[Show abstract][Hide abstract] ABSTRACT: Common genetic variations in toll-like receptor 2 (TLR2), an innate pathogen recognition receptor, may influence the development of atopic diseases. So far, very little is known about the role of rare TLR2 mutations in these diseases.
We investigated the functional properties of six rare amino acid changes in TLR2 (and one amino acid change in a TLR2 pseudogene) and studied their effect on atopic sensitization and disease.
We identified rare TLR2 mutations leading to amino acid changes from databases. Functional effects of TLR2 variants were analyzed by NF-kappaB-dependent luciferase reporter assay and interleukin-8 enzyme linked immunosorbent assay in vitro. The frequency of these mutations was determined in a random sample of the general population (n = 368). Association with atopic diseases were studied in a cross sectional German study population (n = 3099).
Three out of six mutations in the TLR2 gene altered receptor activity in vitro. Out of these, only the minor allele of R753Q occurred reasonably frequent in the German population (minor allele frequency 3%). The risk to develop atopy increased by 50% in carriers of the 753Q allele (P = 0.021) and total (P = 0.040) as well as allergen specific serum IgE levels (P = 0.011) were significantly elevated.
The rare but functionally relevant mutation R753Q in TLR2 may significantly affect common conditions such as atopic sensitization in the general population.
[Show abstract][Hide abstract] ABSTRACT: Mainly Gram-negative and Gram-positive bacterial infections, but also other infections such as with fungal or viral pathogens, can cause the life-threatening clinical condition of septic shock. Transgression of the host immune response from a local level limited to the pathogen's place of entry to the systemic level is recognised as a major mode of action leading to sepsis. This view has been established upon demonstration of the capacity of specific pathogen-associated molecular patterns (PAMPs) to elicit symptoms of septic shock upon systemic administration. Immune stimulatory PAMPs are agonists of soluble, cytoplasmic, as well as/or cell membrane-anchored and/or -spanning pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs). However, reflection of pathogen-host crosstalk triggering sepsis pathogenesis upon an infection by a host response to challenge with an isolated PAMP is incomplete. Therefore, an experimental model more reflective of pathogen-host interaction requires experimental host confrontation with a specific pathogen in its viable form resulting in a collective stimulation of a variety of specific PRRs. This chapter describes methods to analyse innate pathogen sensing by the host on both a cellular and systemic level.
No preview · Article · Feb 2009 · Methods in Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: Gram-negative bacterial infection is a major cause of sepsis and septic shock. An important inducer of inflammation underlying both syndromes is the cellular recognition of bacterial products through pattern recognition receptors (PRRs), including Toll-like receptors (TLRs). We identified a novel antagonistic mAb (named 1A6) that recognizes the extracellular portion of the TLR4-MD-2 complex. If applied to mice before infection with clinical isolates of Salmonella enterica or Escherichia coli and subsequent antibiotic therapy, 1A6 prevented otherwise fatal shock, whereas application of 1A6 after infection was ineffective. In contrast, coapplication of 1A6 and an anti-TLR2 mAb up to 4 h after infection with Gram-negative bacteria, in combination with the start of antibiotic therapy (mimicking clinical conditions), provided robust protection. Consistent with our findings in mice, dual blockade of TLR2 and TLR4 inhibited TNF-alpha release from human peripheral blood mononuclear cells upon Gram-negative bacterial infection/antibiotic therapy. Both murine splenocytes and human PBMCs released IFN-gamma in a TLR4-dependent manner, leading to enhanced surface TLR2 expression and sensitivity for TLR2 ligands. Our results implicate TLR2 as an important, TLR4-driven sensor of Gram-negative bacterial infection and provide a rationale for blockade of both TLRs, in addition to antibiotic therapy for the treatment of Gram-negative bacterial infection.
Full-text · Article · Aug 2008 · Journal of Experimental Medicine
[Show abstract][Hide abstract] ABSTRACT: Overactivation of the immune system upon acute bacterial infection leads to septic shock. Specific bacterial products potently stimulate immune cells via toll-like receptors (TLRs). Gram-negative bacteria induce a predominantly TLR4-driven signal through LPS release. To neutralize LPS signaling in experimental models of sepsis, we generated mAbs toward the TLR4/myeloid differentiation protein-2 (MD-2) complex. The binding properties of an array of selected rat mAbs differed in respect to their specificity for TLR4/MD-2 complex. The specificity of one such mAb, 5E3, to murine TLR4 was confirmed by its recognition of an epitope within the second quarter of the ectodomain. 5E3 inhibited LPS-dependent cell activation in vitro and prevented proinflammatory cytokine production in vivo following LPS challenge in a dose-dependent manner. Furthermore, 5E3 protected mice from lethal shock-like syndrome when applied using both preventative and therapeutic protocols. Most notably, in the colon ascendens stent peritonitis model of polymicrobial abdominal sepsis, administration of a single dose of 5E3 (50 mug) protected mice against mortality. These results demonstrate that neutralizing TLR4/MD-2 is highly efficacious in protecting against bacterial infection-induced toxemia and offers TLR4/MD-2 mAb treatment as a potential therapy for numerous clinical indications.
Full-text · Article · Dec 2007 · The Journal of Immunology
[Show abstract][Hide abstract] 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 (Myr3CSK4) 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 Myr3CSK4 challenge, whereas TLR2–/–/TLR4d/d cells were unresponsive. Through interferon-γ (IFNγ) 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 Myr3CSK4 challenge-induced fatal shock. d-Galactosamine-sensitized mice expressing defective TLR4 or lacking TLR4 expression acquired susceptibility to eLPS-driven
toxemia upon IFNγ priming, whereas double deficient mice did not. Immunization toward ovalbumin using Myr3CSK4 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 Myr3CSK4 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.
Full-text · Article · Jun 2007 · Journal of Biological Chemistry