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(Clinical Chemistry. 2002;48:1661-1667.)
© 2002 American Association for Clinical Chemistry, Inc.

Polymorphisms of TLR4: Rapid Genotyping and Reduced Response to Lipopolysaccharide of TLR4 Mutant Alleles

Christopher Schmitt1,1, Andreas Humeny2,1, Cord-Michael Becker2, Kay Brune1 and Andreas Pahl1a

Departments of
1 Experimental and Clinical Pharmacology and Toxicology and
2 Biochemistry, Emil-Fischer-Center, University of Erlangen-Nürnberg, Fahrstrasse 17, D-91054 Erlangen, Germany.

aAddress correspondence to this author at: Institut für Pharmakologie und Toxikologie, Universität Erlangen, Fahrstrasse 17, D-91054 Erlangen, Germany. Fax 49-9131-206119; e-mail pahl{at}pharmakologie.uni-erlangen.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Pathogen recognition receptors such as Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns, lead to the activation of innate immunity. Genetic variations in these receptors may lead to an altered host immune response to pathogens.

Methods: We developed homogeneous fluorescence-based PCR assays as well as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) genotyping assays to detect TLR4 polymorphisms. These assays were compared with restriction fragment length polymorphism (RFLP) analysis. Peripheral blood monocytes from donors with differing genotypes were prepared and exposed to bacterial products in vitro. The abundance of mRNAs of the proinflammatory cytokines interleukin (IL)-1ß, IL-6, and tumor necrosis factor-{alpha} from these monocytes were monitored by real-time reverse transcription-PCR.

Results: By our homogeneous PCR method, the allele frequencies were 5.6% for the TLR4 Asp299Gly and 6.0% for the TLR4 Thr399Ile polymorphism in 116 healthy German Caucasians. Nine incorrect genotype calls were detected in the RFLP analysis and two in the TaqMan genotype analysis. MALDI-TOF-MS allowed clear detection of all TLR4 alleles. Monocytes from donors homozygous for the TLR4 mutant alleles Asp299Gly and Thr399Ile were lipopolysaccharide hyporesponsive and exhibited median effective concentrations (EC50s) approximately fourfold higher than those of monocytes carrying wild-type or heterozygous alleles. In contrast, a TLR2 agonist elicited similar responses in monocytes irrespective of the TLR4 genotype.

Conclusions: Homogeneous fluorescence-based PCR assays provide a specific and sensitive method for high-throughput genotyping of TLR4 mutations. The newly developed PCR and MALDI-TOF-MS assays may be useful to evaluate the presence of TLR4 polymorphisms in patients to predict susceptibility to bacterial infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Sepsis is a contributing factor in more than 100 000 deaths/year in the US, and the annual incidence is nearly 500 000 cases (1). In general, the septic response occurs when microbial pathogens achieve an intolerable burden and cause coordinated activation of innate immune receptors throughout the host. Lipopolysaccharide (LPS; 2 also called endotoxin), a component of the outer membrane of gram-negative bacteria, is the most potent and well-characterized gram-negative bacterial signal molecule for innate immune receptors (2). After forming a complex with the LPS-binding protein, LPS interacts with a glycosylphosphatidylinositol-anchored membrane molecule, CD14 (3)(4). Together with a class of pattern recognition receptors called Toll-like receptors (TLRs) at the surface of monocytes/macrophages and neutrophils, this complex induces second-messenger and signal transduction pathways (5). These signals in turn activate transcription factors, mainly nuclear factor-{kappa}B and cytokines (3)(6). As a result, LPS triggers a wide variety of cellular responses, including the production of cytokines and chemokines, release of arachidonic acid metabolites, and generation of reactive oxygen and nitrogen intermediates, that are responsible for the pathophysiologic reactions (7)(8)(9)(10)(11)(12). Monocytes play an essential role in infection and inflammation by mediating the effects of LPS and producing these mediators.

LPS belongs to a group of pathogen-associated molecular patterns (PAMPs), and Janeway and Medzhitov (5) proposed the existence of pathogen recognition receptors, which recognize PAMPs and lead to the activation of innate immunity. The first pathogen recognition receptors identified was the Drosophila receptor Toll (13). This receptor was shown to be essential for protective immunity to fungal infections in flies (13). In a seminal paper, a human homolog was identified and shown to confer LPS responsiveness to human cells (14). Since then, numerous Toll homologs have been discovered in organisms as disparate as plants, insects, and mammals (15)(16)(17)(18)(19). Ten human Toll-like receptors have been identified, which are characterized by extracellular leucine-rich repeats and an intracellular domain that is homologous to the signaling domain of the interleukin (IL)-1R (14). Two of the mammalian TLRs, TLR2 and TLR4, have been demonstrated to have critical roles in innate immunity. Transfection of these receptors confers responsiveness to a variety of bacterial cell wall components on cells that do not usually respond to them (20)(21)(22)(23). In mice, gene knock-out studies have indicated that TLR4, but not TLR2, is required for LPS responsiveness (24)(25), whereas TLR2 is essential for responses to several gram-positive PAMPs (21)(26)(27)(28)(29). Human TLR2 and TLR4 have been shown to recruit and activate IL-1 receptor-associated kinase in response to a variety of PAMPs, including LPS, leading to activation of nuclear factor-{kappa}B and c-Jun N-terminal kinase and the secretion of cytokines (22)(23)(28)(30)(31)(32)(33).

Recently, it has been shown that TLR4 mutations lead to a LPS-hyporesponsive phenotype in either human primary airway epithelial cells or alveolar macrophages (34). The A896G substitution leads to replacement of a conserved aspartic acid residue with glycine at amino acid 299. This missense mutation (Asp299Gly) in the fourth exon of TLR4 probably alters the structure of the extracellular domain of this receptor. An additional missense mutation was found in the TLR4 gene, which replaces a nonconserved threonine with an isoleucine at amino acid 399 (Thr399Ile) in the extracellular domain of the TLR4 receptor. Asp299Gly and Thr399Ile mutations of TLR4 have been shown to underlie the variability in airway responsiveness to inhaled LPS in humans.

Because of the potential usefulness of the TLR4 polymorphisms in risk stratification of patients vulnerable to endotoxin-induced disease (e.g., gram-negative sepsis), we developed single-nucleotide polymorphism (SNP) detection methods for genotyping these polymorphisms. These new methods were compared with the restriction fragment length polymorphism (RFLP) analysis with regard to sensitivity, specificity, time, throughput, and costs. In addition, we used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for genotyping of TLR4 polymorphisms. Finally, we show the influence of these polymorphisms on the response of human monocytes to microbial stimuli.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
materials
RPMI 1640 was obtained from Life Technologies. Oligonucleotides were synthesized by MWG and Applied Biosystems. Histopaque 1077 was purchased from Sigma. LPS was extracted from Salmonella abortus equi S-form and was phenol-purified (purchased from Bioclot).

genotyping by TaqMan PCR
Genomic DNA was extracted from 200 µL of peripheral blood anticoagulated with EDTA with the QIAamp DNA Blood Mini Kit (QIAGEN) according to the manufacturer’s instructions. After extraction, the DNA concentration was measured photometrically and the DNA was diluted to a concentration of 5 ng/µL. RFLP analysis was performed as described (35). Primer and hybridization probes were designed with Primer Express Ver. 1.5 (Applied Biosystems; Table 1 ). The size of the PCR product for the Asp299Gly polymorphism was 187 bp, and the size of the product for Thr399Ile was 178 bp.


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  		Table 1. Primers and probes for allelic discrimination and quantitative real-time RT-PCR.

Two oligonucleotide probes for each SNP were synthesized. The oligonucleotide corresponding to the wild type was labeled with the fluorescent dye 6-carboxyfluorescein (FAM), and the oligonucleotide corresponding to the mutation was labeled with VICTM at the 5' end. The 3' end of the probe carried a dark quencher that suppressed the fluorescence of the reporter dyes. During PCR, fluorescence developed when the oligonucleotide hybridized to perfectly matching DNA and the exonuclease activity of Taq polymerase separated the quencher from the reporter dye. After PCR, the fluorescence yield for the two different dyes was measured and presented in a two-dimensional graph.

Amplification was performed in a final volume of 5 µL containing 0.8 µL of DNA solution at a concentration of 5 ng/µL, 0.1 µL of each primer (100 pmol/µL), 0.1 µL of each probe (100 pmol/µL), 2.5 µL of TaqMan Universal PCR Master Mix (Perkin-Elmer), and 1.3 µL of distilled water. In every assay, controls for the wild type and mutations were included. Reaction mixtures were loaded into 384-well plates and placed in an ABI Prism Sequence Detector 7900 (Applied Biosystems). The PCR conditions were as follows: initial denaturation at 95 °C for 10 min, followed by 45 cycles of denaturation (95 °C for 15 s), annealing, and extension in one step (60 °C for 60 s).

genotyping by maldi-tof-ms
PCR products were generated as described for TaqMan PCR. PCR fragments were purified with magnetic beads (genopure dsTM) as specified by the supplier (Bruker Daltonik GmbH). Purified, dried PCR products were used for the primer extension reaction after addition of extension mixture (10 µL) containing 12 pmol of extension primer (TLR4 Asp299Gly SNP: reverse, 5'-TTA AAT AAG TCA ATA ATA-3'; TLR4 Thr399Ile SNP: forward, 5'-AAA GTG ATT TTG GGA CAA-3'; TLR2 Arg753Gln SNP: reverse, 5'-TGG TGT TCA TTA TCT TC-3'; MWG Biotech), 1–2 U of Thermosequenase, 2 nmol of dTTP, and 2 nmol of dideoxy-CTP (ddCTP) in 1x Thermosequenase reaction buffer (Pharmacia). Extension reactions were performed at 96 °C for 2 min; 40 cycles of 94 °C for 30 s and 39 °C for 30 s; and finally 72 °C for 3 min. Primer extension products were purified with magnetic beads (genopure oligoTM) as described by the supplier (Bruker Daltonik). MALDI-TOF-MS analysis was performed as described previously (36). MALDI-TOF-MS-based genotyping results were confirmed by independent sequence analysis.

isolation of monocytes and in vitro stimulation
This study was conducted according to the International Declarations of Helsinki and Tokyo. Peripheral blood monocytic cells from donors (age, 23–37 years) of different genotypes were isolated by density gradient centrifugation over Histopaque 1077, washed twice in Hanks buffer, and resuspended in RPMI 1640. Cells were resuspended at 106 cells/mL and incubated in 200-µL volumes in 96-well tissue culture plates. Monocytes were isolated by adherence for 2 h in a humidified 5% CO2 incubator at 37 °C; nonadherent cells were removed by changing the medium. Monocytes were stimulated with different concentrations of LPS or the synthetic bacterial lipoprotein Pam3CysSerLys4 for 4 h.

rna isolation and quantitative reverse transcription-pcr
RNA was prepared from frozen lysates by use of RNeasy 96 (QIAGEN), and reverse transcription-PCR (RT-PCR) was performed with a QIAGEN OneStep RT-PCR Kit. Cytokines expression was determined in relation to ß-actin by real-time PCR using fluorogenic TaqMan assays on an ABI Prism 7900. Primers and probes are listed in Table 1Up . The quantity of mRNA was calculated by use of the {Delta}Ct method. Briefly, the fluorescence signal threshold was defined such that each PCR reaction was in the exponential phase. The Ct value was the cycle at which the fluorescence signal of the reaction exceeded this value. Ct values were proportional to the starting copy number. Ct values of cytokines were normalized to the Ct value of a housekeeping gene (ß-actin) with the same RNA probe. Results are presented as 2{Delta}Ct ({Delta}Ct = Ctß-actin -Ctcytokine).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
genotyping of tlr4 asp299gly and tlr4 thr399ile by TaqMan PCR
We developed a homogeneous PCR method based on minor-groove-binding DNA probes. This assay was completed in 100 min. Among 116 healthy German Caucasians tested, 11 were heterozygous for Asp299Gly and Thr399Ile, 1 was heterozygous only for Thr399Ile, and 1 was homozygous for both SNPs. This represents allele frequencies of 5.6% for Asp299Gly and 6.0% for Thr399Ile and a cosegregation of mutant alleles of 92.9%.

comparison of genotyping methods
For 105 donors, we compared our proposed new method with results of the usual RFLP analysis. Identical genotyping results were obtained for 99 samples (94.3%) for Asp299Gly and 100 samples (95.2%) for Thr399Ile. Genotype differences were resolved by direct sequencing of PCR products. Although all calls were correct for TaqMan genotyping of Asp299Gly, RFLP analysis produced six incorrect calls for this SNP. Five of these errors were false positives, and one was false negative (Table 2 ). With RFLP analysis, three genotype calls for Thr399Ile were incorrect; two of these errors were false positives, and one was false negative. In the TaqMan analysis of this SNP, two genotype calls were false negatives (Table 2 ).


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  		Table 2. Errors in RFLP and TaqMan genotype calls.

genotyping by maldi-tof-ms
The genomic regions of TLR4 that include the polymorphic nucleotides were amplified from genomic DNA by PCR, and purified products were subjected to allele-specific primer extension reactions. The primers were designed to anneal to the target DNA directly adjacent to the polymorphic sides. After MALDI-TOF-MS-compatible sample preparation, the extended primers were subjected to MALDI-TOF-MS for analysis of the TLR4 genotypes.

The reverse extension primer for genotyping the Asp299Gly SNP had a predicted molecular mass of 5515 Da. In case of the A allele, a dTTP was incorporated, whereas the following ddCTP was expected to terminate the primer extension reaction, yielding an extended primer of 6092 Da. When the allelic A-to-G exchange was present, the primer was terminated directly by a ddCTP, producing a mass of 5788 Da. Fig. 1A depicts the MALDI-TOF-MS spectra of the three possible TLR4 genotypes. For analysis of the Thr399Ile SNP by MALDI-TOF-MS, the forward extension primer (5588 Da) was extended by dTTP and terminated by ddCTP when the T allele was present (6165 Da), whereas the primer was directly terminated by ddCTP when the C allele (5861 Da) was present. Fig. 1B shows the spectra for these MALDI-TOF-MS TLR4 genotyping results. All MALDI-TOF-MS-based genotyping results were confirmed by conventional DNA sequence analysis.



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  		Figure 1. Genotyping of TLR4 alleles by MALDI-TOF-MS.

After PCR and purification of the amplified DNA fragments, an allele-specific primer extension reaction was performed. Purified samples were subjected to MALDI-TOF-MS analysis to determine different genotypes. (A), TLR4 Asp299Gly. MALDI-TOF-MS spectra of the homozygous A/A (Sample 1), homozygous G/G (Sample 2), and heterozygous A/G (Sample 3) genotypes are shown. (B), TLR4 Thr399Ile. MALDI-TOF-MS spectra of the homozygous C/C (Sample 1), homozygous T/T (Sample 2), and heterozygous T/C (Sample 3) genotypes are shown.

phenotype of tlr4 alleles
The phenotypes of these SNPs have been demonstrated for alveolar cells only (34). We therefore sought to determine the phenotype of the peripheral monocytes. We prepared monocytes from donors with different TLR4 genotypes and exposed them in vitro to different doses of LPS or Pam3CysSerLys4, a TLR2 agonist. In addition, all donors were genotyped for the TLR2 Arg753Gln polymorphism (37). All donors carried TLR2 wild-type alleles only. Depending on the TLR4 genotype, we observed different responses to LPS stimulation (Fig. 2A ). Monocytes homozygous for the Asp299Gly mutation showed strongly reduced response to LPS as measured by tumor necrosis factor-{alpha} (TNF{alpha}) production. Monocytes from wild types and heterozygotes showed similar responses to LPS. When we stimulated the cells with the TLR2 agonist Pam3CysSerLys4, no differences between the TLR4 genotypes were observed (Fig. 2B ). These results were confirmed by calculating the median effective concentrations (EC50s) for LPS and Pam3CysSerLys4 for the different genotypes and for TNF{alpha}, IL-1ß, and IL-6 (Table 3 ). The EC50s of monocytes homozygous for the TLR4 mutation were approximately fourfold higher for each cytokine compared with the EC50s for monocytes from patients with the heterozygous or wild-type genotype. No significant differences in the EC50s for all three cytokines could be observed for the TLR2 agonist.



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  		Figure 2. Dose response of monocytes from donors of different genotypes to TLR agonists.

Human primary monocytes from different donors were stimulated with different LPS (A) or Pam3CysSerLys4 (B) concentrations. After 4 h, cells were lysed and RNA was prepared. TNF{alpha} mRNA concentrations were determined by real-time RT-PCR. Concentrations were normalized to ß-actin, and unstimulated cells were set to 1. Each point and bar represents the mean ± SE of three different donors. For mut/mut ({blacktriangleup}), only one donor was available. This experiment is representative of three different experiments. wt, wild type;mut, mutant.


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  		Table 3. Effect of Asp299Gly on EC50s for TLR4 and TLR2 agonists on monocytes.1


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The discovery of the family of TLRs as pattern recognition receptors for PAMPs was a milestone in the understanding of innate immunity. Their function in the first line of defense against pathogens makes TLRs likely candidates as the cause of individual variations in resistance to microbial infection. Our study demonstrates that monocytes from donors with mutant alleles of the LPS receptor TLR4 are hyporesponsive to LPS but not to TLR2 agonists. The establishment of reliable assays suitable for high-throughput genotyping makes it possible to study the role of TLR4 mutations in various disease settings.

Although RFLP analysis has been widely used for TLR4 genotyping, this method is time-consuming and requires multiple manual steps. Recently, minor-groove- binding DNA probes have been shown to possess superior sequence specificity, making them ideal candidates for detecting single-base mismatches (38). Using homogeneous PCR with TaqMan probes, we were able to establish a rapid assay to genotype known TLR4 mutations in a high-throughput manner. The observed allele frequencies in our study population were in good agreement with allele frequencies found in other populations. Allele frequencies of 7.9% and 6.6% were found in a population from Iowa, and a frequency of 3.3% was reported for the parental chromosomes of the Centre d‘Etude du Polymorphisme Humain (34). We compared our assay with a published RFLP assay (35). For the Asp299Gly SNP, our TaqMan assay produced no incorrect genotype calls. In contrast, RFLP analysis produced six incorrect genotype calls. For the Thr399Ile SNP, RFLP analysis produced three incorrect genotype calls, and the TaqMan assay produced two incorrect genotype calls. Thus, the error rate is much lower for the TaqMan assay. Our newly developed TaqMan assays were more reliable and faster as the standard RFLP assay.

MALDI-TOF-MS represents a key technology to answer a variety of proteomic and genomic questions [reviewed in Ref. (39)]. Because of its high molecular resolution and high accuracy of mass determination, MALDI-TOF-MS-based genotyping of SNPs and deletions offers a promising alternative to conventional genotyping methods (36)(40)(41). In contrast to electrophoretic and chromatographic methods, MALDI-TOF-MS determines the exact molecular mass of oligonucleotides, which represents an inherent physical property independent of further analytical variables. In addition to RFLP and TaqMan, the two SNPs in the TLR4 gene were analyzed in this report by MALDI-TOF-MS and confirmed by DNA sequence analysis. Especially in heterozygous situations, the high molecular resolution of MALDI-TOF-MS permitted a clear determination of the genotypes. As shown for thrombotic risk alleles, the high sensitivity of MALDI-TOF-MS ensures the detection of heterozygous genotypes in the presence of a 10-fold excess of homozygous genomic DNA, offering the potential for DNA pooling (36). Moreover, Ross et al. (42) demonstrated a limit of 2% for detecting a heterozygous DNA in the presence of an excess of homozygous DNA and a limit for quantification of 5–10%. In addition to the method-inherent advantages described above, MALDI-TOF-MS genotyping possesses the potential for high throughput because it is compatible with automated sample preparation, data collection, and post-data processing and therefore can serve as an analytical platform technology (39)(43).

The LPS hyporesponsiveness of alveolar epithelial cells and alveolar macrophages from donors carrying TLR4 mutations has been shown (34). We extended this finding to peripheral monocytes by showing that monocytes from a donor carrying two mutant TLR4 alleles have fourfold higher EC50s for LPS stimulation of proinflammatory cytokines. In contrast, EC50s for a TLR2 agonist were comparable regardless of the TLR4 genotype. To exclude other genetic factors for the observed differences, we also genotyped donors for TLR2 mutations. The only reported SNP leading to an amino acid substitution in the TLR2 coding region is Arg753Gln (37). All donors carried only wild-type TLR2 alleles with regard to this SNP. The Arg677Trp reported by Kang and Chae (44) was not found in their control group or by an extensive TLR2 mutation screening reported on the internet (45). In addition, this screening revealed no other frequent SNP in the TLR2 coding region. Furthermore, we genotyped donors for the CD14 (-159C/T) polymorphism (C. Schmitt and A. Pahl, submitted for publication). Stratifying donors by these data could not explain our observed EC50 difference because CD14 alleles were evenly distributed among the different TLR4 genotype cohorts. In line with this, no association of the CD14 (-159C/T) polymorphism with TNF{alpha} production has been found (46). Finally, the age of the donors did not differ significantly among our cohorts. Altogether, other known possible genetic factors explaining the different phenotype of monocytes dependent on the TLR4 genotype can be excluded. However, other unknown genetic factors leading to variations in cytokine expression cannot be excluded, e.g., after stimulation with the TLR2 agonist, monocytes with the heterozygous TLR4 alleles produce twice as much TNF{alpha} as monocytes with the homozygous wild-type or mutated TLR4 alleles. This may be caused by TNF{alpha}promoter polymorphisms (47)(48). Because we found similar EC50 differences for all three different cytokines, a relevant difference among monocytes of different TLR4 genotypes are evident. This functional phenotype demonstrates the importance of determination of the described TLR4 mutations.

In conclusion, our results provide evidence that a sequence polymorphism in TLR4 is associated with an endotoxin-hyporesponsive phenotype in human peripheral monocytes. We developed rapid and accurate assays suitable for rapid genotyping in the clinic. A preliminary study detected an association between TLR4 mutations and graft-vs-host disease (49). Because of the observed phenotypes in human monocytes, these mutations may be of diagnostic value for the response of the human immune system to microbial pathogens. Currently, we are investigating the influence of this polymorphism in the outcome of infectious diseases such as sepsis.


   Acknowledgments
 
This work was supported by the Bundesministerium für Bildung und Forschung (BMBF), the Deutsche Forschungsgemeinschaftl (DFG), and the German-Israeli Foundation for Scientific Research and Development (GIF). We gratefully acknowledge the skillful technical assistance of Claudia Sass. We thank Bruker Daltonik GmbH (Bremen, Germany) for providing the Biflex III MALDI-TOF mass spectrometer. We are grateful to Dr. Markus Kostrzewa (Bruker Saxonia Analytik GmbH, Leipzig, Germany) for helpful discussions and thank Eva Lorenz for providing control DNA samples.


   Footnotes
 
1 Both authors contributed equally.

2 Nonstandard abbreviations: LPS, lipopolysaccharide; TLR, Toll-like receptor; PAMP, pathogen-associated molecular pattern; IL, interleukin; SNP, single-nucleotide polymorphism; RFLP, restriction fragment length polymorphism; MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; FAM, 6-carboxyfluorescein; ddCTP, dideoxy-CTP; RT-PCR, reverse transcription-PCR; TNF{alpha}, tumor necrosis factor-{alpha}; and EC50, median effective concentration.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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