The Toll-like receptor 4 (TLR4) is essential for initiating the innate response to lipopolysaccharide (LPS) from Gram-negative bacteria by acting as a signal-transducing receptor (1, 2). Through interactions with both CD14 and myeloid differentiation 2, LPS binds to TLR4 and initiates a complex intracellular signaling pathway, resulting in the activation of nuclear transcription factors and the subsequent production of proinflammatory cytokines (3-5).
There is growing evidence indicating that the genetic variation in the TLR4 gene is intimately associated with changes of responsiveness to Gram-negative bacteria or LPS. Studies have demonstrated that the hyporesponsiveness or the nonresponsiveness of C3H/HeJ and C57BL/10ScCr mouse strains to LPS challenge, because of the defect of the TLR4 gene (6-8), causes a decreased ability to clear Klebsiella pneumonia from the lung and a profound increase in mortality when compared with mice having a functional TLR4 receptor (9). In this case, the TLR4 gene (Tlr4) has been recognized as LPS gene Lps, being responsible for the modulation of the bioeffects of LPS (10). In humans, an increasing number of studies provide evidence suggesting that the genetic variation in the TLR4 gene may be associated with susceptibility to infectious and noninfectious diseases (11-23). Although more than 100 single nucleotide polymorphisms (SNPs) have been identified to date in the TLR4 gene (http://www.genecards.org/cgi-bin/carddisp.pl?gene=TLR4&search=TLR4&snp), TLR4/A896G and T1196C are the polymorphisms mostly often described (11-23). These 2 polymorphisms are located in the coding region and cause the substitution of the conserved aspartic acid to glycine at amino acid 299 (Asp299Gly) and the replacement of threonine by isoleucine at amino acid 399 (Thr399Ile), respectively (17). However, conflicting reports indicate that there is no clinical relevance for the TLR4/299/399 SNPs (24-31) and that there is no association of cytokine production with ex vivo LPS stimulation (24, 26). Yet, little is known about the biologic significance of other polymorphisms in the TLR4 gene.
In view of the central role of TLR4 in the LPS transmembrane signaling, we sequenced the whole TLR4 gene (referred to GenBank, Accession No. AF177765) using genomic DNA samples collected from 27 Han Chinese. We identified 14 genetic variants, with 6 in the 5′-flanking, 6 in the introns, and 2 in the 3′ untranslated region (3′ UTR). We did not observe the Asp299Gly and Thr399Ile polymorphic sites in the same samples. Using an in vitro transfection model, we did not find any functional effect of genetic variants in the TLR4 promoter (32).
The 3′ UTR of a gene has been recognized to be responsible for post-transcriptional regulations, including mRNA stability, subcellular targeting, and translation of many transcripts (33, 34). Several studies indicated that genetic variations in 3′ UTR could affect the target gene expression and are associated with the development of diseases (35-39). We identified two SNPs (T10764C and G11367C) in the 3′ UTR of TLR4 gene. Our data indicated that only the G11367C was a common SNP with minor allele frequency of more than 10%. We therefore investigated the functional significance of the TLR4/11367 polymorphism by observing its association with TLR4 expression and leukocyte activation in response to ex vivo LPS stimulation and its direct effect on 3′ UTR post-transcriptional regulation. Meanwhile, the occurrence of the TLR4/896 polymorphism in Chinese Han population was reexamined in a relatively large group in this study.
MATERIALS AND METHODS
A total of 370 healthy volunteers (men, 213; women, 157), with median age of 35 years (range, 20-58 years), were recruited for this study. They are Han Chinese and live in Chongqing district. The protocol for this study was approved by the Ethical and Protocol Review Committee of the Third Military Medical University. An informed consent was obtained from each subject.
The TLR4/G11367C polymorphism was genotyped using single-tube bidirectional allele-specific amplification (40). Two specific primer pairs were designed: forward outer primer (F1) 5′-GTC ATT CCA AAG TTA TTG CCT ACT AAG-3′, reverse outer primer (R1) 5′-GTG ATA TCT CAT TGT GGT TTT TAT TTT C-3′,forward inner primer (F1′) 5′-TAAACCCGGGGTGACCTCATGAAATCAG-3′, and reverse inner primer (R1′) 5′-TCTGAAAAAAATAAACTTCTGCTGC TAG-3′. The F1′ and the R1 were used to amplify a 398-base pair (bp) DNA fragment containing G allele; the F1 and the R1′ were used to amplify a 257-bp DNA fragment containing C allele; the F1 and the R1 were used to amplify a 599-bp DNA fragment as internal control. Polymerase chain reaction (PCR) was performed using 40-ng template DNA, 0.4-μM F1, 0.4-μM R1, 0.2-μM F1′, 0.8-μM R1′, 3-mM MgCl2, 200-μM dNTP, and 0.5 U Hotstar Taq polymerase (Qiagen, Hilden, Germany). The PCR conditions were as follows: 2 min at a temperature of 95°C, followed by 30 cycles of 40-s duration at 95°C, 1 min at 68°C, and 40 s at 72°C. For touchdown reactions, the annealing temperature was 70°C for the first cycle, decreasing by 0.5°C per cycle until the annealing temperature reached 60°C, then continuing at 60°C in the annealing step of the remaining cycles. The PCR products were electrophoresed in a 2% agarose gel with subsequent staining by means of ethidium bromide. The genotype was determined according to electrophoresis bands, which were further confirmed by means of DNA sequencing with 10 random samples (Takara Biotech, Dalian, China).
In view of the reported functional significance of the TLR4 Asp299Gly/Thr399Ile polymorphisms and their tight linkage disequilibrium (17), the TLR4/Asp299Gly (896) polymorphism was redetermined using PCR-restriction fragment length polymorphism method. The PCR primers were 5′-ATT TAG AAA TGA AGG AAA CTT GGA AA-3′ and 5′-CCA AGA AGT TTG AAC TCA TGG TAA TA-3′. The PCR conditions were as follows: temperature of 94°C for 5 min, followed by 30 cycles at a temperature of 94°C for 30 s, 56°C for 40 s, and 72°C for 40 s. The PCR products were digested with BccI (New England Biolabs, Beverly, Mass) at 37°C for 2 h. The genotype was then determined on the basis of fragment size after agarose gel electrophoresis.
Ex vivo stimulation of whole blood with LPS
A human whole-blood assay was used as described by Majetschak et al. (41). In brief, aliquots of whole blood collected from the healthy volunteers were mixed at a ratio of 1:1 using Roswell Park Memorial Institute (RPMI) 1640 culture medium and were incubated with 100 ng/mL LPS (Escherichia coli O26:B6; Difco Laboratories, Detroit, Mich) in a sample mixer at 37°C for 4 h.
Flow cytometric analysis
The expression of TLR4 on the surface of peripheral blood mononuclear cells was measured by fluorescence-activated cell sorter. Briefly, LPS-activated peripheral blood mononuclear cell samples at a density of 1 × 106 cells/mL were stained for 30 min on ice with saturating amounts of fluorescein isothiocyanate-conjugated antihuman TLR4 monoclonal antibody (SeroTe, Oxford, UK). The cells were then washed and incubated with phycoerythrin-labeled antihuman CD14 monoclonal antibody (SeroTe) at 4°C in the dark. The cells were then fixed with 1% paraformaldehyde in phosphate-buffered saline solution at 4°C in the dark. After washing, the proportion of TLR4-positive cells among the CD14-positive cells was determined using a flow cytometer (Coulter Epics XL; Beckman-Coulter, Marseille, France) equipped with an argon laser at a wavelength of 488 nm. With the use of XL2 software (Beckman-Coulter), data was acquired in a minimum of 8,000 peripheral blood monocytes identified by forward scatter and positive CD14 labeling. The TLR4 expression on monocytes was presented as mean fluorescence intensity (MFI). Because white blood cells nonspecifically bind immunoglobulins through Fc receptors, the isotype-matched murine immunoglobulins (fluorescein isothiocyanate-conjugated monoclonal immunoglobulin G2b and phycoerythrin-conjugated monoclonal immunoglobulin G2a) with no reactivity against the antigen of interest were used for threshold adjustment of negative fluorescence in all cases. The observed nonrelevant signal using isotype control was considered to represent background staining and had to be eliminated.
Tumor necrosis factor α assay
The supernatants were separated from the whole blood after ex vivo LPS stimulation. The tumor necrosis factor α (TNF-α) levels in the supernatants were determined by using enzyme-linked immunosorbent assay according to the manufacturer's instructions (R&D System, Minneapolis, MN).
The full-length 3′ UTR of the TLR4 gene with 1126 base pairs from position 10361 to 11487 (42, 43), created by PCR amplification of genomic DNA, was obtained from a subject homozygous for G allele at position 11367. The primers were 5′TCT AGA AGA GGA AAA ATA AAA ACC TCC TGA GGC-3′ (forward), 5′TCT AGA TAG TTC TCC TGG CAG GA-3′ (reverse). To facilitate the insertion of the 3′ UTR of TLR4 gene into the 3′ UTR sequence of the luciferase gene in the pGL3-promoter plasmid (Promega, Madison, Wis), an Xba I site (as indicated by underline in the primers) was introduced to the 5′ end of the forward and reverse primers. After digestion with Xba I, the 3′ UTR fragment was directly inserted into the Xba I site of the pGL-3 promoter vector (pGL3-TLR4/3′ UTR 11367G plasmid). The pGL3-TLR4/3′ UTR 11367C plasmid was then created using QuikChange site-directed mutagenesis kit (Takara Biotech). The primers were 5′-TCT AGA CCT CAT GAA ATG ACT TGC AGC AGA AG-3′ (forward) and 5′-TCT AGA TCA CCC CGG CTT TAT CAG CCC ATA TG-3′ (reverse), in which the underlined and the bold italic letters refer to the Xba I sites and the altered nucleotide, respectively. The plasmid constructs were verified by means of direct sequencing analysis (Takara Biotech).
Human embryonic kidney (HEK) 293 cells were cultured in RPMI 1640 medium (HyClone, Logan, Utah) containing 10% fetal calf serum, glutamine, penicillin-streptomycin mixture, and sodium bicarbonate at a temperature of 37°C in a humidified 5% of CO2 air atmosphere. After incubation for 24 h, the cultured cells were harvested and cotransfected with the constructed vector and Renilla luciferase reporter plasmid pRL-CMV using Lipofectamine 2000 system (Invitrogen, Carlsbad, Calif). The transfected cells were maintained in growth medium for 24 h. At the end of each experiment, the luciferase activity of the transfected cells was measured using the Luciferase assay system (Promega), in accordance with the supplier's protocol, on a Luminoskan Ascent luminometer (Thermo LabSystems, Helsinki, Finland). The transfection efficiency was normalized by measuring the luciferase activity of the control plasmid pRL-CMV. The luminescence experiments were performed in triplicate with each transfection. Three independent transfections were performed for each constructed vector.
Luciferase mRNA expression
The total RNA was isolated from the transfected cells using TRIzol reagent (Invitrogen), in accordance with the manufacturer's instructions. After the cDNA was prepared using the avian myeloblastosis virus reverse transcriptase (Takara Biotech) and the oligo-dT primer, it was purified using a PCR purification kit (Takara Biotech) and then dissolved in Tris-EDTA buffer (Tris, 10 mM; EDTA, 1 mM; pH, 8.0) at 10 μg/mL. Glyseraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the reference gene; the primer sequences for the GAPDH gene were CGG GAA ACT GTG GCG TGA T (F) and CAA AGG TGG AGG AGT GGG T (R). The primers for the luciferase gene were CTC TGC CTC ATA GAA CTG CC (F) and AAT GTA GCC ATC CAT CCT TGT (R). Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed using a SYBR ExScript RT-PCR kit (Perfect Real Time) (Takara Biotech) on quantitative PCR (iCycler optical module 584BR; Bio-Rad, Hercules, CA). The PCR cycling conditions included the denaturation at a temperature of 95°C for 3 min and 45 cycles of 15-s duration, 30 s at 60 °C, and 30 s at 72°C. The relative expression intensity of luciferase mRNA was normalized as the ratio of luciferase to GAPDH.
The genotype distribution was tested for departure from Hardy-Weinberg equilibrium using chi-square analysis. The association between the G11367C polymorphism and the TLR4 protein expression or plasma TNF-α levels was determined using one-way analysis of variance (ANOVA). Three genetic models, such as allele dose model, dominant model, or recessive model, were used. For dominant effect, we compared the C variant allele carriers (heterozygous and homozygous genotypes) with the noncarriers (G allele homozygous genotype), whereas for recessive effect, subjects homozygous for the C allele were compared with heterozygous carriers and noncarriers. Allele dose was defined as the number of copies of C allele in the genotype. We performed linear regression analysis to quantify the allele dose effect. The luciferase activities and their mRNA expression were compared using independent-sample t test. The results were considered significant at P < 0.05. All statistical analyses were performed using SPSS version 11.0.
Allele frequencies and genotype distribution
In this study, we reexamined the allele frequency and the genotype distribution of the TLR4/11367 polymorphism in a relatively large group (n = 370 Han Chinese). Our data show that the frequency of G or C allele was 85.3% and 14.7%, respectively. The genotype distribution was in agreement with the Hardy-Weinberg equilibrium (P = 0.963). We did not observe genetic variation at position 896 in the same population.
Association of the TLR4/11367 polymorphism with TLR4 protein expression
To examine whether there is a functional linkage between TLR4/11367 polymorphism and TLR4 protein expression, we examined the expression of TLR4 on the surface of peripheral leukocytes to ex vivo LPS stimulation in subjects with different genotypes. The TLR4 expression was shown to be well associated with the 11367 polymorphism, showing a significant difference in case of recessive (P = 0.003) and allele dose-dependent (P = 0.008) effects (genotype MFI: GG, 15.66 ± 3.93; GC, 11.99 ± 3.67; CC, 10.01 ± 2.95; Fig. 1).
Association of TLR4/11367 polymorphism with TNF-α production
In addition to TLR4 expression, the LPS-induced activation of peripheral leukocytes was also reduced in subjects with the 11367C allele (Fig. 2). There was a significant difference in plasma TNF-α levels in the case of dominant effect (P = 0.001). The association of TLR4/11367 polymorphism with TNF-α production was significantly allele dose dependent, as indicated by linear regression analysis (P = 0.002, with 32.40 ± 1.87 ng/mL in GG, 15.98 ± 1.08 ng/mL in GC, and 11.25 ± 0.79 ng/mL in CC genotypes).
Effect of TLR4/11367 polymorphism on post-transcriptional regulation
To elucidate the direct effect of the TLR4/11367 polymorphism on the post-transcriptional regulation of 3′ UTR, we prepared two plasmid constructs by inserting full-length sequence of TLR4 3′ UTR into the pGL3-promoter. The constructs contained G or C allele at position 11367 of the TLR4 3′ UTR. The activity and the mRNA expression of luciferases in transfected HEK 293 cells were determined by means of reporter gene assay and real-time quantitative RT-PCR, respectively. Our results showed that the luciferase activity level was significantly lower in cells transfected with construct containing 11367 C allele than those transfected with construct containing 11367 G allele (0.019 ± 0.003 vs. 0.38 ± 0.07; P < 0.001). Luciferase mRNA expression was shown to be 6.3-fold lesser in cells transfected with construct containing 11367 C allele than those transfected with construct containing 11367G allele (8.1 ± 2.7 vs. 50.9 ± 16.9; P < 0.001) (Table 1).
The identification of gene polymorphisms implicated in the susceptibility to common polygenetic and multifactorial diseases is a new and very exciting topic of medical research. In this study, we investigated the functional role of the TLR4/G11367C polymorphism, which is a novel SNP we identified in the 3′ UTR of TLR4 gene in Chinese Han population. A cohort of 370 healthy Han Chinese living in Chongqing district was recruited for this study. The genotyping results are consistent with our previous findings of sequencing (32), confirming that the TLR4/11367 polymorphism is a common SNP in Chinese Han population.
The TLR4/A896G polymorphism, in tight linkage disequilibrium with the T1196C, although not being identified in our previous sequencing study (32), was redetermined using PCR-restriction fragment length polymorphism method in the present study with respect to its positive functionality in many association studies (11-23). Yet, we did not observe this genetic variation in the cohort of 370 Han Chinese. In agreement with our results, Hang et al. (44) and Guo et al. (45) also did not detect both TLR4/896 and 1196 polymorphisms in Chinese Han population in Shanghai and Wuhan districts, respectively. In addition, both TLR4/896 and 1196 polymorphisms have been shown not to exist in Japanese population (46, 47). Our study, together with reports from other authors (44-47), suggests a possibility that both TLR4/896 and 1196 polymorphisms may be rare in Asian populations. In Western populations, the frequencies for the TLR4/896 and 1196 polymorphisms are generally less than 10% (11-23).
To investigate the functional significance of TLR4/G11367C polymorphism, we examined the TLR4 expression on peripheral leukocytes and the activation of these cells in response to ex vivo LPS stimulation in samples from 90 subjects of the 370 healthy volunteers. Our results revealed that the 11367C variant allele was associated with decreased expression of TLR4 protein on the surface of peripheral leukocytes in an allele d ose-dependent manner. There was also an allele dose-dependent association of 11367C variant allele with reduction in LPS-induced activation of peripheral leukocytes, as shown by significantly lower levels of plasma TNF-α in subjects with the 11367C allele. The expression defect of the TLR4 gene has been demonstrated to be responsible for functional hyporesponsiveness or nonresponsiveness to LPS stimulation in both animal and human studies (6-10). Our study provides evidence suggesting that the novel variant of TLR4/G11367C polymorphism may play a functional role in regulating the TLR4 expression and the activation of innate immune cells. Because of limited volume of blood sample we could take from each volunteer, we could not simultaneously perform Western blot analyses to check the TLR4 expression at total protein level. The mechanism mediating the effect of the TLR4/11367 polymorphism on TLR4 protein expression remains to be elucidated.
To exclude the potential effect of other TLR4 polymorphisms in linkage disequilibrium with the TLR4/11367 polymorphism and to determine the possible mechanism for the effect of this polymorphism on the target gene expression, we inserted the TLR4 full-length 3′ UTR, which contained 11367 G or C allele, into the 3′ UTR sequence of the luciferase gene. As expected, the luciferase activity and its mRNA levels are significantly lower in cells transfected with 11367C allele than in cells transfected with 11367G allele. It has been demonstrated that the 3′ UTR of a gene is responsible for post-transcriptional regulations, such as mRNA stability and translation of many transcripts (33, 34). Several studies have shown that genetic variations in 3′ UTR could affect the mRNA stability of a target gene (48-50). Inasmuch as the differences in luciferase activity and the mRNA levels between the cells transfected with 11367C and 11367G alleles, to some extent, reflect the effect of the TLR4/11367 polymorphism on the translation of the TLR4 mRNA and its stability, our data suggest that G→C variation at position 11367 in the 3′ UTR of the TLR4 gene may cause decreased mRNA stability and/or translational repression. Additional experiments performed using transcription inhibitor actinomycin D would provide further evidence to support this possibility. Transcript-specific translational control is generally directed by transacting proteins that bind to structural elements in the 3′ UTR of the target mRNA (51, 52). Thus, another possible mechanism for post-transcriptional suppression induced by the 11367 polymorphism might be caused by the altered affinity of DNA/protein interactions.
The TLR4 is the pivotal signaling receptor for LPS and plays a central role in the innate immune response to infection (1-3). The increased expression of TLR4 protein has been demonstrated to be an important mechanism for the development of inflammatory diseases (53, 54). It has been reported that the 3′ UTR polymorphisms are associated with the development of diseases (35-39). Our study provides evidences supporting the possibility that the TLR4/11367 polymorphism may be associated with a decreased risk of inflammatory diseases in the studied Chinese Han population. Further clinical studies are warranted to confirm the clinical relevance of this polymorphism.
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