathrin dependent endocytosis in ATII and NCI-H441 cells, which was dependent on Ca(2+) entry. The internalized material accumulated in non-acidic granules distinct from surfactant containing lamellar bodies (LB). LB exocytosis was not observed under these conditions. Our study demonstrates that 2-APB/CPZ induces Ca(2+) entry which unlike ATP- or stretch-induced Ca(2+) entry in ATII cells does not activate exocytosis but an opposing endocytotic mechanism.

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.

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-kappaB activation. LTA internalization, but not NF-kappaB 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.

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.

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.

High sensitivity pyrogen testing in water and dialysis solutions.

BACKGROUND: The dialysis patient is confronted with hundreds of litres of dialysis solution per week, which pass the natural protective barriers of the body and are brought into contact with the tissue directly in the case of peritoneal dialysis or indirectly in the case of renal dialysis (hemodialysis). The components can be tested for living specimens or dead pyrogenic (fever-inducing) contaminations. The former is usually detected by cultivation and the latter by the endotoxin-specific Limulus Amoebocyte Lysate Assay (LAL). However, the LAL assay does not reflect the response of the human immune system to the wide variety of possible pyrogenic contaminations in dialysis fluids. Furthermore, the test is limited in its sensitivity to detect extremely low concentrations of pyrogens, which in their sum result in chronic pathologies in dialysis patients. The In vitro Pyrogen Test (IPT) employs human whole blood to detect the spectrum of pyrogens to which humans respond by measuring the release of the endogenous fever mediator interleukin-1beta. Spike recovery checks exclude interference. The test has been validated in an international study for pyrogen detection in injectable solutions. METHODS: In this study we adapted the IPT to the testing of dialysis solutions. RESULTS: Preincubation of 50 ml spiked samples with albumin-coated microspheres enhanced the sensitivity of the assay to detect contaminations down to 0.1 pg/ml LPS or 0.001 EU/ml in water or saline and allowed pyrogen detection in dialysis concentrates or final working solutions. CONCLUSIONS: This method offers high sensitivity detection of human-relevant pyrogens in dialysis solutions and components.

IL-10 release requires stronger toll-like receptor 4-triggering than TNF: a possible explanation for the selective effects of heterozygous TLR4 polymorphism Asp(299)Gly on IL-10 release.

The toll-like receptor 4 Asp(299)Gly polymorphism results in an inactive receptor. Heterozygosis is associated with reduced LPS-inducible IL-10 protein and IL-10 mRNA from blood leukocytes and isolated monocytes, while numerous other mediators are not affected. We could exclude that this effect is due to the differences in the kinetics of IL-10 release, in the expression of total surface TLR4 or in LPS-binding to monocytes between subjects heterozygous for the Asp(299)Gly polymorphism or homozygous carriers of the wild-type allele. Furthermore, we could show that IL-10 induction in general requires stronger LPS-triggering than TNF and is more sensitive to LPS inhibitors. The lower number of responsive wild-type TLR4 receptors on monocytes of heterozygotes may explain why only IL-10 release is affected.

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.

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.

An in vitro pyrogen safety test for immune-stimulating components on surfaces.

Due to the discovery of novel surgical techniques and new materials medical devices are increasingly used. Contact of these biomaterials with human tissue or blood commonly leads to inflammation of varying degrees, sometimes resulting in severe health problems. Possible causes are limited biocompatibility or pyrogenic contamination of the material. We adapted the recently validated in vitro pyrogen test (IPT), based on human whole blood cytokine release, to test the safety of biomaterials. Human whole blood is brought into direct contact with the surface of the test material and the release of the pro-inflammatory cytokine IL-1beta is measured. This procedure represents a human-relevant assay allowing the detection of pyrogens of different origins, e.g. Gram-negative (lipopolysaccharide, LPS) or Gram-positive (lipoteichoic acid, LTA), peptidoglycan (cell wall components of most bacteria) and fungal zymosan by direct material contact. The sensitivity of the test system allows a starting concentration of 10 pg/ml for LPS, 10 ng/ml for zymosan and 1 microg/ml for LTA and peptidoglycan from different strains. Furthermore, we have shown that the test for solid materials can be carried out with cryo-preserved blood, which results in an even lower detection limit.