Pattern recognition receptors (PRRs) are primary key sensors of invading pathogens. It has been demonstrated that PRRs confer specificity to the innate immune response through their extracellular domain to recognize different pathogen-associated molecular patterns of microbes, such as CD14, Toll-like receptor 4 (TLR4), myeloid differentiation 2 (MD-2) responsible for the recognition of bacterial lipopolysaccharide (1), TLR5 for bacterial flagellin (2), and TLR9 for bacterial DNA (3). Toll-like receptor 2, however, has been shown to mediate the innate immune response to pathogen-associated molecular patterns from a large variety of pathogens, including gram-positive or gram-negative bacteria, mycobacteria, fungi, viruses, and parasites (4). Growing evidence has revealed that overexpression of TLR2 could confer responsiveness to various kinds of bacterial components, such as lipoprotein, peptidoglycan, lipoteichoic acid, and even lipopolysaccharide (4, 5). Toll-like receptor 2-deficient mice showed impaired responsiveness to invading pathogens (6). Therefore, TLR2 has been recognized as one of the key PRRs and plays a pivotal role in initiating the innate response to microbial pathogens.
Clinical observations have revealed that the susceptibility to microbial pathogens varies between individuals (7). Unequivocal evidence shows that genetic factors may be important determinants for the interindividual differences in susceptibility to infection (7, 8). The essential role of PRRs in the initiation of immune responses makes them interesting candidates for genetic analysis. Our results, and those of others, have indicated that genetic polymorphisms within some PRR genes, such as CD14, TLR4, MD-2, TLR5, and TLR9 could significantly influence the expression of inflammatory cytokines and are well associated with the susceptibility to, and outcome from, sepsis in critically ill patients (9-12). In addition, the genetic variations of PRRs might affect Chlamydia pneumoniae growth in human macrophages in vitro (13). Therefore, some genetic polymorphisms of the PRR genes have been considered to be used as relevant risk estimates for organ dysfunction and sepsis in critically ill patients.
Although a total of 20 single-nucleotide polymorphisms (SNPs) have been identified within the entire TLR2 gene as shown by HapMap database (www.hapmap.org), two SNPs (rs5743704 and rs5743708) situated in the exon 3 of the TLR2 gene are the polymorphisms most often described (14-22). The two polymorphisms could lead to amino acid replacement at positions 677 and 753, respectively, and have been shown to affect transmembrane signaling of the TLR2 (14) and to be well associated with susceptibility to microbial infections, such as staphylococcal infections (15), tuberculosis (16), gram-negative sepsis (17), and leprosy (18). Moreover, the Arg677Trp and Arg753Gln polymorphisms are associated with the risk of noninfectious inflammatory diseases, such as acute reactive arthritis (19) and ischemic heart disease (20). Despite the previously mentioned findings, a growing body of literature shows numerous inconsistencies concerning the clinical relevance of these two polymorphisms and their effect on TLR2-mediated cellular activation (21, 22). Recently, there are a few reports about other polymorphisms (rs3804100, +596, +2408, and the GT repeat microsatellite polymorphism in intron 2) in the TLR2 gene in relation to disease susceptibility (23, 24). Taken together, only a few TLR2 gene polymorphisms have been addressed in each of previous studies. Little is known about the global biosignificance of the genetic variants within the whole gene.
Trauma is a major public health problem worldwide, ranking as the leading cause of death in people younger than 45 years (25). One of the most serious complications of major trauma is the sequential dysfunction of vital organs, which in majority of cases is associated with severe sepsis. Toll-like receptor 2 expression is upregulated in trauma (26) and might be a mechanism for that injury that primes the innate immune system (27). Toll-like receptor 2 deficiency could reduce the expression of cytokines and chemokines and recruitment of T cells after trauma (6). Therefore, TLR2 signaling has been considered to play a critical role in orchestrating the innate immune response and the development of sepsis and subsequent organ dysfunction after trauma. To our knowledge, little is known about the clinical relevance of TLR2 polymorphisms in trauma patients.
In the present study, we identified haplotype tag SNPs (htSNPs) within the entire TLR2 gene in an attempt to comprehensively assess common genetic variations within and around the TLR2 gene and assess them in relation to development of sepsis and multiple organ dysfunction (MOD) in patients with major trauma.
MATERIALS AND METHODS
Study design and data collection
A total of 410 unrelated patients with major trauma were recruited in this study. All of the participants were Han Chinese and lived in the Chongqing district. The trauma patients were admitted to the Department of Trauma Surgery in the Daping Hospital and the Chongqing Emergency Medical Center between January 1, 2005, and January 5, 2009. They were enrolled in the study if they met the following criteria: (1) aged 18 to 65 years, (2) expected Injury Severity Score (ISS) greater than 16 combined with the presence of at least one life-threatening injury and at least one additional severe injury in another part of the body, and (3) probability of survival greater than 48 h. Patients were not eligible if they had penetrating injuries or preexisting cardiovascular, respiratory, renal, hepatic, hematologic, or immunologic diseases. The ISS was performed according to the abbreviated injury scale by independent evaluators (The Abbreviated Injury Scale: 2005 revision. Association for the Advancement of Automotive Medicine, Barrington. Ill, 2005 [http://www.AAAM.org]). All patients requiring surgical intervention received standard surgical care and postoperative intensive care unit treatment. The protocol for this study was approved by the Ethical and Protocol Review Committee of the Third Military Medical University, and informed consent was obtained from the subjects or the patient's next of kin. Patient confidentiality was preserved according to the guidelines for studies of human subjects.
Selection of haplotype tag SNPs
The full sequence of the human TLR2 gene (accession no. NC_00064) observed in the current study included 10 kb upstream of the transcription start site, all exons and introns, and 4 kb downstream of the stop codon (35.872 kb total), which was pinpointed to chromosome 4, position 154814890-154850761 (data retrieved from GenBank in the Web site of NCBI).
Genetic variation data for the entire TLR2 gene was obtained from the HapMap project for 45 Chinese Han Beijing (CHB) population (www.hapmap.org). From this database, a total of 8 SNPs were identified within the entire TLR2 gene (Table 1). Six SNPs out of them show a minor allele frequency (MAF) more than 0.05. To select representative tag SNPs from the 6 SNPs with MAF of greater than 0.05 within the TLR2 gene, haplotype blocks were constructed throughout the whole TLR2 gene, using Haploview, version 4.0 (Broad Institute of MIT and Harvard, Cambridge, Mass), a software package that provides computation of linkage disequilibrium (LD) statistics and population haplotype patterns from genotype data. Haplotype blocks represent regions with little evidence of historical recombination between common SNPs. The history of recombination between a pair of SNPs can be estimated with the use of the normalized measure of allelic association D′ (value of D prime between the two loci). The criteria for SNPs to construct a haplotype block is that all SNPs in one region must be in strong LD with D′ greater than 0.98 for the upper 95% confidence bound and greater than 0.7 for the lower bound (28).
A maximally informative htSNP was then selected from each block using software Tagger program (http://www.broad.mit.edu/mpg/haploview). This algorithm selects a subset of variants that captures all known common genetic variations in a gene based on an LD threshold of r2 = 0.8. The inverse of r2 represents the ratio of sample size needed to detect an indirect association with an unassayed SNP to direct association at the same power.
Genotyping of htSNPs
Blood specimens were collected in tripotassium EDTA sterile tubes from trauma patients immediately after admission to avoid the effect of blood transfusion. The preparation of genomic DNA was done as previously described (10). The htSNPs were detected by the polymerase chain reaction-restriction fragment length polymorphism method. The primers for amplification were derived from published genomic sequences (NCBI accession no. NC_000004; Table 2). The PCR conditions were run at 94°C for 5 min followed by 30 cycles of 30 s at 94°C, 30 s at given annealing temperature, 45 s at 72°C, and finally, 72°C for 8 min. After digestion with specific restriction endonucleases (New England Biolabs, Beverly, Mass), the SNPs were then genotyped by fragment size obtained after agarose gel electrophoresis, which were further confirmed by DNA sequencing of the TLR2 gene with 10 random samples (Takara Biotech, Dalian, China). The genotyping was performed in a blinded fashion so that those analyzing the genotype data did not know any other experimental results.
Ex vivo bacterial lipoprotein stimulation of whole blood
A human whole-blood assay was used as described in our previous report (10). In brief, aliquots of whole blood collected from the trauma patients immediately after admission (within 12 h after trauma) were mixed 1:1 with RPMI 1640 culture medium and incubated with 100 ng/mL bacterial lipoprotein (Pam3Cys-Ser-[Lys]4.3HCl; EMC Microcollections, Tuebingen, Germany) in a sample mixer at 37°C for 4 h. After 4 h of incubation, the supernatants were collected after centrifugation and stored at −80°C until used.
Assay of TNF-α, IL-8, and IL-10 production
The supernatant levels of tumor necrosis factor-α (TNF-α), interleukin 8 (IL-8), and IL-10 were assayed by a sandwich enzyme-linked immunosorbent assay according to the manufacturer's instructions (Quantikine, R & D Systems, Minneapolis, Minn).
The patients with major trauma were prospectively monitored after admission by physicians who did not know the genotypes. Sepsis was defined if patients met all of the following criteria: clinical evidence of infection, body temperature greater than 38.5°C or less than 36.5°C, and leukocyte count greater than 10 × 109/L or less than 4 × 109/L. When patients were diagnosed with sepsis, the assessments of respiratory (Po2 /Fio2 ratio), hepatic (serum bilirubin level), renal (serum creatinine level), cardiovascular (pressure-adjusted heart rate), and hematologic (platelet count) functions were made. The organ function was then scored using the method of Marshall et al. (29) and calculated as a single daily value during the intensive care unit stay. Neurological scoring was not performed because every patient was sedated. The MOD scores and sepsis were determined by individuals who did not know the genotypes.
Allele frequencies for each SNP were determined by gene counting. Genotype distribution of each SNP was tested for departure from Hardy-Weinberg equilibrium (HWE) using chi-square analyses. The extent of pairwise LD between polymorphisms was determined by the Haploview (version 4.0) software. The association between polymorphisms and TNF-α, IL-8, or IL-10 levels was determined using one-way analysis of variance. The association between polymorphisms and MOD scores was performed using analysis of covariance testing with age, gender ratio, and injury severity to adjust for possible confounding effects. Three genetic models (allele-dose, dominant, and recessive) were used. For dominant effect, we compared the variant allele carriers (variant homozygous plus heterozygous genotypes) versus noncarriers (wild-type homozygous genotype); whereas for recessive effect, subjects homozygous for the variant allele were compared with heterozygous carriers plus noncarriers (wild-type homozygotes). Allele dose was defined as the number of copies of minor allele in the genotype. We performed linear regression analysis to quantify the allele dose effect. The association of genotypes with sepsis morbidity rate was determined by chi-square analysis. Odds ratios with 95% confidence intervals were calculated by multiple logistic regression analyses to estimate the relative risk of sepsis.
Selection of htSNPs
HapMap database revealed that there are a total of 8 SNPs within the full TLR2 gene identified in Chinese Han Beijing population (www.hapmap.org) (Table 1). Six of them had an MAF of more than 5%. They constructed two haplotype blocks (Fig. 1): block 1, from −5819 to 16,506, included three SNPs (rs13150331, rs1898830, rs11938228); block 2 (19,216-19,969) included two SNPs (rs3804099 and rs3804100). Based on the analysis of haplotypes of SNPs in each block and tagging threshold of r2, rs1898830 and rs3804099 were selected out as htSNPs of block 1 and block 2, respectively. The SNP rs7656411 in 3′-flanking region, although not forming any haplotype block with other SNPs, was still included in this study because of its highly frequent polymorphic site (41.1%). Taken together, a total of three htSNPs (rs1898830, rs3804099, and rs7656411) were selected in this cohort. They were located in the intron 1 (+3013), exon 3 (+19,216), and 3′-flanking (+22,215) regions, respectively, and might represent the genetic variations of the entire TLR2 gene and the possible biological significance of the TLR2 genetic variations.
Allele frequencies and genotype distribution of the three htSNPs among trauma patients
The MAF of the rs1898830 SNP among the 410 trauma patients in this cohort was quite similar to that among the 45 healthy CHBs shown in HapMap data (39.9% vs. 36.7%). The MAF of the rs3804099 SNP was 26.5%, being significantly lower (P = 0.042) than in the CHBs (36.7%). The minor allele of the rs7656411SNP, although its frequency was quite similar (47.4% vs. 41.1%) in our study cohort and CHBs, was opposite, being G in our cohort and T in CHBs. The genotype distributions of the three htSNPs were all in agreement with the HWE (P > 0.05; Table 3), indicating that the allele and genotype frequencies of these htSNPs in the population remain constant-that is, they are in equilibrium from generation to generation.
Association of the three htSNPs with the capacity of peripheral leukocytes to produce cytokines
Supposing that the three htSNPs might be functional, we hypothesized that they may affect the TLR2-mediated activation of peripheral leukocytes. There were no significant differences in age, gender ratio, or ISS scores among patients stratified according to the different genotypes of the three htSNPs (P > 0.05; Table 4). Both rs1898830 and rs3804099 SNPs were shown to be well associated with the capacity of peripheral leukocytes to produce anti-inflammatory and proinflammatory cytokines in response to bacterial lipoprotein stimulation. For the rs1898830, the patients with the variant G allele showed significant increases in the production of IL-10 (P = 0.013 in case of recessive effect, Fig. 2A), IL-8 (P = 0.014 in case of dominant effect, Fig. 2B), and TNF-α (P = 0.007 in case of dominant effect, Fig. 2C). Data from linear regression analysis indicated that the association of the rs1898830 with IL-8 production was significantly allele dose dependent (P = 0.008). For the rs3804099, cytokine production was significantly increased in patients with the variant C allele comparing with those with the wild-type T allele, showing a significant difference in IL-10 (P = 0.002 in case of dominant effect and P = 0.038 in case of recessive effect, Fig. 2A), IL-8 (P = 0.004 in case of dominant effect, Fig. 2B), and TNF-α production (P = 0.005 in case of dominant effect, Fig. 2C). Data from linear regression analysis indicated that the association of this SNP was significant in allele dose dependence with the production of all three cytokines, respectively (IL-10, P = 0.003; IL-8, P = 0.045; TNF-α, P = 0.039). No significant association was observed among patients with different genotypes of the rs7656411 (Fig. 2).
Association of the three htSNPs with development of sepsis and MODS in patients with major trauma
All the patients survived at least 48 h after admission and completed genotyping. Patients were severely injured (mean ISS, 25.8 ± 8.4), mostly young (mean age, 38.4 ± 13.9 years). One hundred fifty-six patients (38.0%) developed sepsis. Two hundred fifty-five (62.2%) patients developed organ dysfunction, among whom 160 (39.0%) had two or more organ dysfunctions. For the rs1898830 and rs7656411, there were no significant differences in sepsis morbidity rate or MOD scores among patients with different genotypes. With respect to the rs3804099, the patients with the C variant allele showed a significantly higher sepsis morbidity rate and MOD scores than those with the T allele (P = 0.004 for sepsis in case of dominant effect and P = 0.027 for MOD scores in case of recessive effect). In addition, the percentage of the patients with MOD scores greater than or equal to 6 was also significantly higher in the patients with the C variant allele than that in those with the T allele (8/32, 25% for the CC genotype; 20/154, 13% for the TC genotype; 25/224, 11.1% for the TT genotype; P = 0.034 in case of recessive effect). Data from multiple logistic regression analyses indicated that the rs3804099 was significantly associated with a higher risk of sepsis (odds ratio, 1.65; 95% confidence interval, 1.139-2.390; P = 0.008) after adjusting for possible confounders, including age, gender ratio, and ISS (Table 4).
Combination effects of the three htSNPs
According to the association with cytokine production (Fig. 2), the alleles of the three htSNPs were classified into hyperresponsive and hyporesponsive ones. The hyporesponsive alleles were rs1898830A, rs3804099T, and rs7656411T, respectively. The collective influences of the hyporesponsive alleles were then analyzed. A total of 378 patients, showing all genotypes for the three polymorphic loci, were divided into three groups based on the number of hyporesponsive haplotype ATT (in an order of rs1898830A, rs3804099T, and rs7656411T). There were no significant differences in age, gender ratio, or ISS scores among the three groups. As we expected, the haplotype ATT was significantly associated with a lower production of IL-10, IL-8, and TNF-α in response to bacterial lipoprotein stimulation. Data from liner regression analyses indicated that the association of the haplotype ATT with the IL-10 and IL-8 production was significantly haplotype dose dependent (P = 0.033, P = 0.001, respectively). Accordingly, the haplotype ATT was significantly associated with a lower sepsis morbidity rate, showing that the sepsis morbidity rate was significantly lower in the patients with two ATT haplotypes when comparing with those with zero ATT haplotype (25% vs. 42.1%, P = 0.036). But there was no significant difference in MOD scores among the three groups (5.4 ± 2.9 for two ATT haplotypes, 4.4 ± 2.2 for one ATT haplotype, and 4.8 ± 2.3 for zero ATT haplotype). In addition, the percentage of the patients with MOD scores greater than or equal to 6 were similar among the three groups (14.6% for the two ATT haplotypes, 12.2% for one ATT haplotype, and 13.5% for the zero ATT haplotype).
The present study investigates the potential clinical relevance of the genetic variation within the entire TLR2 gene by means of htSNPs. Three SNPs are selected as htSNPs to represent the whole genetic variations within the TLR2 gene in Chinese Han population through construction of haplotype blocks. They are rs1898830, rs3804099, and rs7656411, respectively. The TLR2 Arg677Trp and Arg753Gln SNPs, although being most studied among the reported gene polymorphisms in the TLR2 gene, do not exist in CHB (www.hapmap.org) and other Chinese Han populations (30, 31). It suggests the possibility that both SNPs might be at least rare polymorphisms the in Chinese Han population. In fact, the TLR2 Arg677Try SNP is often not found and that the frequency for the TLR2 Arg753Gln SNP is generally less than 10% in Western populations (14-22).
Supposing the functional significance of the three htSNPs, we first investigated the effects of these htSNPs on TLR2-mediated activation of peripheral leukocytes. Toll-like receptor 2 is a PRR mainly responsible for the recognition and activation of gram-positive bacteria (4). To this end, bacterial lipoprotein was used as a stimulus in our study. Among the three htSNPs we selected, the variant alleles of both rs1898830 and rs3804099 polymorphisms are significantly associated with increased production of IL-10, IL-8, and TNF-α by peripheral blood leukocytes in response to bacterial lipoprotein stimulation. The rs1898830, although located in intron 1, is in strong LD with the rs13150331 in the 5′-flanking region in the same haplotype block. The association of the rs1898830 with TLR2-meidated cellular activation might be caused by the effect of the rs13150331 on transcriptional activities of the TLR2 gene promoter. The rs3804099 is located in exon 3 and induces a synonymous variation (Asn199Asn). The association of this SNP with TLR2-induced activation of leukocytes might be caused by other unidentified causal polymorphism, which is in strong LD with the rs3804099. In addition, our results reveal that the wild-type haplotype ATT (in an order of rs1898830A, rs3804099T, and rs7656411T) is significantly associated with the lower production of IL-8, TNF-α, and IL-10 in a haplotype dose-dependant fashion. It suggests that there exists a collective effect among the three htSNPs (Table 5).
Toll-like receptor 2 plays a central role in the development of sepsis and MODS in major trauma patients (6, 7, 26). The three htSNPs of the TLR2 gene were then investigated in relation to the development of sepsis and MODS in patients with major trauma. A case-control study is a common and convenient association study design for finding genetic bases of diseases. However, a major limitation in this approach is the potential for population stratification when inappropriate patient-control matching occurs, such as if using healthy blood donors as control group in this study. To avoid confounding association, we only selected trauma patients and prospectively followed them to determine whether those who had genetic variants had a higher risk of posttraumatic sepsis and MOD. In addition, the patients recruited in this study were consecutively admitted to the hospitals, and the persons who evaluated the MOD scores and sepsis did not know the genotypes of patients. Our results indicate that only rs3804099 SNP is well associated with sepsis morbidity rate and MOD scores in patents with major trauma. The rs1898830, although being associated with TLR2-mediated activation of leukocytes, seems to have no association with the risk of sepsis and MODS after trauma. In addition, the wild-type haplotype ATT reveals a significant association with a lower sepsis morbidity rate, but not with MOD scores. Although the rs3804099 SNP and the ATT haplotype have a statistical significance in relation to the risk of sepsis and even MOD in trauma patients, this association seems less strong when comparing with their effect on cytokine production. Several reasons might be responsible for this inconsistency. One might be caused by polygenetic and multifactorial involvement in the pathogenesis of sepsis and MOD after trauma. With respect to the PRRs on the surface of immune cells, plenty of PRRs, such as CD14, TLR4, TLR9, and MD2, have been demonstrated to participate in the development of posttraumatic complications (9-11). Therefore, the genetic variants within these PRR genes are closely associated with the development of sepsis and MODS in patients with major trauma (9-11). To this end, the genetic variations within the TLR2 gene, although affecting TLR2-mediated cellular responsiveness, might just have a weak effect on the development of posttraumatic complications. In addition, nonpathogenic factors, such as ischemia and hypoxia occurring immediately after trauma, are also responsible for posttraumatic inflammation (32). Furthermore, TLR2, although regulating the activation of immune cells by a wide range of pathogens, is a PRR mainly responsible for gram-positive bacteria (1). Predominant pathogens inducing traumatic sepsis are gram-negative bacteria, although a mixed infection often occurs in trauma patients (33). This might be a reason why the genetic variations within the TLR2 gene are relatively less associated with posttraumatic complications. Third, the values of power for the rs1898830 and rs7656411 SNPs in this study are shown to be 7.7% and 27.4% for sepsis morbidity rate and 39.2% and 28.5% for MOD scores at a significance of 0.05, whereas those for the rs3804099 are 89.1% and 95.7%, respectively. It indicates that this clinical association study might be underpowered to some extent for the rs1898830 and rs7656411 SNPs because of insufficient numbers of patients. Finally, gender might be also be one of the factors influencing susceptibility to sepsis in trauma patients. Increasing evidence has demonstrated that there are gender differences in disease susceptibility, severity, and outcome in a large variety of diseases. Male gender has been shown to be associated with increased risk for sepsis in some studies (34). Our results also reveal that 12 of the 15 sepsis patients with variant homozygous genotype of the rs3804099 and 11 out of the 12 sepsis patients with two ATT haplotypes are male. It suggests that gender might affect susceptibility to sepsis in trauma patients, although there is no significant difference in sepsis morbidity rate between male and female patients, which might be caused by the small number of patients who are complicated with sepsis.
In summary, our study provides evidence of clinical relevance of the rs1898830, rs3804099, and rs7656411 polymorphisms as htSNPs of the entire TLR2 gene. The rs3804099 SNP and haplotype ATT (in the order of rs1898830, rs3804099, and rs7656411) reveal a close relationship with posttraumatic inflammatory response and the development of sepsis in the Chinese Han population. Future studies in a larger patient population might be needed to further validate their clinical relevance.
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TLR2; haplotype tag polymorphisms; trauma; sepsis; multiple organ dysfunction