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Activation of toll-like receptor (TLR) signaling that initiates an innate immune response to pathogens must be strictly regulated to prevent excessive inflammatory damage in the host. Here, we demonstrate that Mer receptor tyrosine kinase (MerTK) is a negative regulatory molecule in the lipoteichoic acid (LTA)-induced inflammatory response. LTA that activated TLR2 signaling concomitantly induced activation of MerTK signaling in RAW264.7 macrophages, including phosphoinositide 3-kinase (PI3K)/Akt and suppressor of cytokine signaling 3 (SOCS3). Moreover, LTA induced MerTK activation in a time-dependent manner, and LTA-induced MerTK activation was dependent on the ligand Gas6. Additionally, pretreatment with a specific Mer-blocking antibody significantly inhibited LTA-induced phosphorylation of MerTK, while further enhancing LTA-induced phosphorylation of IκB-α and NF-κBp65 as well as production of TNF-α and IL-6. Meanwhile, the antibody blockade of MerTK markedly prevented LTA-induced Akt phosphorylation and SOCS3 expression, both of which were crucial for the inhibition of TLR2-mediated immune response. Collectively, these results suggest, for the first time, that MerTK is an intracellular negative feedback regulator that inhibits the inflammatory response of LTA-stimulated macrophages through the PI3K/Akt pathway and SOCS3 protein. [ABSTRACT FROM AUTHOR]

Abstract: Human endothelial cells (EC) express Toll-like receptor 4 (TLR4), a receptor for lipopolysaccharides (LPS), but little or no TLR2, a lipopeptide receptor. The aim of this study was to investigate to what extent inflammatory stimuli modify the expression by EC of TLR4 and TLR2, of the TLR2 co-receptors TLR1 and TLR6 and of the TLR2-accessory proteins CD14 and CD36. Stimulation of umbilical vein derived EC with TNF-α, LPS or IL-1β for 24h induced a strong increase in TLR2 mRNA but not in TLR1, TLR4 and TLR6 mRNA. Inflammatory activation had little effect on CD14 mRNA, but decreased the expression of CD36 mRNA. TLR2 antigen was readily detected by flow cytometry on activated EC, but not on resting EC. A significant proportion of TLR2 was found to be located intracellularly. By using specific signalling pathway inhibitors we established that the induction of TLR2 by inflammatory stimuli was dependent on NF-κB, p38-MAP kinase and c-Jun kinase. IRAK-1 phosphorylation after treatment with 10μg/ml of lipoteichoic acid (LTA), a TLR2 agonist, was only observed in TNF-α-stimulated EC and not in resting EC. Furthermore, LTA potentiated the increase of the inflammatory markers E-Selectin or IL-8 in EC pre-treated with TNF-α, LPS or IL-1β, but not in resting EC. These results imply that the up-regulated TLR2 is functionally active. Interestingly, LTA had no effect on TLR2 expression, nor maintained TLR2 expression, in activated EC. This suggests that lipopeptide responses of EC are dependent on the continued presence of inflammatory cytokines, provided by other cell types, or LPS. In conclusion, inflammatory stimuli induce a high TLR2 expression in EC, which in turn enables the cells to strongly respond to lipopeptides. The up-regulation of TLR2 may be of relevance for the vascular effects of Gram-positive bacteria. [Copyright &y& Elsevier]

Abstract: 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 TNFα 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-κB 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 TNFα 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-κB activation in pbMEC by S. aureus or LTA. In contrast, E. coli and LPS activate strongly NF-κB 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-κB 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-κB activation in MEC thereby severely weakening the immune response in the udder. [Copyright &y& Elsevier]

Streptococcus mutans is a pathogenic Gram-positive bacterium that is closely associated with dental caries and subsequent pulpal inflammation. Although lipoteichoic acid (LTA) is considered a major virulence factor of Gram-positive bacteria, little is known about the innate immunity to S. mutans LTA. In this study, we purified LTA from S. mutans (Sm.LTA) through n-butanol extraction, hydrophobic interaction column chromatography, and ion-exchange column chromatography to investigate its immunological properties using murine macrophages. The Sm.LTA preparation had no detectable contamination with endotoxins, proteins, or nucleic acids. Upon exposure to Sm.LTA, the murine macrophage cell-line RAW 264.7 cells produced TNF-α and nitric oxide (NO) in a dose-dependent manner. Sm.LTA preferentially bound to and activated CHO/CD14/TLR2 cells rather than CHO/CD14/TLR4 cells, which are stable transfectants expressing CD14 and TLR2 or CD14 and TLR4, respectively. Sm.LTA could not induce TNF-α or NO production in macrophages derived from TLR2-deficient mice whereas it dose-dependently induced those inflammatory mediators in wild-type macrophages. TLR2-dependent induction of NO by Sm.LTA was also confirmed in RAW 264.7 cells using specific antibodies blocking TLR2. Furthermore, Sm.LTA deacylated by alkaline hydrolysis neither stimulated TLR2 nor induced TNF-α or NO production, suggesting that Sm.LTA lipid moieties are crucial for the immuno-stimulatory activity of Sm.LTA. Unlike Staphylococcus aureus LTA, which has potent immuno-stimulating activity, Sm.LTA showed a modest induction of NO production comparable to LTAs of other oral bacteria Enterococcus faecalis and Lactobacillus plantarum. In conclusion, our results suggest that the Sm.LTA interacts with TLR2 through the lipid moiety for the induction of inflammatory mediators in macrophages., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Among the non-carbohydrate components of glycans, the addition of phosphocholine (ChoP) to the glycans of pathogens occurs more rarely than acetylation or methylation, but it has far more potent biological consequences. These arise from ChoP's multiple interactions with host proteins, which are important at all stages of the infection process. These stages include initial adherence to cells, encountering the host's innate immune system and then the adaptive immune system. Thus, in the initial stages of an infection, ChoP groups are an asset to the pathogen, but they can turn into a disadvantage subsequently. In this review, we have focussed on structural aspects of these phenomena. We describe the biosynthesis of the ChoP modification, the structures of the pathogen glycans known to carry ChoP groups and the host proteins that recognize ChoP., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)