Toll-related receptors and the control of antimicrobial peptide expression in Drosophila.
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Insects defend themselves against infectious microorganisms by synthesizing potent antimicrobial peptides. Drosophila has appeared in recent years as a favorable model to study this innate host defense. A genetic analysis of the regulation of the antifungal peptide drosomycin has demonstrated a key role for the transmembrane receptor Toll, which prompted the search for mammalian homologs. Two of these, Toll-like receptor (TLR)2 and TLR4, recently were shown to play a critical role in innate immunity against bacteria. Here we describe six additional Toll-related genes (Toll-3 to Toll-8) in Drosophila in addition to 18-wheeler. Two of these genes, Toll-3 and Toll-4, are expressed at a low level. Toll-6, -7, and -8, on the other hand, are expressed at high levels during embryogenesis and molting, suggesting that, like Toll and 18w, they perform developmental functions. Finally, Toll-5 is expressed only in larvae and adults. By using chimeric constructs, we have tested the capacity of the signaling Toll/IL-1R homology domains of these receptors to activate antimicrobial peptide promoters and found that only Toll and Toll-5 can activate the drosomycin promoter in transfected cells, thus demonstrating specificity at the level of the Toll/IL-1R homology domain. In contrast, none of these constructs activated antibacterial peptide promoters, suggesting that Toll-related receptors are not involved in the regulation of antibacterial peptide expression. This result was independently confirmed by the demonstration that a dominant-negative version of the kinase Pelle can block induction of drosomycin by the cytokine Spaetzle, but does not affect induction of the antibacterial peptide attacin by lipopolysaccharide. (+info)
Recognition of CpG DNA is mediated by signaling pathways dependent on the adaptor protein MyD88.
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The innate immune system evolved to recognize conserved microbial products, termed pathogen-associated molecular patterns (PAMPs), which are invariant among diverse groups of microorganisms. PAMPs are recognized by a set of germ-line encoded pattern recognition receptors (PRRs). Among the best characterized PAMPs are bacterial lipopolysaccharide (LPS), peptidoglycan (PGN), mannans, and other constituents of bacterial and fungal cell walls, as well as bacterial DNA. Recognition of bacterial DNA is the most enigmatic of these, as it depends on a particular sequence motif, called the CpG motif, in which an unmethylated CpG present in a particular sequence context accounts for a potent immunostimulatory activity of CpG DNA. Receptor(s) of the innate immune system that mediate recognition of CpG DNA are currently unknown. Here, we report that recognition of CpG DNA requires MyD88, an adaptor protein involved in signal transduction by the Toll-like receptors (TLRs), essential components of innate immune recognition in both Drosophila and mammals [1,2]. Signaling induced by CpG DNA was found to be unaffected in cells deficient in TLR2 or TLR4, suggesting that some other member of the Toll family mediates recognition of bacterial DNA. (+info)
A novel synthetic acyclic lipid A-like agonist activates cells via the lipopolysaccharide/toll-like receptor 4 signaling pathway.
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ER-112022 is a novel acyclic synthetic lipid A analog that contains six symmetrically organized fatty acids on a noncarbohydrate backbone. Chinese hamster ovary (CHO)-K1 fibroblasts and U373 human astrocytoma cells do not respond to lipopolysaccharide (LPS) in the absence of CD14. In contrast, exposure to ER-112022 effectively induced activation of CHO and U373 cells under serum-free conditions. Expression of CD14 was not necessary for cells to respond to ER-112022, although the presence of soluble CD14 enhanced the sensitivity of the response. Several lines of evidence suggested that ER-112022 stimulates cells via the LPS signal transduction pathway. First, the diglucosamine-based LPS antagonists E5564 and E5531 blocked ER-112022-induced stimulation of CHO-K1, U373, and RAW264.7 cells. Second, ER-112022 was unable to activate C3H/HeJ mouse peritoneal macrophages, containing a mutation in Toll-like receptor (TLR) 4, as well as HEK293 cells, an epithelial cell line that does not express TLR4. Third, ER-112022 activated NF-kappaB in HEK293 cells transfected with TLR4/MD-2. Finally, tumor necrosis factor release from primary human monocytes exposed to ER-112022 was blocked by TLR4 antibodies but not by TLR2 antibodies. Our results suggest that ER-112022 and the family of lipid A-like LPS antagonists can functionally associate with TLR4 in the absence of CD14. Synthetic molecules like ER-112022 may prove to be valuable tools to characterize elements in the LPS receptor complex, as well as to activate or inhibit the TLR4 signaling pathway for therapeutic purposes. (+info)
Lipopolysaccharide induces physical proximity between CD14 and toll-like receptor 4 (TLR4) prior to nuclear translocation of NF-kappa B.
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CD14, a GPI-linked protein, plays a pivotal role in LPS-mediated signaling by potentiating leukocyte adherence, activation, and cytokine production. Recent studies have identified the Toll-like receptor 4 (TLR4) as a membrane cofactor in LPS-mediated transmembrane signaling in cytokine induction, although the mechanism responsible for this cooperation is unknown. Using fluorescence resonance energy transfer (RET) techniques, we demonstrate that LPS triggers a physical association between CD14 and TLR4. Because LPS stimulation upregulates CD14 and TLR4 expression, it was necessary to control for the possibility that these newly expressed molecules were associated with one another independent of LPS stimulation. Although the calcium ionophore A23187 increased the expression of CD14 and TLR4, they did not exhibit energy transfer. However, following A23187 treatment, LPS promoted physical proximity between CD14 and TLR4. Therefore, we suggest that a close interaction between CD14 and TLR4 participates in LPS signaling, leading to nuclear translocation of NF-kappaB. (+info)
Activation of toll-like receptor 2 on human dendritic cells triggers induction of IL-12, but not IL-10.
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Mammalian Toll-like receptors (TLRs) are required for cell activation by bacterial lipoproteins (bLP) and LPS. Stimulation of monocytes with bLP and LPS results in a TLR-dependent induction of immunomodulatory genes leading to the production of pro-inflammatory cytokines. In this paper, we compared the expression and response of TLRs on monocytes and dendritic cells (DC). TLR2, but not TLR4, was detected on peripheral blood monocytes and DC, in lymphoid tissue CD1alpha+ DC as well as on in vitro monocyte-derived DC. Upon stimulation with bLP or LPS, monocytes produced IL-12 and IL-10 at similar levels, whereas monocyte-derived DC produced comparable levels of IL-12, but little IL-10. Greater than 90% of the bLP-induced production of IL-12 was blocked by anti-TLR2 mAb. Thus, DC express TLR2 and activation of this receptor by bLP provides an innate mechanism by which microbial pathogens preferentially activate cell-mediated immunity. (+info)
Cutting edge: TLR2-deficient and MyD88-deficient mice are highly susceptible to Staphylococcus aureus infection.
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Toll-like receptor (TLR) family acts as pattern recognition receptors for pathogen-specific molecular patterns. We previously showed that TLR2 recognizes Gram-positive bacterial components whereas TLR4 recognizes LPS, a component of Gram-negative bacteria. MyD88 is shown to be an adaptor molecule essential for TLR family signaling. To investigate the role of TLR family in host defense against Gram-positive bacteria, we infected TLR2- and MyD88-deficient mice with Staphylococcus aureus. Both TLR2- and MyD88-deficient mice were highly susceptible to S. aureus infection, with more enhanced susceptibility in MyD88-deficient mice. Peritoneal macrophages from MyD88-deficient mice did not produce any detectable levels of cytokines in response to S. aureus. In contrast, TLR2-deficient macrophages produced reduced, but significant, levels of the cytokines, and TLR4-deficient macrophages produced the same amounts as wild-type cells, indicating that S. aureus is recognized not only by TLR2, but also by other TLR family members except for TLR4. (+info)
Gene expressions of Toll-like receptor 2, but not Toll-like receptor 4, is induced by LPS and inflammatory cytokines in mouse macrophages.
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Toll-like receptors (TLRs) are a family of mammalian homologues of Drosophila Toll and play important roles in host defense. Two of the TLRs, TLR2 and TLR4, mediate the responsiveness to LPS. Here the gene expression of TLR2 and TLR4 was analyzed in mouse macrophages. Mouse splenic macrophages responded to an intraperitoneal injection or in vitro treatment of LPS by increased gene expression of TLR2, but not TLR4. Treatment of a mouse macrophage cell line with LPS, synthetic lipid A, IL-2, IL-15, IL-1beta, IFN-gamma, or TNF-alpha significantly increased TLR2 mRNA expression, whereas TLR4 mRNA expression remained constant. TLR2 mRNA increase in response to synthetic lipid A was severely impaired in splenic macrophages isolated from TLR4-mutated C3H/HeJ mice, suggesting that TLR4 plays an essential role in the process. Specific inhibitors of mitogen-activated protein/extracellular signal-regulated kinase kinase and p38 kinase did not significantly inhibit TLR2 mRNA up-regulation by LPS. In contrast, LPS-mediated TLR2 mRNA induction was abrogated by pretreatment with a high concentration of curcumin, suggesting that NF-kappaB activation may be essential for the process. Taken together, our results indicate that TLR2, in contrast to TLR4, can be induced in macrophages in response to bacterial infections and may accelerate the innate immunity against pathogens. (+info)
Toll-like receptor 4, but not toll-like receptor 2, is a signaling receptor for Escherichia and Salmonella lipopolysaccharides.
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Two members of the mammalian Toll-like receptor (TLR) family, TLR2 and TLR4, have been implicated as receptors mediating cellular activation in response to bacterial LPS. Through the use of mAbs raised against human TLR2 and TLR4, we have conducted studies in human cell lines and whole blood to ascertain the relative contribution of these receptors to LPS induced cytokine release. We show that the contribution of TLR2 and TLR4 to LPS-induced cellular activation correlates with the relative expression levels of these two TLRs in a given cell type. In addition, we have found that significant differences in cell stimulatory activity exist between various smooth and rough LPS types that cannot be ascribed to known LPS structural features. These results suggest that impurities in the LPS may be responsible for some of the activity and this would be in agreement with recently published results of others. Upon repurification, none of the commercial LPS preparations activate cells through TLR2, but continue to stimulate cells with comparable activity through TLR4. Our results confirm recent findings that TLR4, but not TLR2, mediates cellular activation in response to LPS derived from both Escherichia coli and Salmonella minnesota. Additionally, we show that TLR4 is the predominant signaling receptor for LPS in human whole blood. (+info)