Abstract

Background.Experimental data suggest that cytomegalovirus (CMV) initiates innate immunity through the activation of Toll-like receptor 2 (TLR2). To assess the clinical relevance of this experimental observation, we assessed the association between the specific single-nucleotide polymorphism that results in the substitution of arginine for glutamine in position 753 of TLR2 (the TLR2 Arg753Gln polymorphism) and CMV replication and disease after liver transplantation.

Methods.Ninety-two liver transplant recipients with chronic hepatitis C were screened for the presence of the TLR2 Arg753Gln polymorphism. CMV load was determined in serially collected blood samples using CMV DNA polymerase chain reaction. Kaplan-Meier estimation and univariable and multivariable stepwise Cox proportional hazard models were used to assess associations.

Results.The degree of CMV replication, as measured by CMV load, was significantly higher in patients who were homozygous (mean maximum viral load, 37,059 copies/mL) and heterozygous (mean maximum viral load, 29,718 copies/mL) for this polymorphism, compared with patients without the TLR2 Arg753Gln polymorphism (mean maximum viral load, 3252 copies/mL; P = .003). Kaplan-Meier survival analysis demonstrated an association between being homozygous for the TLR2 Arg753Gln polymorphism and CMV disease (P = .04). A multivariate Cox proportional hazard model demonstrated a trend towards a higher risk of CMV disease among patients who were homozygous for the TLR2 Arg753Gln polymorphism (hazard ratio, 1.91 [95% confidence interval, 0.91–3.40]; P = .08) after adjusting for patient age, CMV serostatus, and allograft rejection.

Conclusions.TLR2 Arg753Gln polymorphism is possibly associated with CMV replication and disease after liver transplantation. This novel clinical observation supports the potential role of TLR2 in the immunologic control of CMV infection in humans.

The discovery of the Toll-like receptors (TLRs) resulted in a better understanding of the molecular mechanisms of pathogen recognition and innate immunity against infectious agents [1]. Stimulation of TLRs by pathogen-associated molecular patterns activates intracellular signaling pathways and leads to the induction of antimicrobial genes and inflammatory cytokines, which help to control the early stages of infection until the pathogen-specific adaptive arm of the immune response is established [1]. Currently, 10 TLR molecules have been described in humans. These pathogen-recognition receptors recognize specific ligands that range from lipopolysaccharide (LPS) in gram-negative bacteria, peptidoglycan and lipoteichoic acid in gram-positive bacteria, bacterial flagellin, CpG DNA, viral nucleic acids, and the components of mycobacteria, yeast, viral, and parasitic pathogens [1, 2].

The ability of TLRs to signal the presence of invading pathogens in the internal milieu and orchestrate an immune response to them characterizes their role in the pathogenesis of infectious diseases [3–10]. Experimental data demonstrated that TLR4-deficient mice have an impaired ability to respond to LPS [11, 12]. In humans, the presence of missense mutations that affect the extracellular domain of TLR4 results in a blunted immune response to inhaled LPS [4] and predisposes an individual to severe respiratory syncitial virus–related bronchiolitis [13]. Similarly, TLR2-deficient mice are highly susceptible to infections with Staphylococcus aureus [14]—an observation that concurs with a higher risk of staphylococcal [9] and mycobacterial infection [7, 15] among humans with functional TLR2 polymorphisms.

Little is known about the role of TLRs in the pathogenesis of cytomegalovirus (CMV) infection in humans. Recent experimental observations indicate that TLRs participate in innate immunity against CMV infection. In in vitro experiments using murine and human cells, stimulation of TLR2 with CMV results in the translocation of nuclear factor-κ B and the secretion of cytokines [16, 17]. However, the clinical relevance of these experimental observations has not been investigated. We hypothesize that, if TLR2 participates in the control of CMV infection through the release of antiviral cytokines and the regulation of adaptive immunity, the presence of functional polymorphisms in TLR2 would impede this immune response and clinically lead to higher levels of CMV replication and symptomatic CMV disease. We tested this concept using the human model of liver transplantation—a clinical situation characterized by the common occurrence of CMV infection and disease. In particular, we compared the occurrence of CMV disease in patients with or without a specific single-nucleotide polymorphism that results in the substitution of arginine for glutamine in position 753 of TLR2 (the TLR2 Arg753Gln polymorphism). This specific polymorphism is of interest because it is the most commonly reported mutation in TLR2 [18], and its presence has been described to result in a functional defect to stimulation with the prototypic TLR2 ligand bacterial lipopeptide [19]. As our data suggest, this specific single-nucleotide polymorphism in TLR2 is potentially associated with CMV replication and disease after liver transplantation.

Patients and Methods

Study population and blood samples.The study population consisted of a cohort of 92 consecutive patients who underwent liver transplantation for chronic hepatitis C during the period 1991–2000. This cohort was chosen solely on the the basis of availability of blood samples for testing. This cohort has been previously investigated to assess predictors of allograft failure and mortality after undergoing liver transplantation for chronic hepatitis C [20]. Peripheral blood samples obtained from these patients were stored at -80°C prior to this study. The study was approved by the Institutional Review Board of the Mayo Foundation (Rochester, MN).

Study outcomes and clinical follow-up.The outcome of interest for this study was CMV disease and viral load. CMV disease was defined as the presence of clinical signs and symptoms compatible with the diagnosis accompanied by the isolation of CMV from blood or tissue samples [21]. The medical records of all patients were reviewed for demographic and clinical characteristics, immunosuppressive treatment, acute allograft rejection, and donor and recipient CMV serostatus. Patients' immunosuppressive drug regimens varied over time and, depending on the transplant period, consisted of cyclosporine, azathioprine, and prednisone (from 1991 to 1994); tacrolimus, prednisone, and mycophenolate mofetil or azathioprine (from 1994 to 1998); and tacrolimus, mycophenolate mofetil, and prednisone (from 1999 to 2000). All patients were observed until the time of CMV disease or death, or up to a maximum period of 2 years after undergoing liver transplantation.

Assessment of CMV replication.Serum samples that were collected weekly for the first 6 weeks and at months 4 and 12 after liver transplantation was performed were retrieved for CMV DNA quantification. Genomic DNA was extracted from 200 µL of serum using the IsoQuick nucleic acid extraction kit (ORCA Research) according to the manufacturer's guidelines. All samples were subjected to CMV DNA quantification by real-time PCR using the LightCycler instrument (Roche Molecular Biochemicals), as previously described [22]. Because all patients had an undetectable CMV load at the time of transplantation, any subsequent detection of CMV DNA was indicative of CMV replication.

Detection of the TLR2 polymorphism.To perform nucleic acid extraction, peripheral blood samples (200 µL of whole blood or 106 PBMCs) were subjected to nucleic acid extraction using the Isoquick Nucleic Acid Method (ORCA Research), according to the manufacturer's instructions. The extracted nucleic acid was eluted in 100 µL of sterile DNAse- and RNAse-free water. Five µL of the eluted DNA was used for detection of TLR polymorphism using the real-time PCR assay performed on a LightCycler instrument, according to methods previously described [18, 23], with several modifications. The PCR results were confirmed by gene sequencing of representative samples.

To detect the TLR2 Arg753Gln polymorphism, 5 µL of extracted DNA was mixed with 15 µL of PCR solution consisting of LightCycler FastStart master mix (Roche Molecular Biochemicals), 0.25 µmol/L of each primer (sense primer 5′-AGTGAGCGGGATGCCTACT-3′ and antisense primer 5′-GACTTTATCGCACTCTCAGATTTAC-3′), 4 mmol/L magnesium chloride, 9.5 µL water, 0.2 µmol/L TLR2 sensor probe (5′-CAAGCTGCAGAAGATAATGAACACCAAG-3′-FL), and 0.4 µmol/L TLR2 anchor probe (LC Red 640–5′-CCTACCTGGAGTGGCCCATGGACG-3′). Initial denaturation was performed at 95°C for 10 min, followed by 45 cycles of denaturation (95°C for 0 sec, 20°C per sec), annealing (55°C for 10 sec), and extension (72°C for 18 sec). Melting curve analysis involved 1 cycle at 95°C for 0 sec and 53°C for 30 sec, followed by an increase of temperature to 80°C at a slope of 0.1°C per sec. For melting curve analysis, the wild-type TLR2 had a melting peak of 61.8°C, whereas the single nucleotide polymorphism that resulted in the Arg753Gln substitution had a melting peak of 66.3°C.

Statistical analysis.Data analysis was performed using descriptive statistics, including mean, median, SD, 95% CI, and range. Comparison of proportions was performed using Fisher's exact test. Kaplan-Meier estimation was used to describe the survival curve. Univariable and multivariable stepwise Cox proportional hazard models were used to assess associations between covariates and CMV disease. To describe the relationship between TLR polymorphism and CMV replication, the maximum viral load was obtained from each patient and compared among patients with or without TLR polymorphisms using the analysis of variance. Statistical significance was set at P ⩽ .05.

Results

The study population consisted of 92 patients who underwent liver transplantation for chronic hepatitis C. The mean (±SD) age of the patients was 49.5 ± 8.6 years. The majority of patients (60.6%) were male. The primary outcome of CMV disease occurred in 25 patients (27.2%) at a median time of 37 days (mean ± SD, 57 ± 67 days) after transplantation.

All 92 patients were tested for the TLR2 Arg753Gln polymorphism [18, 23], which resulted in a functionally defective receptor [19]. Among the 92 patients, 12 (13%) had at least 1 allele that had the TLR2 Arg753Gln polymorphism; 7 patients (8%) were heterozygous, and 5 (5%) were homozygous for this polymorphism.

TLR2 polymorphism is associated with CMV disease.Homozygosity, but not heterozygosity, for the TLR2 Arg753Gln polymorphism was associated with CMV disease. CMV disease occurred in 3 (60%) of 5 patients who were homozygous for the TLR2 Arg753Gln polymorphism, in 1 (14%) of 7 patients who were heterozygous for the TLR2 Arg753Gln polymorphism, and in 20 (25%) of 80 patients who did not have the TLR2 Arg753Gln polymorphism. Survival analysis using Kaplan Meier estimation demonstrated a possible association between CMV disease and homozygosity (but not heterozygosity) for the TLR2 Arg753Gln polymorphism (P = .04, by the log-rank test) (figure 1).

Figure 1

Kaplan-Meier estimation of cytomegalovirus (CMV) disease–free survival in liver transplant recipients with homozygosity or heterozygosity for the specific single-nucleotide polymorphism that results in the substitution of arginine for glutamine in position 753 of Toll-like receptor 2 (the TLR2 Arg753Gln polymorphism), compared with wild-type homozygotes.

Figure 1

Kaplan-Meier estimation of cytomegalovirus (CMV) disease–free survival in liver transplant recipients with homozygosity or heterozygosity for the specific single-nucleotide polymorphism that results in the substitution of arginine for glutamine in position 753 of Toll-like receptor 2 (the TLR2 Arg753Gln polymorphism), compared with wild-type homozygotes.

In a univariable Cox proportional hazard model, a trend was observed between homozygosity for the TLR2 Arg753Gln polymorphism and CMV disease (hazard ratio, 1.79 [95% CI, 0.87–3.05]; P = .10). In this model, CMV disease was significantly more common among patients who experienced acute allograft rejection (risk ratio [RR], 1.85 [95% CI, 1.22–2.90]; P = .003); patients who received steroid boluses (for treatment of acute allograft rejection; RR, 1.37 [95% CI, 1.08–1.74]; P = .009); and CMV-seronegative patients who received liver allografts from CMV-seropositive donors (RR: 1.79 [95% CI, 1.003–3.04]; P = .04). A nonsignificant trend towards a higher risk of CMV disease was observed with patient age (RR, 1.04 [95% CI, 0.92–1.007]; P = .09). None of the immunosuppressive drugs were significantly associated with an increased risk of CMV disease, including azathioprine, mycophenolate mofetil, and prednisone. Use of cyclosporine, which involved 38% of patients, resulted in a trend towards an association with CMV disease (RR, 1.45 [95% CI, 0.971–2.193]; P = .07), whereas use of tacrolimus, which involved 71% of patients, was associated with lower risk of CMV disease (RR, 0.60 [95% CI, 0.399–0.899]; P = .01).

In the subsequent multivariable Cox proportional hazard model, using a stepwise approach that accounted for patient age, acute allograft rejection, and CMV mismatch status, there was a marginal association between homozygosity for the TLR2 Arg753Gln polymorphism and CMV disease (HR, 1.91 [95% CI, 0.91–3.40]; P = .08) (table 1). No significant change in the possible association was observed when use of cyclosporine or tacrolimus was included in the statistical model (data not shown).

Table 1

Multivariate Cox proportional hazard model assessing the associations between acute allograft rejection, cytomegalovirus (CMV) mismatch status, patient age, and homozygosity for the specific single-nucleotide polymorphism that results in the substitution of arginine for glutamine in position 753 of Toll-like receptor 2 (the TLR2 Arg753Gln polymorphism), with the outcome of CMV disease.

Table 1

Multivariate Cox proportional hazard model assessing the associations between acute allograft rejection, cytomegalovirus (CMV) mismatch status, patient age, and homozygosity for the specific single-nucleotide polymorphism that results in the substitution of arginine for glutamine in position 753 of Toll-like receptor 2 (the TLR2 Arg753Gln polymorphism), with the outcome of CMV disease.

TLR2 polymorphism is associated with CMV replication.To further examine the association between TLR2 and CMV, we correlated the TLR2 Arg753Gln polymorphism with the degree of CMV DNAemia. As shown in table 2, the presence of the TLR2 Arg753Gln polymorphism was significantly associated with higher levels of CMV DNA in the peripheral blood. The mean maximum CMV DNA load was significantly higher among patients who were homozygous (37060 copies/mL [95% CI, 12,575–61,545 copies/mL]) and heterozygous (29,719 copies/mL [95% CI, 9025–50,412 copies/mL]) for the TLR2 Arg753Gln polymorphism, compared with those without the polymorphism (3252 copies/mL [95% CI, 0–9412 copies/mL]; P = .003). Among the subgroup of patients who developed CMV disease, the maximum CMV load was significantly higher among those who were homozygous (61,733 copies/mL [95% CI, 10,153–113,313 copies/mL]) and heterozygous (166,100 copies/mL [95% CI, 76,761–255,439 copies/mL]) for the polymorphism, compared with those who did not have the polymorphism (12,923 copies/mL [95% CI, 0–33,419 copies/mL]; P = .004).

Table 2

Mean maximum cytomegalovirus (CMV) levels in patients with or without the specific single-nucleotide polymorphism that results in the substitution of arginine for glutamine in position 753 of Toll-like receptor 2 (the TLR2 Arg753Gln polymorphism).

Table 2

Mean maximum cytomegalovirus (CMV) levels in patients with or without the specific single-nucleotide polymorphism that results in the substitution of arginine for glutamine in position 753 of Toll-like receptor 2 (the TLR2 Arg753Gln polymorphism).

Discussion

This study provides clinical evidence that would support the potential role of TLR2 in pathogenesis of CMV disease in humans. Liver transplant recipients with a single-nucleotide TLR2 Arg753Gln polymorphism in at least 1 allele had significantly higher levels of CMV replication, as measured by CMV viral load. Moreover, patients with homozygosity for the TLR2 Arg753Gln polymorphism had a higher incidence of CMV disease. These novel observations concur with in vitro experimental data on the TLR2-CMV interaction. Therefore, these observations provide insight into the pathogenesis of CMV infection by implying a potential role of TLR2 in the immunologic control of CMV infection.

The involvement of TLR2 in the immune response against CMV in humans was initially suggested by an in vitro experimental model [16, 17]. Cells that lacked TLR2 expression did not secrete cytokines during stimulation with a CMV virion or antigens [16, 17]. Moreover, when TLR2-deficient cells were experimentally manipulated to express human TLR2 in vitro and were subsequently stimulated with CMV, the cells acquired responsiveness, as manifested by the secretion of proinflammatory cytokines [16, 17]. Recently, it was further suggested that this is mediated through the recognition of the viral envelope glycoprotein B by TLR2 [16, 17]. Our observation regarding the possible association between a functional polymorphism in TLR2 and CMV replication and disease supports—and potentially provides clinical relevance to—these experimental observations. More importantly, from the pathogenesis standpoint, our observations possibly imply that the earliest stage of encounter between the host (i.e., the innate immune cells via TLR sensing) and the pathogen (i.e, CMV) must be integral to the optimal control of this pathogen.

The single nucleotide polymorphism that resulted in the Arg753Gln mutation in TLR2 has been shown to result in a functionally-defective receptor, at least against bacterial lipopeptides. In a recent study, cells that were transfected with a TLR2 Arg753Gln polymorphism did not respond optimally to bacterial lipopeptide [19]. Indeed, this particular mutation has been significantly associated with a higher predisposition to the development of bacterial sepsis [9]. The present study concurs with these clinical observations by demonstrating the relationship between homozygosity for the TLR2 Arg753Gln polymorphism and CMV replication and clinical disease. Accordingly, we hypothesize that the functional defect in the TLR2 mutant may have impeded the innate immune response against CMV (a TLR2 ligand), thereby resulting in symptomatic disease. Interestingly, our study further implies that the presence of a single functional wild-type allele may be sufficient for controlling CMV infection [19], because patients who were heterozygous for the TLR2 Arg753Gln polymorphism behaved phenotypically identically to those without the polymorphism (i.e., the patients did not have a significantly higher risk for CMV disease, compared with wild-type homozygotes). This clinical observation concurs with experimental data demonstrating the ability of cells obtained from individuals with heterozygosity for the TLR2 Arg753Gln polymorphism to respond when stimulated with a TLR2 ligand [19]. Nonetheless, our clinical data also suggest that the immune response against CMV among patients with heterozygosity for the TLR2 Arg753Gln polymorphism may be attenuated, because these patients exhibited a significantly higher degree of CMV replication.

The potential clinical implications of our observations should encourage the development of more studies to confirm our findings. We recognize and emphasize that our conclusions are limited by the relatively small number of study patients and the marginal statistical associations that were observed. In addition, our study population was homogenous, consisting only of liver transplant recipients with underlying chronic hepatitis C. Hence, our results may only apply to hepatitis C virus–infected liver transplant recipients. Accordingly, we suggest that other studies be performed to confirm our observations in liver transplant recipients who do not have chronic hepatitis C, in other solid-organ and hematopoietic stem cell transplant recipients, and in nontransplant-related immunocompromised patients, such as those with AIDS and infants with congenital CMV infection.

In conclusion, this study demonstrates the potential relationship between CMV and the TLR2 Arg753Gln polymorphism in liver transplant recipients. Our study showed that patients with homozygosity for the TLR2 polymorphism had higher levels of CMV replication and clinical CMV disease. On the other hand, patients with heterozygosity for the polymorphism are not at a higher risk of CMV disease, although they tend to have high levels of CMV replication. To our knowledge, these observations provide clinical evidence that possibly supports the role of TLR2 in the pathogenesis of CMV disease in humans. Because of the potential clinical implications, we encourage the development of further studies to confirm our observations.

Acknowledgments

We thank Ross Dierkhising for statistical analysis and consultation and Teresa Hoff for manuscript preparation.

Financial support.Department of Medicine and the Transplant Center Scholarly Award by the William J. von Liebig Transplant Center, Mayo Clinic, Rochester, MN (to R.R.R.).

Potential conflicts of interest.All authors: no conflicts.

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Present affiliation: Eli Lilly and Company, Indianapolis, Indiana (C.V.P).
Presented in part: World Transplant Congress, Boston, Massachusetts, July 2006 (abstract 3337) and the Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, California, September 2006 (abstract V-1085).

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