Human Cytomegalovirus Attenuates Interleukin-1 and Tumor Necrosis Factor Alpha Proinflammatory Signaling by Inhibition of NF-B Activation
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《病菌学杂志》
Vaccine and Gene Therapy Institute Department of Molecular Microbiology and Immunology
Department of Cell and Developmental Biology, Oregon Health Science University, Portland, Oregon
Department of Pediatrics, University of Alabama, Birmingham, Alabama
ABSTRACT
Viral infection is associated with a vigorous inflammatory response characterized by cellular infiltration and release of the proinflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-). In the present study, we identified a novel function of human cytomegalovirus (HCMV) that results in inhibition of IL-1 and TNF- signaling pathways. The effect on these pathways was limited to cells infected with the virus, occurred at late times of infection, and was independent of cell type or virus strain. IL-1 and TNF- signaling pathways converge at a point upstream of NF-B activation and involve phosphorylation and degradation of the NF-B inhibitory molecule IB. The HCMV inhibition of IL-1 and TNF- pathways corresponded to a suppression of NF-B activation. Analysis of IB phosphorylation and degradation suggested that HCMV induced two independent blocks in NF-B activation, which occurred upstream from the point of convergence of the IL-1 and TNF- pathways. We believe that the ability of HCMV to inhibit these two major proinflammatory pathways reveals a critical aspect of HCMV biology, with possible importance for immune evasion, as well as establishment of infection in cell types persistently infected by this virus.
INTRODUCTION
Interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-) signaling pathways of the innate immune response are critical antimicrobial responses that have been highly conserved (37). In higher vertebrates, these pathways have evolved to serve as a critical interface between the innate and adaptive immune system (25). IL-1 and TNF- signaling is an area of extensive research that has resulted in a considerable understanding of the signaling events involved in these pathways (1, 9). Activation of IL-1 and TNF- pathways is initiated by proinflammatory cytokines IL-1/ and TNF- binding their cognate receptors following release from activated macrophages and T lymphocytes at sites of infection. Signal transduction via IL-1 and TNF- pathways involves activation of pathway-specific receptor proximal kinases and ubiquitin E3 ligases. IL-1 and TNF- signaling pathways converge more distally at the level of mitogen-activated protein kinase kinase kinases (MAP3Ks), leading ultimately to the activation of MAPKs (p38, c-Jun NH2-terminal kinase [JNK], and extracellular signal-regulated kinase) and the transcription factor nuclear factor-B (NF-B) (47, 55). Together, MAPKs and NF-B induce expression of inflammatory chemokines (e.g., IL-8, IL-6, growth-regulated oncogene alpha [GRO-], and monocyte chemoattractant protein 1 [MCP-1]), as well as adhesion and costimulatory molecules that recruit and activate various aspects of the innate and adaptive immune response.
MAPKs are activated by phosphorylation on specific threonine and tyrosine residues in a conserved "activation loop" of the MAPK kinase domain (34). Following activation, MAPKs induce the expression of IL-1- and TNF--responsive genes by phosphorylating a number of transcription factors, including Jun, Fos, and ATF family members. NF-B is a critical transcription factor that serves as a major modulator of inflammation, immune regulation, differentiation, and apoptosis (19, 24). NF-B induces target gene expression by binding to NF-B binding sites in the promoter region of NF-B-responsive genes. The NF-B molecule is a homo- or heterodimer of Rel family members, with the most common form being comprised of a heterodimer between RelA (p65) and p50. In nonactivated cells, NF-B resides in an inactive cytoplasmic complex with an inhibitory molecule, IB, which masks the NF-B nuclear localization sequence. NF-B activation results from the phosphorylation, ubiquitination, and subsequent proteosomal degradation of IB. IB degradation appears to represent the rate-limiting step in activation of NF-B, and following release from IB, NF-B translocates to the nucleus to directly induce target gene expression (24).
The importance of the IL-1 and TNF- pathways for infectious organisms can be seen by the large number of bacteria (13) and viruses (23) that have evolved mechanisms to modulate (both activate and suppress) these signaling pathways. Cytomegaloviruses (CMVs), a family of ubiquitous DNA viruses, have coevolved with their host (36), which has resulted in a considerable level of coadaptation between host and virus. A critical aspect of this coevolution appears to be the ability of CMV to modulate components of the IL-1 and TNF- signaling pathways. To date, most studies have focused on the activation of these pathways by human cytomegalovirus (HCMV) infection. At early times after infection, numerous studies have shown that HCMV activates signaling components of both IL-1 and TNF-, including NF-B (32, 45, 56, 57, 59) and various MAPKs (29, 30, 43, 56). This activation occurs in two distinct phases, with initial activation being mediated by virus attachment and entry (6, 12, 57). A second phase of activation occurs following virus entry and is mediated, at least in part, by major transactivators of the virus, IE1 and IE2 (10, 45, 59). These activation events have been proposed as necessary for the production of a cellular environment conducive to initial viral replication. IL-1 and TNF- pathway activation may also facilitate virus dissemination through recruitment of HCMV-susceptible cells by increasing the secretion of chemokines (IL-8, IL-6, RANTES, and MCP-1) (2, 4, 12, 18, 20, 22, 40, 41).
More recent studies are beginning to reveal an ability of HCMV to suppress IL-1 and TNF- signaling pathways at later times of infection (3, 22). Specifically, HCMV has been shown to decrease TNF--mediated signaling, which corresponded to a down-modulation of TNFR1 surface expression and decreased activation of the MAPK JNK (3). Similarly, HCMV has been shown to block both IL-1- and TNF--induced expression of the chemokine MCP-1 at the level of transcription (22). The precise mechanisms involved in these inhibitory effects of HCMV on the IL-1 and TNF- pathways are not completely resolved, and the breadth of the suppression on the proinflammatory response has not been investigated. However, these observations suggest a requirement for suppression of proinflammatory signaling pathways at later stages of the HCMV replication cycle.
In the current study, we have further examined the ability of HCMV to modulate the IL-1 and TNF- proinflammatory signaling pathways. Our results show that HCMV suppresses both IL-1 and TNF- signaling pathways at late times of infection. This inhibitory effect corresponded to impaired IL-1- and TNF--induced IB phosphorylation and NF-B activation and decreased expression of multiple inflammatory chemokines. Surprisingly, HCMV infection resulted in a differential effect on the IL-1 pathway compared to the TNF- pathway, with a greater effect on IB phosphorylation and NF-B activation observed for the IL-1 signaling pathway. This differential inhibition of IL-1 and TNF- signal transduction suggests that HCMV modulates these pathways at distinct positions upstream from the point of pathway convergence. A number of studies indicate that endothelial cells represent an important site of HCMV persistent replication in vivo (16, 21, 42, 49, 50). The observation that HCMV suppression of IL-1 and TNF- signaling pathways occurs at late times of infection within this cell type suggests that the ability of HCMV to regulate these pathways may represent a critical adaptation to enable virus persistence within the host.
MATERIALS AND METHODS
Cells and virus. HCMV laboratory strains Towne and AD169 were obtained from the American Type Culture Collection (Manassas, VA). HCMV strain TR is a low-passage clinical isolate obtained from an AIDS patient that we have cloned as a bacterial artificial chromosome. TR retains all characteristics of a parental virus, including tropism for endothelial cells and macrophages. HCMV strains were propagated and titrated in normal human foreskin fibroblast (HFF) cells by standard methods (27). HFF cells were cultured at 37°C in an atmosphere of 5% CO2 in complete medium (Dulbecco modified Eagle medium containing 10% fetal bovine serum and supplemented with 4 mM L-glutamine, 200 μg/ml penicillin G, and 200 μg/ml streptomycin sulfate). Primary human umbilical vein endothelial cells (HUVECs) (Cascade Biologics, Portland, OR), retinal (HREC-181) (Cell Systems Corporation, Kirkland, WA) endothelial cells, and telomerase life-extended tAEC-5A endothelial cells were cultured in microvascular endothelial growth medium (EGM-2 MV; Cambrex Bioscience Rockland, Inc., Rockland, ME).
Analysis of IL-1- and TNF--induced chemokine mRNA levels by quantitative real-time reverse transcription-PCR (qRT-PCR). Subconfluent HFF cells cultured in six-well plates were either infected with HCMV at a multiplicity of infection (MOI) of 3 or mock infected. At day 3 postinfection (p.i.), cells were treated with IL-1 (1 ng/ml) (R&D Systems, Inc., Minneapolis, MN) or TNF- (10 ng/ml) (R&D Systems, Inc.). Cytokines were added to the cells directly into the media overlying the cells either with or without changing the media, depending on the experiment. However, in any particular experiment, all wells were treated in an identical fashion other than for the addition of the proinflammatory cytokine. At 1 and 3 h posttreatment, cells were harvested using TRIzol (Invitrogen, Carlsbad, CA), duplicate wells were pooled, and RNA was extracted using the manufacturer's protocol. RNA was quantified by absorbance at an optical density at 260 nm, and 2 μg of DNase-treated RNA was reverse transcribed using OmniScript (Sigma-Aldrich, St. Louis, MO) and oligo(dT) primers (Invitrogen, Carlsbad, CA). The resulting cDNA product was amplified using the ABI Prism 7700 sequence detector in the presence of 2x SYBR green master mix (Applied Biosystems, Foster City, CA) and 0.25 μM of forward and reverse primers, resulting in 120-bp amplicons. For each primer set, gene amplification in 40 cycles of PCR was measured by absolute quantification and then normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) levels to determine induction (n-fold). The denaturing and annealing/extension conditions for each cycle of PCR were 95°C for 15 s and 60°C for 1 min, respectively. The primer sets used for each gene of interest were as follows: GAPDH, 5'-TGACCTCAACTACATGGTTTACATGT-3' and 5'-AGGGATCTCGCTCCTGGAA-3'; IL-8, 5'-CAACACAGAAATTATTGTAAAGCTTTCT-3' and 5'-GAATTCTCAGCCCTCTTCAAAAA-3'; MCP-1, 5'-TCGCTCAGCCAGATGCAAT-3' and 5'-ATGGTCTTGAAGATCACAGCTTCTT-3'; IL-6, 5'-GGATTCAATGAGGAGACTTGCC-3' and 5'-ACAGCTCTGGCTTGTTCCTCAC-3'; and GRO-, 5'-CCGAAGTCATAGCCACACTCA-3' and 5'-TGGATTTGTCACTGTTCAGCATC-3'.
ELISA analysis of IL-8 levels. HFF cells and HUVECs were infected with HCMV at an MOI of 3 or mock infected, followed by treatment with IL-1 or TNF- at day 3 p.i. After 6 h, supernatants were harvested, clarified by centrifugation, and stored at –80°C prior to analysis. The concentration of IL-8 released from cells was determined by enzyme-linked immunosorbent assay (ELISA) using the human IL-8 screening set (Endogen Inc., Rockford, IL) following the manufacturer's protocol. Plates were analyzed at 492 nm using a SpectraMax 190 plate reader (Molecular Devices Corp., Sunnyvale, CA), and levels of IL-8 released were determined by comparison to a standard curve.
Western analysis of viral and cellular proteins. Following IL-1 and TNF- treatment, cell monolayers were washed twice with Dulbecco's phosphate-buffered saline and harvested at the times indicated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer containing a protease inhibitor cocktail (0.5 μg/ml leupeptin, 0.7 μg/ml pepstatin, 2 μg/ml aprotinin, 10 mM phenylmethylsulfonyl fluoride) with 10 mM Na orthovanadate and 50 mM NaF. In designated experiments, the proteosome inhibitor epoxomicin (1 μM) (Peptide Institute, Osaka, Japan) was added to cells 1 h prior to IL-1 or TNF- treatment. Proteins were separated by SDS-PAGE, electrophoretically transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA), and blocked. Viral and cellular proteins were detected using the following primary antibodies (Abs): rabbit anti-immediate-early HCMV IE2 (R638; 1:1,000) (15), rabbit anti-IB (C-15; 1:400) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), mouse anti-phospho-IB (B-9; 1:150) (Santa Cruz Biotechnology, Inc.), rabbit anti-p65 NF-B (C-20; 1:2,000) (Santa Cruz Biotechnology, Inc.), and rabbit anti-p38 (C-20; 1:2,000) (Santa Cruz Biotechnology, Inc.). Primary Abs were detected with species-specific horseradish peroxidase-conjugated secondary Abs (1:1,500 to 1:2,500) (Amersham, Piscataway, NJ) and visualized using West Pico chemiluminescence substrate (Pierce, Rockford, IL).
IF microscopy. After fixation in 4% paraformaldehyde in Dulbecco's phosphate-buffered saline, localization of viral and cellular proteins in cells treated with IL-1 and TNF- was determined by indirect immunofluorescence (IF) microscopy, as previously described (26). Cells were harvested at 6 h posttreatment for analysis of IL-8 and 30 min posttreatment for analysis of NF-B translocation. Primary Abs used were as follows: rabbit anti-IE2 (R638; 1:300), rabbit anti-p65 NF-B (C-20; 1:300), mouse anti-gB (27-156) (1:150) (27), and mouse anti-IL-8 (clone 6217; 1:100) (R&D Systems, Inc.). After incubation with the appropriate fluorescently labeled species-specific secondary Ab, fluorescence was visualized using an Olympus confocal microscope.
RESULTS
HCMV inhibits IL-1- and TNF--mediated induction of multiple inflammatory genes. A unique characteristic of HCMV is the ability of the virus to persist within the host, even in the presence of vigorous inflammatory responses, such as those observed in the tissues of transplanted organs during chronic rejection and transplant vascular sclerosis (28). We hypothesized that HCMV possesses an ability to modulate the innate immune response, which may be critical for persistence of the virus within such harsh proinflammatory environments. To examine whether HCMV was able to modulate the inflammatory environment by interfering with signaling pathways that contribute to cellular responses to soluble mediators of inflammation, we determined the level of inflammatory chemokine induction following treatment of HCMV-infected cells with the two major proinflammatory mediators IL-1 and TNF-. HFF cells were infected with HCMV strain TR at an MOI of 3 or mock infected. At 3 days p.i., cells were treated with either IL-1 (1 ng/ml) or TNF- (10 ng/ml), harvested at 1 and 3 h postinduction, and analyzed for the level of chemokine mRNA induction by quantitative real-time RT-PCR. For these studies, we focused on the induction of four inflammatory chemokines (IL-8, IL-6, MCP-1, and GRO-), which are major mediators of the inflammatory response, being involved in both the recruitment and activation of multiple components of the immune system (5). The level of chemokine induction was normalized to GAPDH, and results are presented as induction (n-fold) compared to basal levels prior to IL-1 or TNF- addition. The GAPDH levels were comparable in HCMV- and mock-infected cells and were not affected by treatment with either IL-1 or TNF- (data not shown). Treatment of mock-infected cells with either IL-1 or TNF- induced mRNA expression of all inflammatory chemokines studied (IL-8, IL-6, MCP-1, and GRO-), consistent with the known positive regulation of the expression of these chemokines by IL-1 and TNF- (Fig. 1). In contrast, cells infected with HCMV showed a decrease in response to both proinflammatory cytokines (IL-1 and TNF-), as observed by the minimal induction of IL-8, IL-6, MCP-1, or GRO-. At the time of harvest, HCMV-infected cells were >90% viable by trypan blue exclusion, and absolute basal levels of IL-8, IL-6, MCP-1, and GRO- prior to IL-1 and TNF- treatment were comparable to those of mock-infected cells (data not shown). These results indicate that the effect of HCMV on chemokine induction in infected cells did not result from either increased cell death or a maximal chemokine expression making cells refractory to IL-1 or TNF- chemokine induction. Together, these results reveal an ability of HCMV to suppress the induction of multiple inflammatory chemokines by the two major proinflammatory cytokines IL-1 and TNF-.
HCMV inhibition of IL-1 and TNF- proinflammatory pathways corresponds to a decreased release of IL-8 from infected cells. To further investigate the characteristics of the HCMV inhibition of proinflammatory pathways, we focused on the effect of HCMV on IL-8 expression. IL-8 was selected as an IL-1- and TNF--inducible gene marker, since this chemokine is an extensively studied prototypical inflammatory chemokine that is highly induced by both proinflammatory cytokines (24) and whose expression following induction with IL-1 and TNF- was observed to be decreased in HCMV-infected cells (Fig. 1). To determine whether the effect of HCMV on IL-8 expression observed by qRT-PCR corresponded to a decrease in IL-8 release, we used an ELISA to measure the levels of IL-8 released into the supernatant of HCMV-infected cells following IL-1 and TNF- induction. Since endothelial cells are a biologically relevant cell type for HCMV infection that represent a site of persistent HCMV infection within the host, the effect of HCMV infection on IL-8 release was analyzed in HUVECs as well as HFF cells. Cells were infected with HCMV TR at an MOI of 3 or mock infected. At day 3 p.i., cells were treated with either IL-1 or TNF-, and the concentration of IL-8 released into the supernatant was quantified at 6 h postinduction. As shown in Fig. 2, treatment of mock-infected cells with either IL-1 or TNF- resulted in a significant release of IL-8 from both HUVECs and HFF cells. However, consistent with the results from the qRT-PCR, both HCMV-infected HUVECs and HCMV-infected HFF cells released reduced levels of IL-8. Together, these results show that the effect of HCMV on proinflammatory cytokine induction of chemokine expression corresponds to a decrease in the release of chemokines from infected cells and that this effect appears to be independent of cell type.
HCMV inhibition of IL-1 and TNF- proinflammatory pathways occurs at later times after infection. Typical of all herpesviruses, HCMV genes can be characterized into three kinetic classes (immediate early [IE], early [E], and late [L]) based on time of viral gene expression. To determine the stage of the viral life cycle that corresponded to the HCMV-mediated inhibition of the proinflammatory pathways, we analyzed the induction of IL-8 in infected cells by IF microscopy. Cells were infected with HCMV TR at an MOI of 0.5 and induced at day 3 p.i. for 6 h with either IL-1 or TNF-. Cells were then fixed and stained for IL-8 as well as HCMV IE (IE2) and L (gB) antigens. The lower MOI utilized in these studies ensured the presence of HCMV-infected cells at different stages of the viral life cycle secondary to the spread of HCMV from infected to surrounding noninfected cells. Figure 3A shows a typical IF micrograph displaying the characteristic IL-8 expression in HCMV-infected cells (HUVECs) following proinflammatory cytokine induction (in this case, IL-1). A similar effect of HCMV on IL-8 expression was observed following TNF- treatment and in a number of different cell types (HFF and primary retinal microvascular [HREC-181] and telomerase life-extended aortic macrovascular endothelial cells [tAEC-5A]) following IL-1 or TNF- treatment (data not shown). In all cases, IL-8 expression was evident in both noninfected and HCMV-infected cells expressing only the IE antigen IE2. In contrast, HCMV-infected cells that had progressed to the stage of E/L gene (gB) expression were negative for IL-8 following induction with either IL-1 or TNF-. These results show that the inhibitory effects of HCMV on the proinflammatory cytokine pathways occurred at later times of HCMV infection, corresponding to the time of gB expression. Since the inhibitory effect was restricted to only a defined population (i.e., gB-expressing cells) within the culture, these results also show that the suppressive effect of HCMV required direct infection of the host cell. This latter observation lessens the likelihood of a paracrine mechanism mediated by a soluble factor released from HCMV-infected cells.
HCMV inhibition of IL-1 and TNF- proinflammatory pathways is HCMV strain independent. To determine whether the HCMV-mediated inhibition of proinflammatory pathways was unique to the TR strain or was a general characteristic of HCMV, the ability of additional HCMV strains to prevent IL-8 induction was assessed by IF microscopy as described above. HFF cells were infected at an MOI of 0.5 with two laboratory strains (AD169 and Towne). Cells were then fixed and stained for IL-8, IE2, and gB after induction with IL-1 and TNF- at day 3 p.i. All viruses exhibited an inhibitory effect on IL-1 (Fig. 3B) and TNF- (data not shown) induction of IL-8. Similar to the TR strain, the effect on IL-8 induction by these viruses corresponded to gB expression at later times of infection. These results indicate that HCMV inhibition of proinflammatory pathways is a general characteristic of HCMV that is conserved between multiple strains.
HCMV inhibition of IL-1 and TNF- pathways corresponds to an inhibition of NF-B activation. The IL-1 and TNF- signaling pathways converge at the level of the MAP3Ks to activate the downstream transcription factor NF-B. In noninduced cells, NF-B is retained in the cytoplasm by an association with the inhibitory molecule IB. Following proinflammatory cytokine induction, IB is phosphorylated, ubiquitinated, and degraded. This targeted degradation of IB releases NF-B to translocate to the nucleus and activate gene expression (for a review, see reference 19). To determine whether the HCMV-mediated inhibition of proinflammatory pathways corresponded to impaired activation of NF-B, NF-B activation was measured in HCMV-infected cells by visualization of NF-B translocation to the nucleus. Figure 4 shows the effect of HCMV infection on the nuclear translocation of NF-B following 30 min of IL-1 induction. In the absence of induction, NF-B was localized to the cytoplasm regardless of HCMV infection status. However, HCMV infection had a significant effect on the subsequent activation of NF-B by IL-1. In both noninfected cells and HCMV-infected cells expressing only the IE marker IE2, treatment with IL-1 caused rapid relocalization of NF-B to the nucleus. In contrast, in HCMV-infected cells expressing the E/L marker gB, NF-B activation was impaired as shown by the continued localization of NF-B to the cytoplasm. These results show that HCMV suppresses the ability of proinflammatory cytokines to activate the transcription factor NF-B. The effects of HCMV on chemokine induction and NF-B activation closely paralleled one another (compare Fig. 3 and 4), with both events requiring direct infection and occurring in infected cells only at later times of infection. This observation suggests that the suppressive effect of HCMV on IL-1 and TNF- pathways is mediated, at least in part, through an inhibitory effect on the nuclear translocation of NF-B.
Interestingly, although HCMV inhibited the nuclear translocation of NF-B following induction with either IL-1 or TNF-, the effect on TNF- activation of NF-B appeared to be less than complete, with an intermediate phenotype being observed (Fig. 5). This differential effect of HCMV on NF-B activation mediated by IL-1 compared to TNF- suggested that HCMV may be modulating these two proinflammatory pathways independently at distinct points upstream (receptor proximal) of the convergence of these two pathways. The IF-based assay used to analyze NF-B nuclear translocation is inherently qualitative. To determine more definitively whether HCMV was modulating these two pathways independently, we analyzed the degradation of IB following induction with IL-1 or TNF- by Western blotting. The rationale behind this experiment was that independent regulation of these two pathways, occurring upstream of the convergence point, would be suggested by distinct characteristics of IB degradation following IL-1 induction compared to TNF- induction. HCMV- or mock-infected cells were induced at day 3 p.i. with either IL-1 or TNF-. Cells were harvested at increasing times postinduction, and the degradation of IB was measured in cell lysates by Western analysis (Fig. 6A). In mock-infected cells, IL-1 and TNF- induction resulted in comparable and rapid degradation of the IB protein. The small shift in size of IB occurring at 5 min postinduction corresponds to phosphorylation of IB, prior to ubiquitination and degradation. Importantly, the comparable rates in loss of IB observed in mock-infected cells (see Fig. 6A) following treatment with either IL-1 or TNF- indicate that the rates in signal transduction resulting in IB degradation are similar for these two pathways in this cell type. Dose-response analysis between cytokine level and IB degradation also indicated that the concentrations of IL-1 and TNF- used were subsaturating (data not shown). HCMV infection had a significant effect on the ability of IL-1 or TNF- to induce IB degradation. However, although HCMV-infected cells displayed a decreased rate of IB degradation following induction with either IL-1 or TNF-, the block in IB degradation was complete following IL-1 but not TNF- treatment. This observation suggests that the effect of HCMV on these signaling pathways is distinct. In the absence of IL-1 or TNF- treatment, basal levels of total IB were comparable in HCMV-infected and mock-infected cells (Fig. 6A) and were independent of virus strain (Fig. 7). These results revealing distinct characteristics of IB degradation suggest that HCMV affects the IL-1 or TNF- pathway independently at a position upstream from the point of signaling pathway convergence.
To further examine the differential effect of HCMV on the IL-1 or TNF- signaling pathway, the experiment whose results are shown in Fig. 6A was repeated in the presence of the highly specific proteosome inhibitor epoxomicin (38), which serves to stabilize the phosphorylated form of IB. In mock-infected cells, IL-1 and TNF- induction resulted in comparable and rapid phosphorylation of the IB protein, as shown by Western analysis using a phospho-IB-specific Ab (Fig. 6B). Stabilization of the phosphorylated form was also observed by the small shift in IB size occurring at 5 min postinduction. Consistent with the inhibitory effect of HCMV on the degradation of IB (Fig. 6A), phosphorylation of IB was also significantly decreased in HCMV-infected cells. However, the characteristics of the effect on IB phosphorylation following IL-1 induction compared to TNF- induction were distinct, with an absence of IB phosphorylation being observed only following IL-1 induction. This absence of IB phosphorylation and degradation following IL-1 induction (compared to a partial block following induction with TNF-) closely paralleled the effect on NF-B nuclear translocation following induction with IL-1 and TNF-, respectively (Fig. 5). Together, these results demonstrate that HCMV suppresses both the IL-1 and TNF- pathways, resulting in impaired inflammatory chemokine induction, which corresponds to inhibition of NF-B activation. HCMV appears to modulate IL-1 or TNF- signaling independently at a level upstream from the point of convergence of these two pathways.
The viral gene(s) responsible for inhibition of IL-1 and TNF- proinflammatory pathways is expressed with L gene kinetics. The observation by IF microscopy that the inhibitory effect of HCMV on proinflammatory pathways and NF-B activation corresponded to times of gB expression suggested that the viral gene(s) mediating these effects was expressed with either E or L kinetics. To precisely define the kinetic class of the viral gene(s) mediating the inhibitory effect, we assessed the effect of foscarnet, an inhibitor of viral L gene expression, on IB degradation. HFF cells were infected at an MOI of 3 with HCMV and maintained in the presence or absence of foscarnet. At 3 days p.i., cells were treated with either IL-1 or TNF- and harvested at increasing times postinduction for Western analysis of IB degradation, as described above. Foscarnet treatment overcame the inhibitory effect of HCMV on both the IL-1- and TNF- induced degradation of IB (Fig. 8). This finding indicates that the viral gene(s) responsible for inhibition of NFB activation, and presumably chemokine induction, is expressed with L gene kinetics.
DISCUSSION
In the current study, we have observed an ability of HCMV to inhibit the IL-1 and TNF- proinflammatory pathways at late times after infection. This inhibitory effect of HCMV resulted in reduced induction of multiple IL-1- and TNF--responsive target genes, including the major inflammatory chemokines IL-6, IL-8, GRO-, and MCP-1. Inhibition of these signaling pathways corresponded to impaired activation of NF-B, a transcription factor essential for expression of target genes through these pathways. Analysis of phosphorylation and degradation of IB, a negative regulator of NF-B, showed that HCMV prevented the normal destruction of IB in response to IL-1 and TNF-, consistent with the observed block in NF-B activation. However, characteristics of the HCMV modulation of IB destruction following treatment with IL-1, compared to treatment with TNF-, suggested that inhibition of these two closely related proinflammatory pathways was mediated by HCMV at distinct positions upstream from the point of pathway convergence.
Although most previous studies have focused on the ability of HCMV to activate components of these pathways at earlier times of infection (29, 30, 32, 43, 45, 56, 57, 59), more recent studies have indicated that HCMV down-modulates aspects of the proinflammatory response, primarily at later times (3, 22). Specifically, HCMV has been shown to inhibit IL-1 and TNF- induction of MCP-1 expression (22) and TNF- activation of JNK (3). The viral genes involved in the inhibition have not been identified; yet, the effect on MCP-1 was mediated at the level of MCP-1 gene transcription. The effect on TNF- activation of JNK corresponded to a down-modulation of TNFR1 surface expression, resulting from an internalization of the receptor from the cell surface. However, since the ability of TNFR1 expression to "rescue" the effect of HCMV on JNK activation was not determined, the precise role of receptor down-modulation in mediating inhibition of the TNF- signaling pathway is unclear. Our finding that HCMV inhibits proinflammatory pathways further suggests an importance for suppression of signaling components, as well as inflammatory mediators, involved in the IL-1 and TNF- pathways at later stages of the HCMV replication cycle.
IL-1 and TNF- signaling involves closely related pathways that are composed of receptor proximal pathway-specific signaling events that converge at the level of MAP3Ks. Following MAP3K activation, a common signaling pathway is utilized for IL-1 and TNF- activation of NF-B (9). In the current study, the differential characteristics of the inhibition of the IL-1 pathway compared to that of the TNF- pathway observed for IB phosphorylation and degradation, as well as for nuclear translocation of NF-B, suggests that the HCMV-mediated inhibition of these pathways occurs receptor proximal from the point of pathway convergence at the MAP3Ks (Fig. 9). A number of possible mechanisms could be responsible for the HCMV-mediated inhibition. One possibility is that inhibition is due to independent down-modulation of IL-1R1 and TNFR1 surface expression. By fluorescence-activated cell sorter analysis, we observed only background levels of IL-1R1 (and TNFR1) surface expression regardless of infection (data not shown), which prevented the assessment of the effect of HCMV infection on IL-1R1 expression. However, since near-undetectable levels of IL-1R1 expression have been shown to result in normal levels of signaling via IL-1R1 (48), a complete block in signal transduction (as we observed for the IL-1 pathway) via receptor down-modulation would be unexpected. It is possible that the more intermediate inhibitory level of HCMV on TNF- signaling is mediated by the previously identified effect of HCMV infection on TNFR1 surface expression (3). In this earlier study, the effect of HCMV on signaling events following TNF treatment was only partial (assayed in an in vitro JNK assay using glutathione S-transferase-Jun as a substrate). Hence, TNFR1 down-modulation as a mechanism for the inhibition of TNF signaling is consistent with our observation of a partial effect on NF-B signaling after TNF treatment, but other explanations could also account for these data.
The comparable kinetics of inhibition, occurring at late times of infection for both IL-1 and TNF- signaling pathways, suggest that inhibition of both pathways may be mediated by a single viral factor. Although the IL-1 and TNF- pathways utilize distinct signaling molecules upstream from the convergence of these pathways, a number of these components are closely related. For example, both utilize members of a family of closely related TRAF (TNFR-associated factor) proteins (9), with the IL-1 and TNF- pathways utilizing TRAF 6 (35) and TRAF 2/5 (52), respectively. TRAFs are a family of ubiquitin E3 ligases that catalyze a number of essential ubiquitination events required for signal transduction (11, 31). TRAFs contain conserved sequence and structural motifs, including an N-terminal RING domain, several zinc finger motifs, and a C-terminal TRAF domain. Hence, the differential effect on IL-1 and TNF- signaling may be the consequence of a single viral factor exhibiting a difference in affinity for individual TRAF family members. A previous study indicated that a viral tegument protein, pp65, was able to suppress the induction of inflammatory genes that occurs in the absence of cytokine addition, which is observed very early (6 h) following infection (7). This inhibition was suggested to be due to an inhibitory effect of pp65 on NF-B (7). However, the UL83 gene that encodes pp65 is expressed with E kinetics (8). In our study, the ability of foscarnet, which prevents L viral gene expression, to overcome the inhibitory effect of HCMV on IB degradation (Fig. 8) suggests that the inhibitory phenotype at late times of infection is independent of pp65. A more recent study has shown that the immediate-early viral transactivator IE2 can dampen the induction of inflammatory genes that accompanies HCMV infection (53). However, in our studies, the normal response of cells expressing IE2 alone in terms of IL-8 induction and activation of NF-B following cytokine treatment, combined with the ability of foscarnet to reverse the inhibitory effect of HCMV on these signaling pathways, indicates that cells expressing IE2 in the absence of E/L genes have intact responses to IL-1 and TNF-.
The similarity between the inhibitory effects of HCMV on IL-1- and TNF--induced inflammatory chemokine expression and NF-B nuclear translocation suggests that the inhibition of chemokine induction results, at least in part, from inhibition of NF-B activation. However, the comparable suppression levels of IL-1- and TNF--induced inflammatory chemokine expression by HCMV infection (Fig. 1) were surprising given the difference in the level of inhibition of HCMV on IL-1 induction compared to that of TNF- induction of NF-B nuclear translocation (Fig. 4 and 5). In addition to NF-B activation, MAPKs have been shown to be critical for maximal induction of chemokine expression via IL-1 and TNF- pathways (24, 33, 51). Consequently, the comparable inhibitory effects of HCMV on chemokine induction via the IL-1 and TNF- pathways may result from additional viral effects on MAPK activity. The effects of HCMV on MAPK activation via IL-1 and TNF- signaling pathways are unclear. In studies investigating the effect of HCMV infection on basal activity of MAPKs (in the absence of proinflammatory cytokine stimulation), infection has been shown to induce activation of both extracellular signal-regulated kinase (43) and p38 (29). In contrast, a recent study identified a biphasic effect of HCMV on expression of the inflammatory chemokine IL-6, with induction at early times of infection being followed by suppression at later times. This suppression corresponded to a decrease in the stability of IL-6 chemokine mRNA transcript (18), which is regulated by p38 MAPK (17). The inhibitory effect of HCMV on TNF- signal transduction, which was correlated to TNFR1 down-regulation, corresponded to a block in JNK activation (3).
The difference between the level of chemokine suppression and inhibition of NF-B nuclear translocation following TNF- treatment may, alternatively, indicate additional levels of HCMV-mediated modulation of NF-B activity. Recent studies have shown that NF-B activity is regulated at multiple levels, with IB-mediated cytoplasmic localization of NF-B representing only one tier of regulation (14, 44, 46, 60). Direct phosphorylation of the NF-B molecule at multiple sites has been shown to both positively and negatively regulate NF-B activity. Phosphorylation of NF-B is mediated by a variety of kinases (54), which could serve as potential targets for additional HCMV-mediated modulation of the pathway.
The apparent differential effect on the IL-1 signaling pathway compared to that on the TNF- signaling pathway is intriguing. This differential effect may reflect the proapoptotic signaling associated with TNF-, but not IL-1, signal transduction pathways in the absence of NF-B activation. Recently, the requirement for NF-B activity was shown to result from NF-B-mediated induction of antiapoptotic molecules that regulate cell survival following activation of the TNF- signaling pathway (39). TNFR1 engagement was shown to result in the formation of two distinct complexes: a prosurvival complex (complex I) and a proapoptotic complex (complex II or death-inducing signaling complex). Complex I formed rapidly following receptor engagement and resulted in NF-B activation. In contrast, complex II assembled more slowly and resulted in apoptosis. Importantly, NF-B activity was shown to be required for the induction of antiapoptotic molecules, primarily FLIPL, which modified the function of complex II and prevented cell death. Hence, in our study, the intermediate effect of HCMV on NF-B activity following TNF- may reflect the requirement for a necessary level of NF-B induction of antiapoptotic molecules to prevent cell death. In contrast, since apoptotic signals are not associated with IL-1 signaling pathways, a complete blockade of NF-B activation can be induced without any negative apoptotic ramifications. In both cases, the virus has evolved to maximize the level of NF-B suppression within the constraints of a requirement to avoid induction of apoptosis.
In the current study, the inhibitory effect of HCMV on IL-1 and TNF- signaling pathways corresponded to an inhibition of NF-B activation. This finding was surprising given the numerous studies showing NF-B activation following HCMV infection. These previous studies have focused primarily on early times of infection and led to the development of a model wherein HCMV mediates a biphasic activation of the NF-B pathway (58). The first phase occurs during virus entry (12, 56, 57) and is mediated by the major viral envelope glycoproteins gB and gH (56, 57). The second phase requires protein synthesis and is mediated by induction of de novo NF-B synthesis (32, 58). Our current study indicates a further complexity in HCMV-mediated modulation of components of the IL-1 and TNF- signaling pathways, with an importance for HCMV down-modulation of these pathways at later times of infection. The requirement for NF-B inhibition at later stages of the HCMV life cycle is probably multifactorial but may be important for functions such as immune evasion and viral persistence. However, given the central role of NF-B in many cellular processes, such as inflammation, immune regulation, differentiation, and apoptosis (19, 24), this HCMV-mediated inhibition of NF-B may also be responsible for various aspects of viral pathology, including developmental and immune dysregulation and tumorigenesis.
ACKNOWLEDGMENTS
We are grateful to Sherry Farley and Veselina Korcheva from the B.E.M. laboratory for technical support and Andrew Townsend at Extreme Images for assistance with graphics illustrations.
This work was supported through funding from the NIH to J.W. and B.E.M. (A1059335), M.A.J. and J.A.N. (AI21640), and W.J.B. (AI35602).
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Department of Cell and Developmental Biology, Oregon Health Science University, Portland, Oregon
Department of Pediatrics, University of Alabama, Birmingham, Alabama
ABSTRACT
Viral infection is associated with a vigorous inflammatory response characterized by cellular infiltration and release of the proinflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-). In the present study, we identified a novel function of human cytomegalovirus (HCMV) that results in inhibition of IL-1 and TNF- signaling pathways. The effect on these pathways was limited to cells infected with the virus, occurred at late times of infection, and was independent of cell type or virus strain. IL-1 and TNF- signaling pathways converge at a point upstream of NF-B activation and involve phosphorylation and degradation of the NF-B inhibitory molecule IB. The HCMV inhibition of IL-1 and TNF- pathways corresponded to a suppression of NF-B activation. Analysis of IB phosphorylation and degradation suggested that HCMV induced two independent blocks in NF-B activation, which occurred upstream from the point of convergence of the IL-1 and TNF- pathways. We believe that the ability of HCMV to inhibit these two major proinflammatory pathways reveals a critical aspect of HCMV biology, with possible importance for immune evasion, as well as establishment of infection in cell types persistently infected by this virus.
INTRODUCTION
Interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-) signaling pathways of the innate immune response are critical antimicrobial responses that have been highly conserved (37). In higher vertebrates, these pathways have evolved to serve as a critical interface between the innate and adaptive immune system (25). IL-1 and TNF- signaling is an area of extensive research that has resulted in a considerable understanding of the signaling events involved in these pathways (1, 9). Activation of IL-1 and TNF- pathways is initiated by proinflammatory cytokines IL-1/ and TNF- binding their cognate receptors following release from activated macrophages and T lymphocytes at sites of infection. Signal transduction via IL-1 and TNF- pathways involves activation of pathway-specific receptor proximal kinases and ubiquitin E3 ligases. IL-1 and TNF- signaling pathways converge more distally at the level of mitogen-activated protein kinase kinase kinases (MAP3Ks), leading ultimately to the activation of MAPKs (p38, c-Jun NH2-terminal kinase [JNK], and extracellular signal-regulated kinase) and the transcription factor nuclear factor-B (NF-B) (47, 55). Together, MAPKs and NF-B induce expression of inflammatory chemokines (e.g., IL-8, IL-6, growth-regulated oncogene alpha [GRO-], and monocyte chemoattractant protein 1 [MCP-1]), as well as adhesion and costimulatory molecules that recruit and activate various aspects of the innate and adaptive immune response.
MAPKs are activated by phosphorylation on specific threonine and tyrosine residues in a conserved "activation loop" of the MAPK kinase domain (34). Following activation, MAPKs induce the expression of IL-1- and TNF--responsive genes by phosphorylating a number of transcription factors, including Jun, Fos, and ATF family members. NF-B is a critical transcription factor that serves as a major modulator of inflammation, immune regulation, differentiation, and apoptosis (19, 24). NF-B induces target gene expression by binding to NF-B binding sites in the promoter region of NF-B-responsive genes. The NF-B molecule is a homo- or heterodimer of Rel family members, with the most common form being comprised of a heterodimer between RelA (p65) and p50. In nonactivated cells, NF-B resides in an inactive cytoplasmic complex with an inhibitory molecule, IB, which masks the NF-B nuclear localization sequence. NF-B activation results from the phosphorylation, ubiquitination, and subsequent proteosomal degradation of IB. IB degradation appears to represent the rate-limiting step in activation of NF-B, and following release from IB, NF-B translocates to the nucleus to directly induce target gene expression (24).
The importance of the IL-1 and TNF- pathways for infectious organisms can be seen by the large number of bacteria (13) and viruses (23) that have evolved mechanisms to modulate (both activate and suppress) these signaling pathways. Cytomegaloviruses (CMVs), a family of ubiquitous DNA viruses, have coevolved with their host (36), which has resulted in a considerable level of coadaptation between host and virus. A critical aspect of this coevolution appears to be the ability of CMV to modulate components of the IL-1 and TNF- signaling pathways. To date, most studies have focused on the activation of these pathways by human cytomegalovirus (HCMV) infection. At early times after infection, numerous studies have shown that HCMV activates signaling components of both IL-1 and TNF-, including NF-B (32, 45, 56, 57, 59) and various MAPKs (29, 30, 43, 56). This activation occurs in two distinct phases, with initial activation being mediated by virus attachment and entry (6, 12, 57). A second phase of activation occurs following virus entry and is mediated, at least in part, by major transactivators of the virus, IE1 and IE2 (10, 45, 59). These activation events have been proposed as necessary for the production of a cellular environment conducive to initial viral replication. IL-1 and TNF- pathway activation may also facilitate virus dissemination through recruitment of HCMV-susceptible cells by increasing the secretion of chemokines (IL-8, IL-6, RANTES, and MCP-1) (2, 4, 12, 18, 20, 22, 40, 41).
More recent studies are beginning to reveal an ability of HCMV to suppress IL-1 and TNF- signaling pathways at later times of infection (3, 22). Specifically, HCMV has been shown to decrease TNF--mediated signaling, which corresponded to a down-modulation of TNFR1 surface expression and decreased activation of the MAPK JNK (3). Similarly, HCMV has been shown to block both IL-1- and TNF--induced expression of the chemokine MCP-1 at the level of transcription (22). The precise mechanisms involved in these inhibitory effects of HCMV on the IL-1 and TNF- pathways are not completely resolved, and the breadth of the suppression on the proinflammatory response has not been investigated. However, these observations suggest a requirement for suppression of proinflammatory signaling pathways at later stages of the HCMV replication cycle.
In the current study, we have further examined the ability of HCMV to modulate the IL-1 and TNF- proinflammatory signaling pathways. Our results show that HCMV suppresses both IL-1 and TNF- signaling pathways at late times of infection. This inhibitory effect corresponded to impaired IL-1- and TNF--induced IB phosphorylation and NF-B activation and decreased expression of multiple inflammatory chemokines. Surprisingly, HCMV infection resulted in a differential effect on the IL-1 pathway compared to the TNF- pathway, with a greater effect on IB phosphorylation and NF-B activation observed for the IL-1 signaling pathway. This differential inhibition of IL-1 and TNF- signal transduction suggests that HCMV modulates these pathways at distinct positions upstream from the point of pathway convergence. A number of studies indicate that endothelial cells represent an important site of HCMV persistent replication in vivo (16, 21, 42, 49, 50). The observation that HCMV suppression of IL-1 and TNF- signaling pathways occurs at late times of infection within this cell type suggests that the ability of HCMV to regulate these pathways may represent a critical adaptation to enable virus persistence within the host.
MATERIALS AND METHODS
Cells and virus. HCMV laboratory strains Towne and AD169 were obtained from the American Type Culture Collection (Manassas, VA). HCMV strain TR is a low-passage clinical isolate obtained from an AIDS patient that we have cloned as a bacterial artificial chromosome. TR retains all characteristics of a parental virus, including tropism for endothelial cells and macrophages. HCMV strains were propagated and titrated in normal human foreskin fibroblast (HFF) cells by standard methods (27). HFF cells were cultured at 37°C in an atmosphere of 5% CO2 in complete medium (Dulbecco modified Eagle medium containing 10% fetal bovine serum and supplemented with 4 mM L-glutamine, 200 μg/ml penicillin G, and 200 μg/ml streptomycin sulfate). Primary human umbilical vein endothelial cells (HUVECs) (Cascade Biologics, Portland, OR), retinal (HREC-181) (Cell Systems Corporation, Kirkland, WA) endothelial cells, and telomerase life-extended tAEC-5A endothelial cells were cultured in microvascular endothelial growth medium (EGM-2 MV; Cambrex Bioscience Rockland, Inc., Rockland, ME).
Analysis of IL-1- and TNF--induced chemokine mRNA levels by quantitative real-time reverse transcription-PCR (qRT-PCR). Subconfluent HFF cells cultured in six-well plates were either infected with HCMV at a multiplicity of infection (MOI) of 3 or mock infected. At day 3 postinfection (p.i.), cells were treated with IL-1 (1 ng/ml) (R&D Systems, Inc., Minneapolis, MN) or TNF- (10 ng/ml) (R&D Systems, Inc.). Cytokines were added to the cells directly into the media overlying the cells either with or without changing the media, depending on the experiment. However, in any particular experiment, all wells were treated in an identical fashion other than for the addition of the proinflammatory cytokine. At 1 and 3 h posttreatment, cells were harvested using TRIzol (Invitrogen, Carlsbad, CA), duplicate wells were pooled, and RNA was extracted using the manufacturer's protocol. RNA was quantified by absorbance at an optical density at 260 nm, and 2 μg of DNase-treated RNA was reverse transcribed using OmniScript (Sigma-Aldrich, St. Louis, MO) and oligo(dT) primers (Invitrogen, Carlsbad, CA). The resulting cDNA product was amplified using the ABI Prism 7700 sequence detector in the presence of 2x SYBR green master mix (Applied Biosystems, Foster City, CA) and 0.25 μM of forward and reverse primers, resulting in 120-bp amplicons. For each primer set, gene amplification in 40 cycles of PCR was measured by absolute quantification and then normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) levels to determine induction (n-fold). The denaturing and annealing/extension conditions for each cycle of PCR were 95°C for 15 s and 60°C for 1 min, respectively. The primer sets used for each gene of interest were as follows: GAPDH, 5'-TGACCTCAACTACATGGTTTACATGT-3' and 5'-AGGGATCTCGCTCCTGGAA-3'; IL-8, 5'-CAACACAGAAATTATTGTAAAGCTTTCT-3' and 5'-GAATTCTCAGCCCTCTTCAAAAA-3'; MCP-1, 5'-TCGCTCAGCCAGATGCAAT-3' and 5'-ATGGTCTTGAAGATCACAGCTTCTT-3'; IL-6, 5'-GGATTCAATGAGGAGACTTGCC-3' and 5'-ACAGCTCTGGCTTGTTCCTCAC-3'; and GRO-, 5'-CCGAAGTCATAGCCACACTCA-3' and 5'-TGGATTTGTCACTGTTCAGCATC-3'.
ELISA analysis of IL-8 levels. HFF cells and HUVECs were infected with HCMV at an MOI of 3 or mock infected, followed by treatment with IL-1 or TNF- at day 3 p.i. After 6 h, supernatants were harvested, clarified by centrifugation, and stored at –80°C prior to analysis. The concentration of IL-8 released from cells was determined by enzyme-linked immunosorbent assay (ELISA) using the human IL-8 screening set (Endogen Inc., Rockford, IL) following the manufacturer's protocol. Plates were analyzed at 492 nm using a SpectraMax 190 plate reader (Molecular Devices Corp., Sunnyvale, CA), and levels of IL-8 released were determined by comparison to a standard curve.
Western analysis of viral and cellular proteins. Following IL-1 and TNF- treatment, cell monolayers were washed twice with Dulbecco's phosphate-buffered saline and harvested at the times indicated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer containing a protease inhibitor cocktail (0.5 μg/ml leupeptin, 0.7 μg/ml pepstatin, 2 μg/ml aprotinin, 10 mM phenylmethylsulfonyl fluoride) with 10 mM Na orthovanadate and 50 mM NaF. In designated experiments, the proteosome inhibitor epoxomicin (1 μM) (Peptide Institute, Osaka, Japan) was added to cells 1 h prior to IL-1 or TNF- treatment. Proteins were separated by SDS-PAGE, electrophoretically transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA), and blocked. Viral and cellular proteins were detected using the following primary antibodies (Abs): rabbit anti-immediate-early HCMV IE2 (R638; 1:1,000) (15), rabbit anti-IB (C-15; 1:400) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), mouse anti-phospho-IB (B-9; 1:150) (Santa Cruz Biotechnology, Inc.), rabbit anti-p65 NF-B (C-20; 1:2,000) (Santa Cruz Biotechnology, Inc.), and rabbit anti-p38 (C-20; 1:2,000) (Santa Cruz Biotechnology, Inc.). Primary Abs were detected with species-specific horseradish peroxidase-conjugated secondary Abs (1:1,500 to 1:2,500) (Amersham, Piscataway, NJ) and visualized using West Pico chemiluminescence substrate (Pierce, Rockford, IL).
IF microscopy. After fixation in 4% paraformaldehyde in Dulbecco's phosphate-buffered saline, localization of viral and cellular proteins in cells treated with IL-1 and TNF- was determined by indirect immunofluorescence (IF) microscopy, as previously described (26). Cells were harvested at 6 h posttreatment for analysis of IL-8 and 30 min posttreatment for analysis of NF-B translocation. Primary Abs used were as follows: rabbit anti-IE2 (R638; 1:300), rabbit anti-p65 NF-B (C-20; 1:300), mouse anti-gB (27-156) (1:150) (27), and mouse anti-IL-8 (clone 6217; 1:100) (R&D Systems, Inc.). After incubation with the appropriate fluorescently labeled species-specific secondary Ab, fluorescence was visualized using an Olympus confocal microscope.
RESULTS
HCMV inhibits IL-1- and TNF--mediated induction of multiple inflammatory genes. A unique characteristic of HCMV is the ability of the virus to persist within the host, even in the presence of vigorous inflammatory responses, such as those observed in the tissues of transplanted organs during chronic rejection and transplant vascular sclerosis (28). We hypothesized that HCMV possesses an ability to modulate the innate immune response, which may be critical for persistence of the virus within such harsh proinflammatory environments. To examine whether HCMV was able to modulate the inflammatory environment by interfering with signaling pathways that contribute to cellular responses to soluble mediators of inflammation, we determined the level of inflammatory chemokine induction following treatment of HCMV-infected cells with the two major proinflammatory mediators IL-1 and TNF-. HFF cells were infected with HCMV strain TR at an MOI of 3 or mock infected. At 3 days p.i., cells were treated with either IL-1 (1 ng/ml) or TNF- (10 ng/ml), harvested at 1 and 3 h postinduction, and analyzed for the level of chemokine mRNA induction by quantitative real-time RT-PCR. For these studies, we focused on the induction of four inflammatory chemokines (IL-8, IL-6, MCP-1, and GRO-), which are major mediators of the inflammatory response, being involved in both the recruitment and activation of multiple components of the immune system (5). The level of chemokine induction was normalized to GAPDH, and results are presented as induction (n-fold) compared to basal levels prior to IL-1 or TNF- addition. The GAPDH levels were comparable in HCMV- and mock-infected cells and were not affected by treatment with either IL-1 or TNF- (data not shown). Treatment of mock-infected cells with either IL-1 or TNF- induced mRNA expression of all inflammatory chemokines studied (IL-8, IL-6, MCP-1, and GRO-), consistent with the known positive regulation of the expression of these chemokines by IL-1 and TNF- (Fig. 1). In contrast, cells infected with HCMV showed a decrease in response to both proinflammatory cytokines (IL-1 and TNF-), as observed by the minimal induction of IL-8, IL-6, MCP-1, or GRO-. At the time of harvest, HCMV-infected cells were >90% viable by trypan blue exclusion, and absolute basal levels of IL-8, IL-6, MCP-1, and GRO- prior to IL-1 and TNF- treatment were comparable to those of mock-infected cells (data not shown). These results indicate that the effect of HCMV on chemokine induction in infected cells did not result from either increased cell death or a maximal chemokine expression making cells refractory to IL-1 or TNF- chemokine induction. Together, these results reveal an ability of HCMV to suppress the induction of multiple inflammatory chemokines by the two major proinflammatory cytokines IL-1 and TNF-.
HCMV inhibition of IL-1 and TNF- proinflammatory pathways corresponds to a decreased release of IL-8 from infected cells. To further investigate the characteristics of the HCMV inhibition of proinflammatory pathways, we focused on the effect of HCMV on IL-8 expression. IL-8 was selected as an IL-1- and TNF--inducible gene marker, since this chemokine is an extensively studied prototypical inflammatory chemokine that is highly induced by both proinflammatory cytokines (24) and whose expression following induction with IL-1 and TNF- was observed to be decreased in HCMV-infected cells (Fig. 1). To determine whether the effect of HCMV on IL-8 expression observed by qRT-PCR corresponded to a decrease in IL-8 release, we used an ELISA to measure the levels of IL-8 released into the supernatant of HCMV-infected cells following IL-1 and TNF- induction. Since endothelial cells are a biologically relevant cell type for HCMV infection that represent a site of persistent HCMV infection within the host, the effect of HCMV infection on IL-8 release was analyzed in HUVECs as well as HFF cells. Cells were infected with HCMV TR at an MOI of 3 or mock infected. At day 3 p.i., cells were treated with either IL-1 or TNF-, and the concentration of IL-8 released into the supernatant was quantified at 6 h postinduction. As shown in Fig. 2, treatment of mock-infected cells with either IL-1 or TNF- resulted in a significant release of IL-8 from both HUVECs and HFF cells. However, consistent with the results from the qRT-PCR, both HCMV-infected HUVECs and HCMV-infected HFF cells released reduced levels of IL-8. Together, these results show that the effect of HCMV on proinflammatory cytokine induction of chemokine expression corresponds to a decrease in the release of chemokines from infected cells and that this effect appears to be independent of cell type.
HCMV inhibition of IL-1 and TNF- proinflammatory pathways occurs at later times after infection. Typical of all herpesviruses, HCMV genes can be characterized into three kinetic classes (immediate early [IE], early [E], and late [L]) based on time of viral gene expression. To determine the stage of the viral life cycle that corresponded to the HCMV-mediated inhibition of the proinflammatory pathways, we analyzed the induction of IL-8 in infected cells by IF microscopy. Cells were infected with HCMV TR at an MOI of 0.5 and induced at day 3 p.i. for 6 h with either IL-1 or TNF-. Cells were then fixed and stained for IL-8 as well as HCMV IE (IE2) and L (gB) antigens. The lower MOI utilized in these studies ensured the presence of HCMV-infected cells at different stages of the viral life cycle secondary to the spread of HCMV from infected to surrounding noninfected cells. Figure 3A shows a typical IF micrograph displaying the characteristic IL-8 expression in HCMV-infected cells (HUVECs) following proinflammatory cytokine induction (in this case, IL-1). A similar effect of HCMV on IL-8 expression was observed following TNF- treatment and in a number of different cell types (HFF and primary retinal microvascular [HREC-181] and telomerase life-extended aortic macrovascular endothelial cells [tAEC-5A]) following IL-1 or TNF- treatment (data not shown). In all cases, IL-8 expression was evident in both noninfected and HCMV-infected cells expressing only the IE antigen IE2. In contrast, HCMV-infected cells that had progressed to the stage of E/L gene (gB) expression were negative for IL-8 following induction with either IL-1 or TNF-. These results show that the inhibitory effects of HCMV on the proinflammatory cytokine pathways occurred at later times of HCMV infection, corresponding to the time of gB expression. Since the inhibitory effect was restricted to only a defined population (i.e., gB-expressing cells) within the culture, these results also show that the suppressive effect of HCMV required direct infection of the host cell. This latter observation lessens the likelihood of a paracrine mechanism mediated by a soluble factor released from HCMV-infected cells.
HCMV inhibition of IL-1 and TNF- proinflammatory pathways is HCMV strain independent. To determine whether the HCMV-mediated inhibition of proinflammatory pathways was unique to the TR strain or was a general characteristic of HCMV, the ability of additional HCMV strains to prevent IL-8 induction was assessed by IF microscopy as described above. HFF cells were infected at an MOI of 0.5 with two laboratory strains (AD169 and Towne). Cells were then fixed and stained for IL-8, IE2, and gB after induction with IL-1 and TNF- at day 3 p.i. All viruses exhibited an inhibitory effect on IL-1 (Fig. 3B) and TNF- (data not shown) induction of IL-8. Similar to the TR strain, the effect on IL-8 induction by these viruses corresponded to gB expression at later times of infection. These results indicate that HCMV inhibition of proinflammatory pathways is a general characteristic of HCMV that is conserved between multiple strains.
HCMV inhibition of IL-1 and TNF- pathways corresponds to an inhibition of NF-B activation. The IL-1 and TNF- signaling pathways converge at the level of the MAP3Ks to activate the downstream transcription factor NF-B. In noninduced cells, NF-B is retained in the cytoplasm by an association with the inhibitory molecule IB. Following proinflammatory cytokine induction, IB is phosphorylated, ubiquitinated, and degraded. This targeted degradation of IB releases NF-B to translocate to the nucleus and activate gene expression (for a review, see reference 19). To determine whether the HCMV-mediated inhibition of proinflammatory pathways corresponded to impaired activation of NF-B, NF-B activation was measured in HCMV-infected cells by visualization of NF-B translocation to the nucleus. Figure 4 shows the effect of HCMV infection on the nuclear translocation of NF-B following 30 min of IL-1 induction. In the absence of induction, NF-B was localized to the cytoplasm regardless of HCMV infection status. However, HCMV infection had a significant effect on the subsequent activation of NF-B by IL-1. In both noninfected cells and HCMV-infected cells expressing only the IE marker IE2, treatment with IL-1 caused rapid relocalization of NF-B to the nucleus. In contrast, in HCMV-infected cells expressing the E/L marker gB, NF-B activation was impaired as shown by the continued localization of NF-B to the cytoplasm. These results show that HCMV suppresses the ability of proinflammatory cytokines to activate the transcription factor NF-B. The effects of HCMV on chemokine induction and NF-B activation closely paralleled one another (compare Fig. 3 and 4), with both events requiring direct infection and occurring in infected cells only at later times of infection. This observation suggests that the suppressive effect of HCMV on IL-1 and TNF- pathways is mediated, at least in part, through an inhibitory effect on the nuclear translocation of NF-B.
Interestingly, although HCMV inhibited the nuclear translocation of NF-B following induction with either IL-1 or TNF-, the effect on TNF- activation of NF-B appeared to be less than complete, with an intermediate phenotype being observed (Fig. 5). This differential effect of HCMV on NF-B activation mediated by IL-1 compared to TNF- suggested that HCMV may be modulating these two proinflammatory pathways independently at distinct points upstream (receptor proximal) of the convergence of these two pathways. The IF-based assay used to analyze NF-B nuclear translocation is inherently qualitative. To determine more definitively whether HCMV was modulating these two pathways independently, we analyzed the degradation of IB following induction with IL-1 or TNF- by Western blotting. The rationale behind this experiment was that independent regulation of these two pathways, occurring upstream of the convergence point, would be suggested by distinct characteristics of IB degradation following IL-1 induction compared to TNF- induction. HCMV- or mock-infected cells were induced at day 3 p.i. with either IL-1 or TNF-. Cells were harvested at increasing times postinduction, and the degradation of IB was measured in cell lysates by Western analysis (Fig. 6A). In mock-infected cells, IL-1 and TNF- induction resulted in comparable and rapid degradation of the IB protein. The small shift in size of IB occurring at 5 min postinduction corresponds to phosphorylation of IB, prior to ubiquitination and degradation. Importantly, the comparable rates in loss of IB observed in mock-infected cells (see Fig. 6A) following treatment with either IL-1 or TNF- indicate that the rates in signal transduction resulting in IB degradation are similar for these two pathways in this cell type. Dose-response analysis between cytokine level and IB degradation also indicated that the concentrations of IL-1 and TNF- used were subsaturating (data not shown). HCMV infection had a significant effect on the ability of IL-1 or TNF- to induce IB degradation. However, although HCMV-infected cells displayed a decreased rate of IB degradation following induction with either IL-1 or TNF-, the block in IB degradation was complete following IL-1 but not TNF- treatment. This observation suggests that the effect of HCMV on these signaling pathways is distinct. In the absence of IL-1 or TNF- treatment, basal levels of total IB were comparable in HCMV-infected and mock-infected cells (Fig. 6A) and were independent of virus strain (Fig. 7). These results revealing distinct characteristics of IB degradation suggest that HCMV affects the IL-1 or TNF- pathway independently at a position upstream from the point of signaling pathway convergence.
To further examine the differential effect of HCMV on the IL-1 or TNF- signaling pathway, the experiment whose results are shown in Fig. 6A was repeated in the presence of the highly specific proteosome inhibitor epoxomicin (38), which serves to stabilize the phosphorylated form of IB. In mock-infected cells, IL-1 and TNF- induction resulted in comparable and rapid phosphorylation of the IB protein, as shown by Western analysis using a phospho-IB-specific Ab (Fig. 6B). Stabilization of the phosphorylated form was also observed by the small shift in IB size occurring at 5 min postinduction. Consistent with the inhibitory effect of HCMV on the degradation of IB (Fig. 6A), phosphorylation of IB was also significantly decreased in HCMV-infected cells. However, the characteristics of the effect on IB phosphorylation following IL-1 induction compared to TNF- induction were distinct, with an absence of IB phosphorylation being observed only following IL-1 induction. This absence of IB phosphorylation and degradation following IL-1 induction (compared to a partial block following induction with TNF-) closely paralleled the effect on NF-B nuclear translocation following induction with IL-1 and TNF-, respectively (Fig. 5). Together, these results demonstrate that HCMV suppresses both the IL-1 and TNF- pathways, resulting in impaired inflammatory chemokine induction, which corresponds to inhibition of NF-B activation. HCMV appears to modulate IL-1 or TNF- signaling independently at a level upstream from the point of convergence of these two pathways.
The viral gene(s) responsible for inhibition of IL-1 and TNF- proinflammatory pathways is expressed with L gene kinetics. The observation by IF microscopy that the inhibitory effect of HCMV on proinflammatory pathways and NF-B activation corresponded to times of gB expression suggested that the viral gene(s) mediating these effects was expressed with either E or L kinetics. To precisely define the kinetic class of the viral gene(s) mediating the inhibitory effect, we assessed the effect of foscarnet, an inhibitor of viral L gene expression, on IB degradation. HFF cells were infected at an MOI of 3 with HCMV and maintained in the presence or absence of foscarnet. At 3 days p.i., cells were treated with either IL-1 or TNF- and harvested at increasing times postinduction for Western analysis of IB degradation, as described above. Foscarnet treatment overcame the inhibitory effect of HCMV on both the IL-1- and TNF- induced degradation of IB (Fig. 8). This finding indicates that the viral gene(s) responsible for inhibition of NFB activation, and presumably chemokine induction, is expressed with L gene kinetics.
DISCUSSION
In the current study, we have observed an ability of HCMV to inhibit the IL-1 and TNF- proinflammatory pathways at late times after infection. This inhibitory effect of HCMV resulted in reduced induction of multiple IL-1- and TNF--responsive target genes, including the major inflammatory chemokines IL-6, IL-8, GRO-, and MCP-1. Inhibition of these signaling pathways corresponded to impaired activation of NF-B, a transcription factor essential for expression of target genes through these pathways. Analysis of phosphorylation and degradation of IB, a negative regulator of NF-B, showed that HCMV prevented the normal destruction of IB in response to IL-1 and TNF-, consistent with the observed block in NF-B activation. However, characteristics of the HCMV modulation of IB destruction following treatment with IL-1, compared to treatment with TNF-, suggested that inhibition of these two closely related proinflammatory pathways was mediated by HCMV at distinct positions upstream from the point of pathway convergence.
Although most previous studies have focused on the ability of HCMV to activate components of these pathways at earlier times of infection (29, 30, 32, 43, 45, 56, 57, 59), more recent studies have indicated that HCMV down-modulates aspects of the proinflammatory response, primarily at later times (3, 22). Specifically, HCMV has been shown to inhibit IL-1 and TNF- induction of MCP-1 expression (22) and TNF- activation of JNK (3). The viral genes involved in the inhibition have not been identified; yet, the effect on MCP-1 was mediated at the level of MCP-1 gene transcription. The effect on TNF- activation of JNK corresponded to a down-modulation of TNFR1 surface expression, resulting from an internalization of the receptor from the cell surface. However, since the ability of TNFR1 expression to "rescue" the effect of HCMV on JNK activation was not determined, the precise role of receptor down-modulation in mediating inhibition of the TNF- signaling pathway is unclear. Our finding that HCMV inhibits proinflammatory pathways further suggests an importance for suppression of signaling components, as well as inflammatory mediators, involved in the IL-1 and TNF- pathways at later stages of the HCMV replication cycle.
IL-1 and TNF- signaling involves closely related pathways that are composed of receptor proximal pathway-specific signaling events that converge at the level of MAP3Ks. Following MAP3K activation, a common signaling pathway is utilized for IL-1 and TNF- activation of NF-B (9). In the current study, the differential characteristics of the inhibition of the IL-1 pathway compared to that of the TNF- pathway observed for IB phosphorylation and degradation, as well as for nuclear translocation of NF-B, suggests that the HCMV-mediated inhibition of these pathways occurs receptor proximal from the point of pathway convergence at the MAP3Ks (Fig. 9). A number of possible mechanisms could be responsible for the HCMV-mediated inhibition. One possibility is that inhibition is due to independent down-modulation of IL-1R1 and TNFR1 surface expression. By fluorescence-activated cell sorter analysis, we observed only background levels of IL-1R1 (and TNFR1) surface expression regardless of infection (data not shown), which prevented the assessment of the effect of HCMV infection on IL-1R1 expression. However, since near-undetectable levels of IL-1R1 expression have been shown to result in normal levels of signaling via IL-1R1 (48), a complete block in signal transduction (as we observed for the IL-1 pathway) via receptor down-modulation would be unexpected. It is possible that the more intermediate inhibitory level of HCMV on TNF- signaling is mediated by the previously identified effect of HCMV infection on TNFR1 surface expression (3). In this earlier study, the effect of HCMV on signaling events following TNF treatment was only partial (assayed in an in vitro JNK assay using glutathione S-transferase-Jun as a substrate). Hence, TNFR1 down-modulation as a mechanism for the inhibition of TNF signaling is consistent with our observation of a partial effect on NF-B signaling after TNF treatment, but other explanations could also account for these data.
The comparable kinetics of inhibition, occurring at late times of infection for both IL-1 and TNF- signaling pathways, suggest that inhibition of both pathways may be mediated by a single viral factor. Although the IL-1 and TNF- pathways utilize distinct signaling molecules upstream from the convergence of these pathways, a number of these components are closely related. For example, both utilize members of a family of closely related TRAF (TNFR-associated factor) proteins (9), with the IL-1 and TNF- pathways utilizing TRAF 6 (35) and TRAF 2/5 (52), respectively. TRAFs are a family of ubiquitin E3 ligases that catalyze a number of essential ubiquitination events required for signal transduction (11, 31). TRAFs contain conserved sequence and structural motifs, including an N-terminal RING domain, several zinc finger motifs, and a C-terminal TRAF domain. Hence, the differential effect on IL-1 and TNF- signaling may be the consequence of a single viral factor exhibiting a difference in affinity for individual TRAF family members. A previous study indicated that a viral tegument protein, pp65, was able to suppress the induction of inflammatory genes that occurs in the absence of cytokine addition, which is observed very early (6 h) following infection (7). This inhibition was suggested to be due to an inhibitory effect of pp65 on NF-B (7). However, the UL83 gene that encodes pp65 is expressed with E kinetics (8). In our study, the ability of foscarnet, which prevents L viral gene expression, to overcome the inhibitory effect of HCMV on IB degradation (Fig. 8) suggests that the inhibitory phenotype at late times of infection is independent of pp65. A more recent study has shown that the immediate-early viral transactivator IE2 can dampen the induction of inflammatory genes that accompanies HCMV infection (53). However, in our studies, the normal response of cells expressing IE2 alone in terms of IL-8 induction and activation of NF-B following cytokine treatment, combined with the ability of foscarnet to reverse the inhibitory effect of HCMV on these signaling pathways, indicates that cells expressing IE2 in the absence of E/L genes have intact responses to IL-1 and TNF-.
The similarity between the inhibitory effects of HCMV on IL-1- and TNF--induced inflammatory chemokine expression and NF-B nuclear translocation suggests that the inhibition of chemokine induction results, at least in part, from inhibition of NF-B activation. However, the comparable suppression levels of IL-1- and TNF--induced inflammatory chemokine expression by HCMV infection (Fig. 1) were surprising given the difference in the level of inhibition of HCMV on IL-1 induction compared to that of TNF- induction of NF-B nuclear translocation (Fig. 4 and 5). In addition to NF-B activation, MAPKs have been shown to be critical for maximal induction of chemokine expression via IL-1 and TNF- pathways (24, 33, 51). Consequently, the comparable inhibitory effects of HCMV on chemokine induction via the IL-1 and TNF- pathways may result from additional viral effects on MAPK activity. The effects of HCMV on MAPK activation via IL-1 and TNF- signaling pathways are unclear. In studies investigating the effect of HCMV infection on basal activity of MAPKs (in the absence of proinflammatory cytokine stimulation), infection has been shown to induce activation of both extracellular signal-regulated kinase (43) and p38 (29). In contrast, a recent study identified a biphasic effect of HCMV on expression of the inflammatory chemokine IL-6, with induction at early times of infection being followed by suppression at later times. This suppression corresponded to a decrease in the stability of IL-6 chemokine mRNA transcript (18), which is regulated by p38 MAPK (17). The inhibitory effect of HCMV on TNF- signal transduction, which was correlated to TNFR1 down-regulation, corresponded to a block in JNK activation (3).
The difference between the level of chemokine suppression and inhibition of NF-B nuclear translocation following TNF- treatment may, alternatively, indicate additional levels of HCMV-mediated modulation of NF-B activity. Recent studies have shown that NF-B activity is regulated at multiple levels, with IB-mediated cytoplasmic localization of NF-B representing only one tier of regulation (14, 44, 46, 60). Direct phosphorylation of the NF-B molecule at multiple sites has been shown to both positively and negatively regulate NF-B activity. Phosphorylation of NF-B is mediated by a variety of kinases (54), which could serve as potential targets for additional HCMV-mediated modulation of the pathway.
The apparent differential effect on the IL-1 signaling pathway compared to that on the TNF- signaling pathway is intriguing. This differential effect may reflect the proapoptotic signaling associated with TNF-, but not IL-1, signal transduction pathways in the absence of NF-B activation. Recently, the requirement for NF-B activity was shown to result from NF-B-mediated induction of antiapoptotic molecules that regulate cell survival following activation of the TNF- signaling pathway (39). TNFR1 engagement was shown to result in the formation of two distinct complexes: a prosurvival complex (complex I) and a proapoptotic complex (complex II or death-inducing signaling complex). Complex I formed rapidly following receptor engagement and resulted in NF-B activation. In contrast, complex II assembled more slowly and resulted in apoptosis. Importantly, NF-B activity was shown to be required for the induction of antiapoptotic molecules, primarily FLIPL, which modified the function of complex II and prevented cell death. Hence, in our study, the intermediate effect of HCMV on NF-B activity following TNF- may reflect the requirement for a necessary level of NF-B induction of antiapoptotic molecules to prevent cell death. In contrast, since apoptotic signals are not associated with IL-1 signaling pathways, a complete blockade of NF-B activation can be induced without any negative apoptotic ramifications. In both cases, the virus has evolved to maximize the level of NF-B suppression within the constraints of a requirement to avoid induction of apoptosis.
In the current study, the inhibitory effect of HCMV on IL-1 and TNF- signaling pathways corresponded to an inhibition of NF-B activation. This finding was surprising given the numerous studies showing NF-B activation following HCMV infection. These previous studies have focused primarily on early times of infection and led to the development of a model wherein HCMV mediates a biphasic activation of the NF-B pathway (58). The first phase occurs during virus entry (12, 56, 57) and is mediated by the major viral envelope glycoproteins gB and gH (56, 57). The second phase requires protein synthesis and is mediated by induction of de novo NF-B synthesis (32, 58). Our current study indicates a further complexity in HCMV-mediated modulation of components of the IL-1 and TNF- signaling pathways, with an importance for HCMV down-modulation of these pathways at later times of infection. The requirement for NF-B inhibition at later stages of the HCMV life cycle is probably multifactorial but may be important for functions such as immune evasion and viral persistence. However, given the central role of NF-B in many cellular processes, such as inflammation, immune regulation, differentiation, and apoptosis (19, 24), this HCMV-mediated inhibition of NF-B may also be responsible for various aspects of viral pathology, including developmental and immune dysregulation and tumorigenesis.
ACKNOWLEDGMENTS
We are grateful to Sherry Farley and Veselina Korcheva from the B.E.M. laboratory for technical support and Andrew Townsend at Extreme Images for assistance with graphics illustrations.
This work was supported through funding from the NIH to J.W. and B.E.M. (A1059335), M.A.J. and J.A.N. (AI21640), and W.J.B. (AI35602).
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