Abstract
Evidence for specific and direct bacterial product recognition through toll-like receptors (TLRs) has been emphasized recently. We analyzed lipopeptide analogues and enterobacterial lipopolysaccharide (eLPS) for their potential to activate cells through TLR2 and TLR4. Whereas bacterial protein palmitoylated at its N-terminal cysteine and N-terminal peptides derived thereof are known to induce TLR2-mediated cell activation, a synthetic acylhexapeptide mimicking a bacterial lipoprotein subpopulation for which N-terminal trimyristoylation is characteristic (Myr(3)CSK(4)) activated cells not only through TLR2 but also through TLR4. Conversely, highly purified eLPS triggered cell activation through overexpressed TLR2 in the absence of TLR4 expression if CD14 was coexpressed. Accordingly, TLR2(-/-) macrophages prepared upon gene targeting responded to Myr(3)CSK(4) challenge, whereas TLR2(-/-)/TLR4(d/d) cells were unresponsive. Through interferon-gamma (IFNgamma) priming, macrophages lacking expression of functional TLR4 and/or MD-2 acquired sensitivity to eLPS, whereas TLR2/TLR4 double deficient cells did not. Not only TLR2(-/-) mice but also TLR4(-/-) mice were resistant to Myr(3)CSK(4) challenge-induced fatal shock. d-Galactosamine-sensitized mice expressing defective TLR4 or lacking TLR4 expression acquired susceptibility to eLPS-driven toxemia upon IFNgamma priming, whereas double deficient mice did not. Immunization toward ovalbumin using Myr(3)CSK(4) as adjuvant was ineffective in TLR2(-/-)/TLR4(-/-) mice yet effective in wild-type, TLR2(-/-), or TLR4(-/-) mice as shown by analysis of ovalbumin-specific serum Ig concentration. A compound such as Myr(3)CSK(4) whose stimulatory activity is mediated by both TLR2 and TLR4 might constitute a preferable adjuvant. On the other hand, simultaneous blockage of both of the two TLRs might effectively inhibit infection-induced pathology.
Highlights
N-terminal cysteine and N-terminal peptides derived thereof are known to induce TLR2-mediated cell activation, a synthetic acylhexapeptide mimicking a bacterial lipoprotein subpopulation for which N-terminal trimyristoylation is characteristic (Myr3CSK4) activated cells through TLR2 and through TLR4
We report on a synthetic lipopeptide carrying a trimyristoylated cysteine at its N terminus that activated TLR4 aside from TLR2
We analyzed lipopeptide analogues differing in the numbers of intramolecular palmitoylations or identities of triacyl moieties [33, 35] for their potential to activate TLR2 and TLR4
Summary
Reagents—LPS from E. coli strain O111:B4 (smooth, carrying a long polysaccharide chain) purified by phenol extraction and gel filtration was from Sigma. Antibody T2.5 antagonizing mTLR2 and hTLR2 or mTLR2-specific isotype control T2.13 were applied to cell cultures at a concentration of 10 g/ml 30 min prior to challenge with TLR agonists [26]. For analysis of serum cytokine concentrations, mice were anesthetized upon systemic challenge by intraperitoneal injection of bacterial products or synthetic analogues. TLR agonists listed above, suspensions of heat-inactivated bacteria, or 200 g of Myr3CSK4 were injected intraperitoneally together with 800 mg/kg D-GalN if indicated [27]. Electromobility Shift Assay—Peritoneal macrophages were challenged by application of bacterial products or synthetic analogues in RPMI 1640 serum containing 2% fetal calf serum (PAA GmbH, Pasching, Austria) for 2 h, and nuclear proteins were analyzed for NF-B recognition element-specific DNA binding [33]. Immunoblot Analysis—Total lysates of 5 ϫ 105 primary macrophages per lane were analyzed by using a purified polyclonal rabbit antiserum raised against a 28-mer peptide representing a subdomain of mTLR2 ectodomain [34]
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