Monocytes heterozygous for the Asp299Gly and Thr399Ile mutations in the Toll-like receptor 4 gene show no deficit in lipopolysaccharide signalling

Abstract

Toll-like receptor 4 (TLR4)-mediated recognition of lipopolysaccharide (LPS) is required for efficient recognition of Gram-negative bacterial infections. Two commonly occurring mutations in the human TLR4 gene (Asp299Gly and Thr399Ile) have recently been shown to be associated with blunted physiological responses to inhaled LPS, and with increased risk of Gram-negative bacteraemia in sepsis patients and reduced risk of atherosclerosis in an Italian population. Here we show that monocytes from individuals heterozygous for both mutations in the TLR4 gene exhibit no deficit in recognition of LPS of Escherichia coli, Neisseria meningitidis, Bacteroides fragilis, Yersinia pestis, Chlamydia trachomatis, Porphyromonas gingivalis, or Pseudomonas aeruginosa. We propose that the relatively high frequency of these mutations in the Caucasian population may reflect modified responses of carriers to alternative TLR4 agonists.

Figure 1.

Detection of wild-type, Asp299Gly, and Thr399Ile alleles in the human TLR4 gene. Genomic DNA of individuals recruited into the current study was amplified according to the method of Lorenz et al. (reference 4). PCR products were digested with the enzymes NcoI (299 allele) or HinfI (399 allele). Lanes 1 and 14, 100 base pair marker. Lanes 2–7, RFLP of the 299 section of TLR4 gene from three wild-type donors and three heterozygotes. Lanes 8–13, RFLP of the 399 section of TLR4 gene from three wild-type donors and three heterozygotes. Top band represents presence of wild-type allele, bottom band indicates presence of mutant allele. Genotypes were confirmed by sequencing.

Figure 2.

IL-1β secretion of wild type and Asp299Gly/Thr399Ile heterozygote TLR4 monocytes in response to LPS and LTA challenge. Human monocytes were challenged with ten-fold serial dilutions of E. coli, N. meningitidis, Y. pestis, C. trachomatis, B. fragilis, P. aeruginosa, P. gingivalis LPS or S. aureus LTA, or medium alone (C). Supernatants were assayed for IL-1β content after 4 h incubation. Open circles represent mean IL-1β secretion ± SEM from three wild-type donors. Filled squares represent mean IL-1β secretion ± SEM from three donors heterozygous for both TLR4 mutations.