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Toll-like receptor 2 (TLR2) serves as a pattern recognition receptor that signals the presence of cytomegalovirus. Herein, we report that R753Q polymorphism paralyzes TLR2-mediated immune signaling in cells exposed to cytomegalovirus glycoprotein B. This immunologic impairment could serve as a biologic mechanism underlying the association between TLR2 R753Q polymorphism and cytomegalovirus disease in humans.
Toll-like receptors (TLR) are germline-encoded innate immune sensors of microbial pathogens . Activation of TLR signaling results in induction of pro-inflammatory cytokines and antimicrobial peptides . Experimental observations suggest that TLR2 is involved in the innate immune response to cytomegalovirus (CMV) [2, 3]. Stimulation of TLR2 by CMV in vitro, specifically by its envelope glycoproteins B (gB) and H , resulted in nuclear factor-kappa B (NF-κB) activation and cytokine secretion [2, 3]. In vivo, mice deficient of TLR2 had higher hepatic and splenic levels of CMV . Moreover, NK cell-mediated control of CMV in mice was dependent on functional TLR2 . To provide clinical relevance to these findings, we reported that transplant recipients with R753Q single nucleotide polymorphism (SNP) in TLR2 had a significantly higher degree of CMV replication and were more likely to develop CMV disease . However, the underlying mechanism why R753Q SNP was associated with CMV disease has not been investigated. We hypothesized that this was due to impaired TLR2-mediated immune recognition of CMV-associated molecular patterns.
To address the hypothesis, we developed an experimental model utilizing stable clones of human embryonic kidney (HEK) 293 cells (a TLR2-deficient cell line [6, 7]) transfected with luciferase-containing NF-kB gene construct. Stable cells expressing TLR2 (HEK293-TLR2) or TLR2-R753Q (HEK293-TLR2-R753Q) were constructed by transfecting HEK293 with human pcDNA3.1-hygro (Invitrogen)-based TLR2 or TLR2-R753Q expression plasmids, respectively, and clonal selection with Hygromycin B (Invitrogen) [6, 7]. Full genetic sequencing of the TLR2 coding region confirmed the change from CGG to CAG at nucleotide position 2257 in HEK293-TLR2-R753Q cells (translating to amino acid substitution from arginine [R] to glutamine [Q] at position 753 [R753Q]) (Gene Bank accession NM-003264); no other genetic polymorphisms were observed. All cells were maintained at 37°C and 5%CO2 in a culture medium as previously described [6, 7]. HEK293-TLR2 cells acquired responsiveness to Staphylococcus aureus peptidoglycan (PGN; Sigma, St. Louis, MO) [6, 7]. All cells were unresponsive to Escherichia coli-derived purified lipopolysaccharide (LPS; Sigma) [6, 7].
A total of 1×106 cells were incubated for 30 minutes with fluorescein isothiocyanate-anti-human (or anti-mouse IgG2a isotype control) TLR2 monoclonal antibody (1.0-μg/million cells; eBioscience, San Diego, CA); this antibody specifically binds to wild-type and mutant TLR2 protein expressed on the cell surface. After washing, cells were fixed with 1% paraformaldehyde, and TLR2-expressing cells were counted using a FACS brand flow cytometer.
A total of 1×106 cells were lysed in 950-μl of Cell Extraction Buffer (Biosource, Camarillo, CA) and 50-μl of Protease Inhibitor Cocktail (Sigma). Protein extracts (10-μg) were separated on NuPage 4-12% Bis-Tris gel and transferred to nitrocellulose membrane using iBlot™ Dry Blotting System (Invitrogen). Protein expression was analyzed using WesternBreeze® Chemiluminescence Western Blot Immunodetection Kit (Invitrogen).
Cell densities of 1×105 HEK293, HEK293-TLR2, or HEK293-TLR2-R753Q cells/100 μl/well were seeded into a 96-well plate (Costar 3595, Corning Incorporated) and incubated overnight at 37°C and 5%CO2. Adherent cells were stimulated with CMVgB (5.0-μg/ml; DevaTal, Inc., Hamilton, NJ), PGN (1.0-μg/ml), LPS (1.0-μg/ml), or tumor necrosis factor (TNF)-α (10-ng/ml; R&D Systems, Minneapolis, MN) for 16 hours at 37°C and 5%CO2.
To assess NF-κB activation, HEK293, HEK293-TLR2 and HEK293-TLR2-R753Q cells were lysed with 20-μl/well of 1× Reporter Lysis Buffer (Promega Corporation, Madison, WI) followed by a single freeze-thaw cycle. The cell lysate was mixed with 100-μl of Promega Luciferase Substrate and chemoluminescence was measured using the Victor instrument (Perkin-Elmer Life and Analytical Sciences, Torrance, CA). To quantify IL-8 secretion, cell-free supernatants of HEK293, HEK293-TLR2, and HEK293-TLR2-R753Q cells were assayed using a single-platform IL-8-specific quantitative sandwich enzyme immunoassay (Quantikine for Human CXCL8/IL-8; R&D Systems).
Cell densities of 3×106 HEK293, HEK293-TLR2, or HEK293-TLR2-R753Q cells/2-ml volume/well were seeded into a 6-well plate (Costar 3516) and incubated overnight at 37°C and 5%CO2. Adherent cells were stimulated with CMVgB for 4 hours at 37°C and 5%CO2. RNA was extracted using RT2qPCR-Grade RNA Isolation Kit (SuperArray Bioscience Corporation, Frederick, MD), and 1.0-μg of RNA was converted to cDNA using RT2Profiler PCR Array System. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using GeneAmp PCR System 2400 (Perkin Elmer, Boston, MA) and genes associated with TLR2 signaling (such as myeloid differentiation factor 88 [MyD88], interleukin-1 receptor associated kinases [IRAK], and TNF receptor-associated factor 6 [TRAF6]) were assessed using the iCycler instrument (Biorad Laboratories, Hercules, CA) .
All experiments were performed in triplicates on at least two different occasions. Results are presented as average (±standard deviation) fold-induction in stimulated over unstimulated cells. Differences between groups were analyzed using a two-tailed Student’s t test, with statistical significance set at P < 0.05.
CMVgB induced a 12-fold higher degree of NF-κB-driven luciferase activity in HEK293-TLR2 cells (Figure 1A). In contrast, no significant NF-κB-driven luciferase activity was observed in TLR2-deficient HEK293 cells (0.7-fold) and HEK293-TLR2-R753Q (1.2-fold) cells during exposure to CMVgB. Accordingly, the degree of NF-κB-driven luciferase activity in response to CMVgB was significantly higher in HEK293-TLR2 compared to TLR2-deficient HEK293 cells (p<.0001) or the mutant HEK293-TLR2-R753Q cells (p<.0001). A similar pattern of cellular response was observed in PGN-stimulated cells (Figure 1A). The lack of cellular activation in HEK293-TLR2-R753Q cells was not due to impaired TLR2 protein expression, as determined by Western Blot analysis and flow cytometry (Figure 1B). Moreover, the lack of NF-κB-driven luciferase activity in HEK293-TLR2-R753Q cells was not due to a global unresponsive state since cells responded to stimulation with a TLR2-independent activator, TNF-α (Figure 1A).
RT-PCR showed no significant upregulation in genes encoding for IL-1β, IL-6, IL-8, and TNF-α in HEK293-TLR2-R753Q cells stimulated for 4 hours with CMVgB (Figure 1C). In contrast, HEK293-TLR2 cells had modest upregulation of genes for IL-1β (4.5-fold) and IL-6 (2-fold), and marked upregulation in genes for IL-8 (85-fold) and TNF-α (90-fold). Furthermore, ELISA demonstrated that HEK293-TLR2 cells secreted high levels of IL-8 (mean±standard deviation, 30801±2017 pg/ml). IL-8 secretion was 30-fold higher in CMVgB-stimulated compared to unstimulated HEK293-TLR2 cells (Figure 1D). In contrast, there was negligible IL-8 secretion in CMVgB-stimulated HEK293 and HEK293-TLR2-R753Q cells. However, in response to TLR2-independent stimulation with TNF-α, HEK293-TLR2-R753Q demonstrated a 23-fold higher degree of IL-8 secretion compared to unstimulated cells.
This experimental study provides in vitro evidence that R753Q SNP paralyzes TLR2-mediated immune signaling in response to CMV. This observation could serve as a putative biologic mechanism underlying the clinical association between TLR2 R753Q SNP and a higher degree of CMV replication and higher incidence of CMV disease in humans.
The human TLR2 gene, which is located in chromosome 4q32, contains at least 89 SNPs, including 17 that modify bases in the coding sequence of exon III . Nine SNPs are non-synonymous and two (R677W and R753Q) were characterized to impair TLR2 function [10-12]. Because TLR2 recognizes a wide repertoire of pathogen-associated molecular patterns from Gram-positive bacteria (PGN), mycobacteria (lipoarabinomannan), parasites (glycophosphatidylinositol), fungi (zymosan), and viruses (envelope proteins), there is considerable interest in investigating the potential association between functional impairment in this receptor and various human infections [9, 13]. In this regard, TLR2 R753Q SNP has been suggested to influence the risk of infection due to S. aureus , Mycobacterium tuberculosis , Borrelia burgdorferi , and Treponema pallidum .
Previously, we reported that patients with TLR2 R753Q SNP were more likely to develop CMV disease after liver transplantation . Our present study provides the biologic explanation for this clinical observation by demonstrating that variant cells with TLR2 R753Q SNP failed to recognize CMVgB . This impaired innate viral recognition may impede development of a more robust antiviral immune mechanism thereby translating to a higher incidence of clinical disease. It is unclear whether R753Q SNP results in an impaired ligand-receptor attachment or impaired engagement of downstream adapter molecules. The location of the R753Q SNP within a group of highly conserved amino acids at the C-terminal of TLR2 (i.e., cytoplasmic Toll-interleukin 1 receptor domain) suggest that R753Q SNP may impede the engagement of MyD88 adapter protein; however, this remains to be demonstrated. Currently, what is known is that, in vitro, R753Q SNP in TLR2 is associated with impaired NF-κB activation during exposure to CMV and bacterial lipoproteins . These findings expand on our previous observations  and those of others  that TLR2 is an important component in the immunopathogenesis of CMV in humans. We emphasize however that the clinical relevance of TLR2 has been suggested only in immunocompromised transplant recipients, and its importance in the pathogenesis of CMV in healthy individuals has yet to be demonstrated.
In conclusion, this study demonstrates that the R753Q SNP impairs TLR2-mediated immune signaling in response to CMV. This specific functional genetic variation, which is reported in 3-12% of humans, has a potential clinical application as a prognostic marker for a heightened risk of CMV disease. As subunit CMV vaccines are being developed clinically, with gB as a major component, it is anticipated that immune responses to vaccination may be impaired in individuals with TLR2 R753Q SNP. These potential clinical implications should encourage the conduct of more studies to confirm our observations, and to assess other functional TLR SNPs and their relevance to human infections.
We thank Ms. Teresa Hoff for manuscript preparation.
Funding Source: This study was funded by the Mayo Clinic Department of Medicine (to RRR), Transplant Center Scholarly Award of the William J von Leibig Transplant Center (to RRR), and Mayo CR20 Award (to RRR). This publication was made possible by Grant Number 1 UL1 RR024150 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH), and the NIH Roadmap for Medical Research. Its contents are solely the responsibility of the authors and do not necessarily represent the official view of NCRR or NIH.
There are no conflicts of interest for all authors.
This study was presented in part during the joint meeting of the Interscience Conference on Antimicrobial Agents and Chemotherapy and the Infectious Disease Society of America, Washington DC, October 2008.