INTRODUCTION

Hepatitis B virus (HBV) infection is one of the major causes of chronic liver inflammation [1]. It is estimated that more than two billion people have been chronically infected by HBV worldwide, and nearly 350 million have become victims of this disease. HBV infection leads to a wide spectrum of outcomes [1]. Although most of the infected individuals can successfully clarify this virus, there are still about 10 % of patients who would develop chronic hepatitis B (CHB) [2]. Among those CHB individuals, some may progress rapidly towards liver failure, a condition referred to as acute-on-chronic liver failure (ACLF) [3]. The outcome of patients with chronic HBV infection is closely related to their immune responses [2].

Toll-like receptors (TLRs) are evolutionary conserved receptors that recognize pathogen-associated molecular patterns and mediate the innate immune responses against invading pathogens [4]. TLRs expressed on immune cells are involved in the uptake and processing of various exogenous and endogenous antigens, such as lipopolysaccharide, viral double-stranded RNA, and unmethylated CpG islands [5]. Upon binding to specific ligands, TLRs promote the maturation of dendritic cells (DCs) and induce production of inflammatory cytokines and activation of adaptive immunity [6–8]. As one of the TLRs’ family members, TLR3 recognizes poly(I:C), a synthetic double-stranded RNA analog, as well as viral double-stranded RNA [5], which is presumably formed during viral infection. TLR3 binding to cognate ligands modulates downstream cytokine and chemokine production through the activation of the transcription factor NF-κB, which translocates to the nucleus to modulate gene expression [5, 6]. A role for TLR3 in viral infection has been suggested based on the demonstration that TLR3 knock-out mice were unable to mount a full response to cytomegalovirus infection [7], perhaps by contributing to cytotoxic T cell response after the initial infection [8]. TLR3 is localized primarily in intracellular vesicles, although some cell surface expression is observed in human embryonic kidney cells [9]. Recent study has shown that TLR3 signaling may inhibit HBV replication in an animal model [10]. The expression of TLR3 on peripheral DCs in CHB patients is significantly lower than in healthy individuals [11]. These researches indicate that TLR3 is closely correlated with HBV infection.

Previous studies have described a human single-nucleotide polymorphism (SNP) in TLR3, C1234T, which causes an amino acid exchange (Leu to Phe) at position 412 [12].

This SNP has been reported to be associated with different diseases such as HIV infection, prostate cancer, non-small cell lung cancer, etc. [13–15]. However, there is no paper reporting TRL3 SNPs and HBV infection. In the current study, we have investigated two TLR3 polymorphisms, A952T and C1234T, and have analyzed their associations with CHB in the Chinese population.

MATERIALS AND METHODS

Study Subjects

This study consisted of 452 CHB cases and 462 controls. All subjects were unrelated Han Chinese and recruited from two hospitals, Beijing 302 Hospital and Weihai 404 Hospital between November 2009 and August 2011. The diagnosis of CHB was made based on HBsAg seropositive, anti-HBs seronegative, and continuously abnormal alanine aminotransferase (ALT) and asparate aminotransferase for more than 6 months. The diagnosis of hepatitis B-related ACLF was made based on the criteria of a history of chronic hepatitis B with serum total bilirubin >10 mg/dL, prothrombin activity <40 %, and recent development of complications. The control population was recruited from people who came for physical examinations in these two hospitals. All the control subjects did not have HBV history. None of them received HBV vaccination by the time of enrollment. People who are relatives or positive for HBsAg were excluded from this study. All the control subjects were matched with patient population in terms of age, sex, and residence area (urban or rural). Written informed consent was obtained from each participant. This study was approved by the institutional review boards of Beijing 302 Hospital and Weihai 404 Hospital.

Polymorphism Detection

Genomic DNA was extracted from peripheral blood lymphocytes using a commercially available kit according to the manufacturer’s instructions (Blood Genomic DNA Miniprep Kit, Axygen Biosciences). The PCR fragment carrying A952T SNP was amplified using the following primer pair: 5′-CGCAATTCCAAGATTATTTCCG-3′ and 5′-GGTACCTCACATTGAAAAGCC-3′. Direct sequencing to detect the A952T SNP was conducted using primer 5′-CTTACAGAGAAGCTATGTTTGG-3′. Similarly, the PCR fragment carrying C1234T polymorphism was amplified using the following primer pair: 5′-TGGGATCTCGTCAAAGCCGTTG-3′ and 5′-AAGTATTTCCCTTGCCTCACTCC-3′. Direct sequencing to detect the C1234T SNP was conducted using primer 5′-TTGCTTAGATCCAGAATGG-3′.

Statistical Analysis

The SPSS statistical software package ver.13.0 (SPSS Inc., Chicago, USA) was used for statistical analysis. Demographic data between the study groups were compared by the chi-square test and by the Student’s t test. The polymorphisms were tested for deviation from Hardy–Weinberg equilibrium by comparing the observed and expected genotype frequencies using the chi-square test. For SNP analysis, genotype and allele frequencies were compared between groups using the chi-square test, and odds ratios (OR) and 95 % confidence intervals (CIs) were calculated using unconditional logistic regression. Haplotypes were reconstructed from genotype data and were statistically analyzed by the SHEsis program (http://analysis.bio-x.cn/myAnalysis.php). p values <0.05 were considered significant.

RESULTS

Characteristics of the Patients and Controls

Table 1 shows the general characteristics of CHB patients and controls. There was no significant difference in regard to age and sex between cases and controls. All the patients were detected HBsAg and anti-HBc IgG positive, whereas the controls were negative for these two items. The CHB patients could be divided into subgroups, without ACLF (251 cases) and with ACLF (201 cases), based on the clinical diagnosis. We then compared basic clinical parameters between these two subsets. As expected, ALT and total bilirubin were significantly higher in ACLF patients (p < 0.001) (Table 1).

Table 1 Clinical characteristics of the study population
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TLR3 Polymorphisms in CHB and Controls

The genotype and allele frequencies of the TLR3 A952T and C1234T polymorphisms in CHB cases and controls are summarized in Table 2. The genotype distributions of these two polymorphisms among the controls were in agreement with the Hardy–Weinberg equilibrium (p > 0.05). The frequency of 953A allele was relatively low in both CHB cases (2.7 %) and controls (2.3 %). As for the C1234T polymorphism, the prevalence of C and T alleles was 67.3 and 32.7 % in patients and 74.6 and 25.4 % in controls (p = 0.0006). The 1234CT and TT genotypes showed significantly higher numbers in CHB cases compared to healthy individuals (OR = 1.42, 95 % CI 1.08–1.86, p = 0.012; OR = 2.31, 95 % CI 1.34–3.96, p = 0.002) (Table 2). Also, we analyzed the linkage disequilibrium (LD) and the possible haplotypes constructed by A952T and C1234T polymorphisms. Data showed that there was no significant LD between these two SNPs (D′ < 0.008). Four haplotypes were detected, and prevalence of AT haplotype was significantly increased in CHB cases than in controls (p = 0.0005) (Table 2). These data suggest that TLR3 C1234T polymorphism is associated with an increased susceptibility to CHB in the Chinese population.

Table 2 The genotype and allele frequencies of TLR3 SNPs between CHB patients and controls
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TLR3 Polymorphisms in CHB Cases with ACLF

Since CHB may progress towards ACLF, which has a rapid disease progression and a high incidence of short- or medium-term mortality of 50–90 %, we therefore investigated whether the TLR3 polymorphisms were correlated with ACLF. The patient group was divided into two subsets based on with or without ACLF (Table 3). The TLR3 A952T SNP did not reveal any difference between these two subsets. However, the 1234CT genotype and 1234T allele showed increased prevalence in patients with ACLF than those without ACLF (OR = 1.55, 95 % CI 1.04–2.29, p = 0.030; OR = 1.43, 95 % CI 1.17–1.75, p = 0.040, respectively) (Table 3). These results indicate that TLR3 C1234T SNP is further correlated with an increased risk of CHB patients with ACLF.

Table 3 Comparison of TLR3 SNPs between CHB-related ACLF and CHB without ACLF
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Stratification Analysis of TLR3 Polymorphisms in CHB Patients with ACLF

In order to understand whether TLR3 polymorphisms were correlated with certain characteristics in patients, we performed stratification analysis in CHB patients with ACLF (Table 4). TLR3 SNPs were compared in ACLF cases based on different age (≥40 or <40), sex (male or female), and HBeAg status (positive or negative). No significant difference was observed between patients with different parameters (p > 0.05) (Table 4). These data demonstrate that A952T and C1234T SNPs may not be associated with age, sex, or HBeAg in CHB patients with ACLF.

Table 4 Stratification analysis of TLR3 SNPs in ACLF patients
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DISCUSSION

In this case–control study, we investigated the role of the TLR3 A952T and C1234T SNPs in susceptibility to CHB in the Chinese population and identified that C1234T was a risk factor for this disease. Further analysis revealed that the TLR3 C1234T polymorphism was associated with increased risk of ACLF in CHB patients. To our knowledge, this is the first study showing the correlation between TLR3 SNPs and CHB, especially CHB-related ACLF. Our results indicate that TLR3 C1234T polymorphism may act as a potential maker for the prognosis CHB.

TLR3 is able to recognize dsRNA, which may be the virus genome or replication intermediate, and plays a key role in host antiviral defenses [5]. Several studies have demonstrated the pivotal position of TLR3 in host responses against a number of viruses (e.g., HIV, West Nile virus, and respiratory syncytial virus) [16–18]. Also, TLR3 has been reported to participate in HCV-induced cellular activation and cytokine production [19]. As for HBV infection, recent research has indicated that TLR3 ligand inhibits HBV replication in the liver of HBV transgenic mice [20]. The expression of TLR3 and INF-beta in DCs is significantly lower in ACLF than in healthy individuals [11]. The protein level of TLR3 in PBMC is dramatically decreased in CHB and ACLF patients [21]. Our results have revealed that a functional TLR3 SNP is associated with increased risk of CHB and ACLF. All these studies indicate that TLR3 may play critical roles against HBV infection and may influence the progression of this disease.

SNPs could potentially be associated with a better or worse clinical outcome for patients with infectious disease. To date, there have been more than 136 SNPs described within the human TLR3 gene; however, only four were predicted to cause changes in the TLR3 protein molecule [22]. Two of these four TLR3 SNPs (A952T [rs5743316] and C1234T [rs3775291]) have been shown to affect TLR3 function [22]. The TLR3 A952T and C1234T SNPs are considered nonsynonymous because the nucleotide changes result in amino acid substitutions from asparagine to isoleucine (at position 284) and leucine to phenylalanine (at position 412), respectively [22]. In vitro experiments have demonstrated a complete functional impairment in TLR3 signaling in the presence of A952T SNP (N284I), whereas a markedly diminished function is observed in the presence of the C1234T SNP (L412F) [22]. Hence, these in vitro findings would support their potential role in the clinical setting. The C1234T polymorphism has been identified in different populations and the T allele is about 25 % in the Chinese population. Studies have demonstrated that the C1234T SNP is associated with different diseases such as HIV infection, prostate cancer, non-small cell lung cancer, etc. [13–15]. Our data showed that 1234CT and TT genotypes were correlated with increased susceptibility to CHB, whereas the 1234CT genotype revealed association with higher risk of ACLF (Tables 2 and 3). The A952T is a relatively rare SNP. The 952T allele is only about 2 % in the Chinese population. We did not find any significant association between this polymorphism and CHB or CALF (Tables 2 and 3). However, due to the low frequency of 952T, it is important to conduct similar analysis in a larger study population.

In summary, our study demonstrated for the first time that the TLR3 C1234T SNP was associated with elevated susceptibility to CHB, especially hepatitis B-related ACLF in the Chinese population. These results may provide support for using TLR3 as a therapeutic target and a prognostic marker in the future.