The innate immune response plays a key role as the primary host defense against invading pathogens including viruses. We have previously shown that treatment of human monocyte-derived macrophages with EBV-encoded dUTPase induces the expression of proinflammatory cytokines through the activation of NF-κB. However, the receptor responsible for EBV-encoded dUTPase-mediated biological effects is not known. In this study, we demonstrate that the purified EBV-encoded dUTPase activates NF-κB in a dose-dependent manner through TLR2 and requires the recruitment of the adaptor molecule MyD88 but not CD14. Furthermore, activation of NF-κB was abrogated by anti-TLR2, anti-EBV-encoded dUTPase blocking Abs and the overexpression of a dominant negative construct of MyD88 in human embryonic kidney 293 cells expressing TLR2. In addition, treatment of human monocyte-derived macrophages with the anti-EBV-encoded dUTPase Ab 7D6 or the anti-TLR2 Ab blocked the production of IL-6 by the EBV-encoded dUTPase. To our knowledge, this is the first report demonstrating that a nonstructural protein encoded by EBV is a pathogen-associated molecular pattern and that it has immunomodulatory functions. Although additional studies are necessary to define the signaling pathways activated by the EBV-encoded dUTPase and to determine its role in modulating immune responses to EBV infection, our results suggest that the dUTPase could be a potential target for the development of novel therapeutic agents against infections caused by EBV.
The innate immune response is an early line of defense, which plays a key role in the protection of a host from invading pathogens including viruses. Viruses, as well as other pathogens, encode for various proteins containing pathogen-associated molecular patterns (PAMP),3 which are recognized by immune sensor molecules that are referred to as pattern recognition receptors (PRR). TLRs, which are PRRs, are responsible for the primary recognition of a broad range of pathogens (1), leading to the initiation of the innate and adaptive immune responses (2, 3, 4).
To better understand the immune responses triggered by viruses, it is critical to identify not only which PRRs are activated during viral infection but also the specific viral components that act as ligands/triggers for the different PRRs, leading to the stimulation of the host immune response. Accumulating evidence indicates that numerous viruses can activate cells through different TLRs. Recognition of members of the Herpesviridae family by the innate immune system involves at least two distinct pathways. One of them is the recognition of intact virions of HSV-1 (5, 6), varicella-zoster virus (VZV; Ref. 7), human CMV (hCMV; Refs. 8, 9, 10), and EBV (11) by the cell surface TLR2. Intracellularly, the recognition of HSV-1, HSV-2, and hCMV CpG-rich dsDNA is mediated by TLR9 (12, 13, 14), whereas the DNA of murine CMV is reportedly recognized by TLR3 and TLR9 (15). Although activation of TLRs might result in a protective antiviral immune response, for some viruses, such as HSV-1, stimulation of TLRs may be detrimental to the host and actually enhance the infection by allowing replication of the virus (5, 16, 17).
EBV is an oncogenic gamma herpesvirus that infects a significant percentage (>90%) of the population worldwide. EBV is similar to other herpesviruses in that after primary infection, the virus establishes latency in memory B cells where it can be reactivated during the lifetime of the individual. Although EBV is considered to be a B-lymphotrophic virus, it is known to infect epithelial cells (18, 19), and there is increasing evidence demonstrating that EBV can infect monocytes and dendritic cells as well as T cells in vitro (12, 20, 21, 22, 23, 24, 25, 26). EBV is the causative agent of infectious mononucleosis and is implicated in the pathogenesis of a variety of lymphoproliferative disorders and human malignancies.
There are numerous reports showing that in vitro acute and chronic EBV infections result in changes in the secretion patterns of TNF-α, IL-1β, IL-6, and IL-10 (20, 21, 27, 28, 29, 30). These studies have focused on the effects of cytokine expression following the interaction of gp350/220 (gp350), the major envelope glycoprotein encoded by EBV with its cellular receptor CR2 (27, 28, 30). Recently, Gaudreault et al. (11) reported that EBV virions were recognized by TLR2 and that this was blocked by the 72A1 Ab, which recognizes an epitope of gp350, suggesting that gp350 acts like a PAMP. Martin et al. (31) also reported that EBV up-regulated the expression of TLR7, that it down-regulated TLR9 expression in naive B cells, and that EBV modulated TLR7 signaling to enhance B cell proliferation and to regulate IRF-5 activity. Overall, these results suggest that there is a virion component, possibly gp350, that plays an important role(s) in the recognition of EBV by macrophages/B cells. However, little is known concerning the role that proteins involved with lytic replication of EBV may have in triggering TLR-mediated responses. We have previously demonstrated that the purified EBV-encoded dUTPase induces the up-regulation of IL-10 and the proinflammatory cytokines TNF-α, IL-1β, IL-6, and IL-8 in human monocyte-derived macrophage (hMDM)-CD14+ cells (32). The increased expression and production of cytokines observed in response to EBV-encoded dUTPase treatment of hMDM were dependent on the activation of NF-κB (33). However, the mechanism(s) by which EBV-encoded dUTPase causes NF-κB activation and subsequent induction of proinflammatory cytokines has not yet been elucidated. In the present study, we demonstrate, using NF-κB luciferase reporter assays and various TLRs stable cell lines as well as TLR expression constructs, that the purified EBV-encoded dUTPase activates NF-κB in a dose-dependent manner through TLR2, but not TLR3, TLR4, or TLR4/MD2, and that the activation of NF-κB requires the recruitment of the adaptor molecule MyD88. Furthermore, EBV-encoded dUTPase-mediated activation of NF-κB was observed in the presence and absence of CD14, suggesting that CD14 is not required for NF-κB induction. To our knowledge, this is the first report demonstrating that a nonstructural protein encoded by EBV functions as a PAMP and is capable of inducing the secretion of cytokines in human PBMCs and hMDM. As such, EBV-encoded dUTPase could potentially modulate the innate immune response in EBV-permissive cells through the TLR2 and MyD88 signal transduction pathway, leading to the activation of NF-κB and the production of various proinflammatory cytokines, including IL-6, and the key anti-inflammatory cytokine IL-10. These two cytokines have received much attention because of their important roles in both innate and adaptive immune responses. IL-6 is the major stimulator of acute-phase responses and is also involved in the control of cytokine and chemokine gene expression, induction of Abs (34), modulation of APC differentiation (35), and the function of regulatory T cells against microbial infections (36). Similarly, stimulation of human macrophages with EBV-encoded dUTPase resulted in the release of the anti-inflammatory and immunosuppressive cytokine IL-10. There is growing interest in elucidating the signaling pathways leading to IL-10 induction because of its key role in inhibiting activated dendritic cells and potentially in shaping Th1 and Th2 cell responses (37). There are also reports suggesting that stimulation of IL-10 production may be a strategy for pathogens to evade a Th1 response, which is more harmful to the virus, and promote a Th2/T regulatory phenotype, a permissive environment for the virus (38, 39). Given the ability of EBV to establish persistent infections in the host, it might be expected that the virus has evolved to develop mechanism(s) to regulate the expression and function of IL-6, IL-10 and TNF-α as part of the virus strategy to evade immune surveillance. Therefore, understanding how EBV-encoded dUTPase is regulating cytokine expression and function may be important in determining immune response outcomes during EBV infection.
Materials and Methods
Blasticidin, hygroGold, zymosan, Pam2CGDPKHPKSF (FSL-1), and N-palmitoyl-S-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-(R)-Cys-(S)-Ser-(S)-Lys4 trihydrochloride (Pam3CSK4) were purchased from Invivogen. Polyinosinic-polycytidylic acid (poly(I:C)) was purchased from Amersham Bioscience, and LPS from Escherichia coli
Human embryonic kidney 293 (HEK293) cell lines stably expressing human TLR2, TLR2/CD14, and TLR3 were purchased from Invivogen. HEK293-hTLR4 and HEK293-hTLR4/MD2 cell lines were gifts from Dr. Douglas Golenbock. All cell lines were maintained in DMEM supplemented with l-glutamine (2 mM), HEPES (10 mM), sodium pyruvate (1%), penicillin (100 μg/ml), streptomycin (100 U/ml), and 10% FBS, plus blasticidin (HEK293-TLR2), hygroGold (HEK293-TLR4; HEK293TLR4/MD2), or a combination of hygroGold and blasticidin (HEK293-TLR2/CD14).
Primary human monocytes were obtained by negative selection (Astarte Biologics) and differentiated into macrophages by culturing cells in DMEM supplemented with 10% calf bovine serum over a 10-day period as previously described (33). Culture medium was changed every 3 days during the differentiation process.
Purification of the EBV-encoded dUTPase
Detailed methods for the purification of the EBV-encoded dUTPase have been previously reported (32, 33). All EBV-encoded dUTPase preparations were tested as described previously (32) and were free of detectable levels of LPS, peptidoglycan (SLP-HS), DNA, or RNA. Protein concentration was determined with a Coomassie Brilliant Blue dye-binding assay (Bio-Rad Laboratories) using BSA as the standard. The purified EBV-encoded dUTPase used in these studies was stored at 4°C at stock concentrations of 0.5 and 1 mg/ml.
Luciferase reporter gene assays
HEK293 cells (2.5 × 105Renilla luciferase activity and expressed as the mean relative stimulation ± SD.
Human TLR2- and EBV-encoded dUTPase blocking experiments
For blocking experiments, HEK293 and TLR2-expressing HEK293 cells were transiently transfected with pNFκB-Luc and pRL-TK reporter vectors as described above. At 24–36 h after transfection, cells were pretreated (10–20 μg/ml) with either anti-human TLR2 mAb (anti-TLR2 mAb; IgG2a, clone TL2.1) or 7D6 anti-EBV-encoded dUTPase Abs and their respective IgG2a or IgG1 isotype control Abs for 1 h at 37°C and subsequently treated with EBV-encoded dUTPase (10 μg/ml) or left untreated for 8 h. After treatment, cell lysates were prepared, and neutralization of TLR2- and EBV-dUTPase-mediated activation of reporter gene activities was determined using the dual-luciferase reporter assay as described above. Data were normalized for transfection efficiency by measuring Renilla luciferase activity and expressed as the mean relative stimulation ± SD.
Human MDM were incubated with anti-TLR2 mAb, 7D6 mAb, anti-TLR6 polyclonal Ab (pAb), or isotype control (IgG2a, IgG1) Abs (10–20 μg/ml) for 1 h before treatment with full-length EBV-encoded dUTPase protein (10 μg/ml) or left untreated. After 24 h, cell culture supernatants from control and treated samples were collected and analyzed for IL-6 using the BD Human Inflammation Cytometric Bead Array Kit, following the recommendation of the manufacturer.
The EBV-encoded dUTPase is not recognized by TLR4 or TLR3
We have previously demonstrated that the EBV-encoded dUTPase induced the expression of proinflammatory cytokines in human macrophages and that the increased cytokine expression was dependent on NF-κB activation (32, 33). Recent reports have indicated that herpesviruses can modulate innate immunity through TLRs (5, 6, 7, 8, 9, 10, 11). To explore the possibility that the EBV-encoded dUTPase could be acting through a TLR, we investigated which TLR might be recognizing the EBV-encoded dUTPase. The initial experiments were performed to address the potential involvement of TLR4, TLR4/MD2, or TLR3 in EBV-encoded dUTPase-mediated activation of NF-κB. For this purpose, HEK293 cells stably expressing TLR4, TLR4/MD2 or TLR3 were transiently transfected with pNF-κB-Luc and the transfection efficiency control pRL-TK reporter vectors, and treated with the EBV-encoded dUTPase; the activation of NF-κB luciferase reporter gene was measured as described in Materials and Methods. Treatment of TLR4-HEK293 cells or TLR4/MD2-expressing cells with the EBV-encoded dUTPase did not result in the activation of the NF-κB promoter (Fig. 1⇓A). Conversely, LPS treatment of TRL4/MD2 expressing cells, a ligand for TLR4, resulted in a 16-fold induction of NF-κB activity when compared with untreated cells. These results confirm our earlier studies, which demonstrated that the ability of the EBV-encoded dUTPase to induce NF-κB activity was not due to contaminating LPS. Likewise, treatment of TLR3-expressing HEK293 cells with the EBV-encoded dUTPase did not cause activation of NF-κB, whereas treatment of these cells with poly(IC), a ligand for TLR3 resulted in a 12-fold increase in luciferase gene reporter activity (Fig. 1⇓B).
The EBV-encoded dUTPase is recognized by TLR2
To determine whether EBV-encoded dUTPase could activate NF-κB through TLR2, HEK293-stable cell lines expressing either TLR2 or TLR2/CD14 were transiently transfected with vectors encoding the NF-κB luciferase reporter gene, transfection control pRL-TK and cotransfected with either empty vector or MyD88 dominant-negative (MyD88DN) construct followed by 8 h of treatment with the EBV-encoded dUTPase (0–10 μg/ml), zymosan (a natural ligand for TLR2), or no treatment. As shown in Fig. 2⇓, treatment of TLR2 (Fig. 2⇓A)- or TLR2/CD14 (Fig. 2⇓B)-expressing cells with various concentrations of the EBV-encoded dUTPase resulted in the stimulation of NF-κB activity in a dose-dependent manner ranging from 2- to 91-fold induction in TLR2-HEK293 (Fig. 2⇓A) and 1.5- to 85-fold induction in TLR2/CD14-HEK293 (Fig. 2⇓B) for 1- and 10-μg/ml EBV-encoded dUTPase concentrations, respectively when compared with that of the untreated control. In addition, only overexpression of NF-κB subunit p65 in wild-type HEK293 cells was able to induce NF-κB promoter activity by 13-fold (Fig. 2⇓C), whereas EBV-encoded dUTPase (10 μg/ml) treatment of these cells failed to do so. Furthermore, the results from this experiment indicate that CD14 is not required for EBV-encoded dUTPase-mediated induction of NF-κB.
We next examined whether the activation of NF-κB by the EBV-encoded dUTPase involved the adaptor molecule MyD88. When TLR2-expressing HEK293 cells were cotransfected with either empty vector or MyD88DN followed by 8 h of treatment with the EBV-encoded dUTPase (10 μg/ml), zymosan, or no treatment, we found that overexpression of the MyD88DN plasmid prevented the activation of NF-κB by both EBV-encoded dUTPase and zymosan treatments (Fig. 3⇓A). Similar findings were observed in the TLR2/CD14-HEK293 stable cell line following EBV-encoded dUTPase treatment (data not shown). Altogether, these data suggest that the dUTPase-mediated activation of NF-κB is TLR2-MyD88 signal mediated.
To further confirm that the EBV-encoded dUTPase is recognized by TLR2, blocking experiments using anti-TLR2 or isotype control Abs were performed. TLR2-expressing HEK293 cells were transiently transfected with the pNF-κB luciferase reporter and transfection control pRL-TK plasmids and incubated with anti-TLR2 or IgG2a isotype control Abs (10–20 μg/ml), 1 h before EBV-encoded dUTPase or zymosan treatment for an additional 8 h. Wheereas EBV-encoded dUTPase lead to the activation of NF-κB in cells preincubated with isotype control Abs, pretreatment of TLR2-expressing HEK293 cells with blocking anti-TLR2 (10 and 20 μg/ml) Abs significantly decreased EBV-encoded dUTPase-mediated activation of NF-κB by 6- and 15-fold, respectively (Fig. 3⇑B). Similarly, pretreatment of TLR2-HEK293 cells with anti-TLR2 Ab resulted in the blockade of NF-κB promoter activity by zymosan (Fig. 3⇑B). These results further demonstrate that the EBV-encoded dUTPase-mediated induction of NF-κB is TLR2 signal dependent.
To conclusively demonstrate that the activation of NF-κB in TLR2-HEK293 cells is in fact mediated by EBV-encoded dUTPase, blocking experiments using the 7D6 Ab specific for EBV-encoded dUTPase were performed. As described above, TLR2-expressing HEK293 cells were transiently transfected with the pNF-κB luciferase reporter and transfection control pRL-TK plasmids and incubated with 7D6 or IgG1 isotype control Abs (10 μg/ml), before treatment of cells with EBV-encoded dUTPase or zymosan for an additional 8 h. Whereas the isotype control Ab pretreatment had no effect on the ability of EBV-encoded dUTPase to stimulate NF-κB reporter activity, the 7D6 Ab effectively blocked/inhibited NF-κB activation by EBV-encoded dUTPase (Fig. 3⇑C). Furthermore, the 7D6 Ab did not interfere with zymosan-mediated activation of NF-κB. These results clearly demonstrate that NF-κB activation in TLR2-expressing HEK293 cells is EBV-encoded dUTPase mediated.
Production of IL-6 by EBV-encoded dUTPase stimulation of hMDM is mediated by TLR2
We have previously demonstrated that EBV-encoded dUTPase stimulates the secretion of proinflammatory cytokines in human PBMCs and hMDM (32, 33); however, it is not known whether this process is TLR2 dependent. To determine whether there is a link between EBV-encoded dUTPase-mediated production of cytokines and activation of TLR2 in hMDM, blocking experiments were performed. hMDM were incubated with anti-TLR2 or isotype control (IgG2a and IgG1) Abs for 1 h followed by treatment with full-length EBV-encoded dUTPase protein (10 μg/ml) or left untreated. After 24 h, cell culture supernatants from control and treated samples were collected and analyzed for IL-6 levels using the BD Human Inflammation Cytometric Bead Assay Kit. We chose to measure IL-6 as a representative marker of EBV-encoded dUTPase induction of this group of cytokines. As shown in Fig. 4⇓, treatment of cells with either the anti-TLR2-mAb or the 7D6 mAb decreased/inhibited the production of IL-6 from hMDM in response to EBV-encoded dUTPase. However, preincubation with the isotype control Abs did not inhibit EBV-encoded dUTPase-mediated stimulation of IL-6 production in hMDM.
TLR2 but not TLR1 or TLR6 is required for EBV-encoded dUTPase-mediated effects
TLR2 functions in combination with TLR1 or TLR6. To determine the possible involvement of these TLRs in EBV-encoded dUTPase signaling, various HEK293 stable cell lines were transiently transfected with NF-κB luciferase reporter, transfection control pRL-TK plasmids and cotransfected with either the pCMV-TLR1 or pCMV-TLR6 expression constructs. Empty pCMV vector was used as a carrier to keep the total amount of DNA-transfected constant. Following transfection, cells were treated with EBV-encoded dUTPase, Pam3CSK4, a TLR1 ligand, or the TLR6 ligand FSL-1 and analyzed for luciferase reporter gene activity. As shown in Fig. 5⇓, transfection of TLR2-expressing HEK293 cells with either TLR1 or TLR6 expression plasmids did not enhance the activation of NF-κB over that of TLR2-expressing HEK293 cells treated with EBV-encoded dUTPase (Fig. 5⇓A), suggesting that neither TLR1 nor TLR6 are required for EBV-encoded dUTPase-mediated induction of NF-κB activity through TLR2. Similar, results were observed in TLR2/CD14-HEK293 cells (data not shown). Conversely, Pam3CSK4 and FSL-1 were able to cause a strong induction of NF-κB activity in TLR2-HEK293 cells transfected with TLR1 and TLR6, respectively (Fig. 5⇓A). Similar findings were observed in the TLR2/TLR6-HEK293-stable cell line following FSL-1 treatment (data not shown). After careful analyses of these data, we made the observation that not only was TLR6 not synergizing with TLR2 in the activation of NF-κB by the EBV-encoded dUTPase but also when overexpressed in HEK293 cells, TLR6 seemed to interfere with the ability of EBV-encoded dUTPase to stimulate NF-κB through TLR2. To explore the possibility that TLR6 could be acting as a negative regulator of TLR2, dose-response experiments in which TLR2-expressing HEK293 cells were transfected with increasing concentrations (0.5–2 μg) of pCMV-TLR6. We noticed that the activation level of NF-κB by the EBV-encoded dUTPase was indeed decreased with increasing concentrations of pCMV-TLR6 expression plasmid (Fig. 5⇓B), which suggests that TLR6 may be acting as a negative regulator of TLR2-mediated activation of NF-κB in response to the EBV-encoded dUTPase. No effect on the ability of EBV-encoded dUTPase to induce NF-κB activation was observed when increasing concentration of TLR1 expression plasmid were transfected in TLR2-HEK293 cells.
To rule out the possibility that the observed negative regulatory effect of TLR6 on EBV-encoded dUTPase-mediated activation of NF- κB could be due to cell death caused by TLR6 overexpression, cell viability assays were performed. For this purpose, TLR2-expressing HEK293 cells were transiently transfected with the pNF-κB luciferase reporter, transfection control pRL-TK plasmids and cotransfected with TLR6 (0.5–2 μg) or empty vector so that the amount of DNA transfected was kept constant in every sample. After 24–36 h, cells were harvested and cell viability was determined by trypan blue exclusion. No cell toxicity/death was found even in cells transfected with 2 μg of TLR6 construct relative to the control cells transfected with the same amount of carrier DNA (data not shown).
To further confirm that TLR6 is a negative regulator of EBV-encoded dUTPase-mediated signaling through TLR2, anti- TLR6 blocking experiments were performed. hMDM were incubated with anti-TLR6-PAb or IgG1 isotype control Abs (10 μg/ml) for 1 h followed by treatment with full-length EBV-encoded dUTPase protein (10 μg/ml) or left untreated. After 24 h, cell culture supernatants from control and stimulated samples were collected and analyzed for the presence of IL-6 by ELISA. As shown in Fig. 5⇑C, preincubation of hMDM with anti-TLR6-PAb followed by treatment with the EBV-encoded dUTPase resulted in enhanced IL-6 production. However, incubation with the isotype control Ab did not have an effect on EBV-encoded dUTPase-mediated stimulation of cytokine secretion in hMDM. These data demonstrate that TLR6 is a negative regulator of EBV-encoded dUTPase-mediated induction of NF-κB through TLR2.
TLRs have been demonstrated to play an important role in modulating innate recognition of viruses not only by serving as pathogen sensors but also by activating signaling pathways that result in the increased production of various cytokines and chemokines and the induction of antiviral immune responses. We reported previously that stimulation of hMDM with the EBV-encoded dUTPase led to the increased expression and secretion of TNF-α, IL-1β, IL-6, and IL-8 as well as IL-10 and that this induction was dependent on NF-κB activation (32, 33). To determine whether the observed EBV-encoded dUTPase-mediated biological effects might be occurring through the activation of a TLR signaling pathway, we performed luciferase reporter gene assays and blocking experiments using various TLR-HEK293-stable cell lines, TLR-expressing vectors, and plasmids expressing different adaptor molecules. Using this model system, we demonstrate that the EBV-encoded dUTPase, a nonstructural protein expressed during lytic replication of EBV, activates NF-κB in a dose-dependent manner through TLR2 and that this activation is dependent on the adaptor molecule MyD88. To our knowledge this is the first demonstration that a nonstructural protein encoded by EBV functions as a PAMP.
Of the TLRs, TLR2 senses the most diverse group of PAMPs including the structural proteins of several viruses (40, 41, 42, 43), as well as the human herpesviruses (5, 6, 7, 8, 9, 10). Although the structural component(s) important for the recognition of HSV-1, HSV-2, and VZV have not been identified, gB and gH of hCMV were reported to interact with the TLR2/TLR1 heterodimer (6), and recently it was suggested that TLR2 senses EBV through the recognition of gp350 (11). Sensing of human herpesviruses by TLR2 results in the activation of an inflammatory response (5, 6, 7, 8, 9, 10); and although this is usually a protective mechanism, in the case of HSV-1 a TLR-2-mediated cytokine response contributes to neuropathogenesis in the host (44, 45, 46).
In this study, we demonstrate that the EBV-encoded dUTPase-mediated biological effects are induced by triggering TLR2 but not TLR1 or TLR6 signaling pathways. Furthermore, our findings also demonstrate that CD14 is not required for the recognition of EBV-encoded dUTPase by TLR2. Although CD14 is a component of the TLR2 complex (47) and may participate in signaling after stimulation by some viruses, such as VZV or hCMV, there was no significant difference in the activation levels of the NF-κB reporter gene in TLR2-expressing HEK293 cells when compared with that of TLR2/CD14-expressing HEK293 cells, suggesting that CD14 is not required for TLR2 activation by the EBV-encoded dUTPase. Similarly, there was no significant difference in the expression levels of the NF-κB reporter gene in TLR2- or TLR2/CD14-expressing HEK293 cells transfected with TLR1 relative to the controls, suggesting that TLR1 is not involved in EBV-encoded dUTPase signaling. Conversely, the overexpression of TLR6 in TLR2- or TLR2/CD14-expressing HEK293 cells resulted in a decreased activation of the NF-κB reporter gene following treatment with the EBV-encoded dUTPase when compared with controls. Although the reason for this interference is unknown, one possibility is that the TLR2-TLR6 complex cannot recognize the EBV-encoded dUTPase and thus the overexpression of TLR6 results in the depletion of functional/active TLR2 due to TLR2-TLR6 complex formation.
It is well established that EBV can productively infect monocytes and macrophages and that following infection there are changes in the expression of various cytokines/chemokines (22, 23, 24, 48). It has been suggested that EBV induces the production of cytokines/chemokines in monocytes/macrophages by at least two distinct mechanisms (11, 23), the first one is through the interaction of EBV envelope glycoproteins, such as gp350, with cellular receptors, and the second mechanism requires virus replication. However, the viral protein(s) responsible for eliciting the increased production of cytokines/chemokines remains unknown. Our data demonstrate conclusively that the EBV-encoded dUTPase activates TLR2 in a dose-response manner at concentrations that are typically used in vitro to study TLR signaling. In addition, our findings suggest that the release of the EBV-encoded dUTPase from infected cells into the extracellular environment allows it to act as a ligand for TLR2 resulting in the triggering of a signaling cascade that activates NF-κB and induces the production of proinflammatory cytokines such as IL-6 and the anti-inflammatory cytokine IL-10, two key regulators in maintaining the balance between pro- and anti-inflammatory immune responses. A similar mechanism has been used to explain how normal cellular endogenous proteins such as heat shock proteins 60 and 70 activate TLR2 and TLR4 (49, 50). There are numerous reports showing that acute and chronic EBV infections result in changes in the secretion patterns of TNF-α, IL-1β, IL-6, and IL-10 (20, 21, 51, 52, 53, 54, 55, 56, 57, 58). EBV is associated with several malignant diseases including nasopharyngeal carcinoma (NPC), chronic lymphocytic leukemia, and several B cell malignancies such as Burkitt’s lymphoma and posttransplant B cell lymphomas (59, 60, 61, 62, 63). In addition, there is evidence linking serum IL-10 and IL-6 levels with prognosis in some of these cancers. Serum levels of both IL-6 and IL-10 have been correlated with disease outcome in chronic lymphocytic leukemia and Hodgkin’s disease patients with higher levels predictive of a poorer health outcome (55, 57). Serum from patients with NPC have elevated levels of IL-10 that is associated with late-stage disease, which suggests that IL-10 may have a potential role in the progression of NPC tumors (56). Furthermore, examination of tumor biopsies showed that NPC tumor cells were positive for IL-10 (56).
In summary, our results indicate that the expression and release of the dUTPase during the lytic replication of EBV could enhance the expression/secretion of the proinflammatory cytokines TNF-α, IL-1β, IL-6, and IL-8, which may contribute to the pathology observed in infections caused by EBV. Furthermore, these results also suggest that the expression and release of the dUTPase during EBV replication may act as an amplification mechanism to attract cells that could become infected by the virus and possibly result in its dissemination to other sites within the host (64, 65). Perhaps more importantly, the EBV-encoded dUTPase could represent a mechanism to dampen the innate and adaptive immune responses to the virus. We have previously shown that the EBV-encoded dUTPase stimulates cytokine IL-10 production in hMDMs (33). IL-10 affects CD8+ T cell function and its increased expression is the primary evasion mechanism used by viruses to establish persistent infections (66). Although additional studies are necessary to define the signaling pathways activated by EBV-encoded dUTPase and to determine how dUTPase modulates immune responses to EBV infections, our results suggest that the dUTPase could be a potential target for the development of novel agents that may be used to treat infections caused by EBV.
We are grateful to Dr. Friedrich Grässer (Institute of Microbiology and Hygiene, Universitätskliniken, Homburg/Saar, Germany) and Dr. Elisabeth Kremmer and Dr. Andrew Flatley (Institute of Molecular Immunology, Hannover Medical School, Hannover, Germany) for providing the 7D6 mAb.
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵1 This work was supported in part by funds from the University of South Carolina Centenary Plan (to M.E.A.) and by Grants AG16321 and DE13749 (National Institutes of Health), The Gilbert and Kathryn Mitchell Endowment, and Ohio State University Comprehensive Cancer Center Core Grant CA16058 (to R.G.).
↵2 Address correspondence and reprint requests to Dr. Maria-Eugenia Ariza, Department of Environmental Health Sciences, Arnold School of Public Health, University of South Carolina, 921 Assembly Street, Columbia, SC 29208. E-mail address:
↵3 Abbreviations used in this paper: PAMP, pathogen-associated molecular patterns; PRR, pattern recognition receptor; Pam3Csk4, N-palmitoyl-S-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-(R)-Cys-(S)-Ser-(S)-Lys4 trihydrochloride; VZV, varicella-zoster virus; hCMV, human CMV; gp350, gp350/220; hMDM, human monocyte-derived macrophage; poly(IC), polyinosinic-polycytidylic acid; pAb, polyclonal Ab; NPC, nasopharyngeal carcinoma.
- Received March 6, 2008.
- Accepted November 12, 2008.
- Copyright © 2009 by The American Association of Immunologists, Inc.