Efficient clearance of virus infections depends on the nature of the host response raised by the infected organism. A proinflammatory cell-mediated immune response is important for elimination of many viruses, including herpesviruses. Macrophages are intimately involved in generation of a proinflammatory response, the initiation of which involves activation of the transcription factor NF-κB. However, the mechanisms of HSV-induced NF-κB activation are poorly understood. In this study we demonstrate that activation of NF-κB by HSV in macrophages is dependent on a functional viral genome and proceeds through a mechanism involving the cellular IκB kinase, as well as the upstream kinases TGF-β-activated kinase 1, mitogen-activated kinase/extracellular signal-regulated kinase kinase 1, and possibly NF-κB-inducing kinase. Furthermore, we show that HSV triggers NF-κB activation by a signaling pathway involving oxidative stress in mitochondria and intracellular calcium, because specific inhibition of mitochondria-derived reactive oxygen intermediates, as well as mitochondrial calcium channels, prevented NF-κB activation. Together, these results point to mitochondria as cellular checkpoints able to initiate NF-κB activation after virus infection and also show that the cellular NF-κB-regulating kinases IκB kinase, TGF-β-activated kinase 1, mitogen-activated kinase/extracellular signal-regulated kinase kinase 1, and possibly NF-κB-inducing kinase, are essential components in the HSV-induced signaling pathway.
During a viral infection, a strong proinflammatory host response is initiated aiming at eliminating the invading pathogen. This involves activation of NK cells and macrophages and generation of proinflammatory mediators, including cytokines and chemokines (1, 2, 3). The transcription factor NF-κB plays a pivotal role in triggering such a proinflammatory response to a range of stimuli, including virus infections (4). This is evidenced by the number and diversity of NF-κB-activating stimuli known to exist (5), including proinflammatory cytokines, Ag stimulation of lymphocytes, bacterial and viral infections as well as various forms of cellular stress.
NF-κB is a dimeric transcription factor composed of homo- or heterodimers of the Rel family proteins, of which p50/p105, p52/p100, p65 (RelA), RelB, and c-Rel have been identified in mammalian cells (5). Among these, p50/p65 is the most frequent dimer and hence considered the prototype. NF-κB is normally present in the cytoplasm in an inactive form bound to a member of the IκB family, usually IκBα (5). In this complex, IκB blocks the nuclear localization signal of NF-κB, and for NF-κB to become activated, the NF-κB-IκB complex needs to be disrupted. This disruption is triggered by specific phosphorylation of serines 32 and 36 on IκBα, which targets IκB for ubiquitination and subsequent degradation by the proteasome complex (5, 6), thereby releasing NF-κB from IκB and allowing nuclear translocation. Once in the nucleus, NF-κB binds specifically to κB sites present in promoters and enhancers of multiple genes and induces expression of various proteins, including cytokines, chemokines, acute phase proteins, MHC molecules, cell adhesion molecules, and antiapoptotic proteins (5, 6).
With a few exceptions, the different NF-κB stimuli all converge on one common kinase, the IκB kinase (IKK)3 (7, 8, 9, 10, 11, 12). This kinase is a key component of NF-κB signaling pathways through its ability to specifically phosphorylate IκBα on serines 32 and 36. IKK is a high m.w. complex containing two closely related kinases termed IKKα (IKK1) and IKKβ (IKK2), which are activated by specific serine phosphorylation, and two additional scaffolding proteins, IKKγ (NF-κB essential modulator) and IKK complex-associated protein (6). Nevertheless, IKKα and IKKβ appear to vary in substrate specificity and have quite different functions. Whereas IKKβ is the kinase subunit responsible for NF-κB activation in response to proinflammatory cytokines, IKKα, in contrast, appears to play a role during embryonic development of the skin and skeletal system (6).
Because most known NF-κB-activating pathways converge on IKK, signal diversity and specificity seem to be achieved upstream of this kinase complex. NF-κB-inducing kinase (NIK) and mitogen-activated kinase/extracellular signal-regulated kinase kinase (MEKK) 1, both belonging to the family of mitogen-activated protein kinase kinase kinases (MAP3K), have been demonstrated to differentially regulate IKK (10, 13). Another MAP3K, TGF-β-activated kinase (TAK) 1, has been shown to act upstream of NIK in IL-1-induced NF-κB activation (14). Other kinases proposed to act upstream of IKK in NF-κB activation include MEKK3 (15), NF-κB-activating kinase (NAK) (16), p38 (17), Akt (18), and Cot (19). In addition, NF-κB activation also requires as yet uncharacterized events sensitive to antioxidants, because reactive oxygen intermediates (ROIs) seem to be indispensable for activation of NF-κB by most stimuli (20).
In recent years much has been learned about receptor-mediated NF-κB activation through the TNFR superfamily and Toll-like receptors (21). Additionally, intracellular activators of NF-κB, including viruses, are known to exist, whereas their mechanism of action is less well-characterized. Thus for many viruses, it remains to be elucidated at which point signaling converges with classical NF-κB pathways, and how this signaling is triggered. Recently, NF-κB activation by HSV has been demonstrated to proceed through IKK (12). However, cellular components upstream of IKK have not been identified. In this study we report that activation of NF-κB by HSV in macrophages is critically dependent upon the presence of IKKβ and the upstream kinases TAK1 and MEKK1, and possibly NIK. We further demonstrate that HSV infection targets mitochondria, resulting in generation of ROIs and signaling by intracellular calcium, and that these events are essential for activation of NF-κB.
Materials and Methods
Mice and cell culture
The mice used were 7- to 8-wk-old female C57BL/6 mice from M&B (Ry, Denmark). Activated peritoneal cells (PCs) were induced by injection of 2 ml of 10% thioglycollate into the peritoneal cavity. Five days later PCs were harvested by lavage of the peritoneal cavity with cold PBS supplemented with 2% FCS and 20 IU/ml of heparin. The cells were washed once in RPMI 1640 medium + 5% FCS and seeded at a density of 2 × 106 cells per well in 24-well tissue culture plates and left overnight before further treatment. The murine macrophage cell line J774A.1 was maintained in DMEM with 1% Glutamax I (Life Technologies, Grand Island, NY), supplemented with antibiotics, and 5% FCS. J774A.1-derived cell lines (see below) were maintained as the parental cell line in the presence of 300 μg/ml G418 (Roche, Mannheim, Germany). For experiments, the cells were seeded at a density of 3.5 × 105 cells per cm2 and left 16–20 h before further treatment. Vero cells were maintained in MEM supplemented with antibiotics and 10% FCS.
Virus and LPS
HSV-1 (17+ strain) and HSV-2 (MS strain) were produced in Vero cells as previously described (2). Just before usage, virus was thawed and used as infectious virus or inactivated by UV irradiation for 15 min. LPS (Escherichia coli) was purchased from Sigma-Aldrich (St. Louis, MO).
Murine IL-6, IL-12 p40, and RANTES were detected by ELISA. Maxisorp plates were coated overnight at 4°C with primary Ab (2 μg/ml anti-IL-6 (BD PharMingen, San Diego, CA), 6 μg/ml anti-IL-12 p40 (BD PharMingen), or 0.5 μg/ml anti-RANTES (PeproTech, Rocky Hill, NJ)) in coating buffer (15 mM Na2CO3; 35 mM NaHCO32SO4. Between each step the plates were washed three times with PBS containing 0.05% v/v Tween 20. The results were quantified by reading at 450 nm.
Isolation of nuclear proteins
To isolate nuclear proteins, the cell monolayer was washed twice with ice-cold PBS, scraped off the plate, and spun down (2000 × g for 1 min). The cells were resuspended in a hypotonic buffer (20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.2 mM EDTA, 0.2 mM PMSF, 0.2 mM leupeptin, 0.2 mM pepstatin A, 0.1 mM Na3VO4) and left on ice for 15 min. Nonidet P-40 was added to 0.6% and the mixture was vortexed 15 s and centrifuged at 10,000 × g for 1 min. The supernatant was removed and the pellet was washed once in the hypotonic buffer. Extraction buffer (20 mM HEPES, pH 7.9, 20% glycerol, 1.5 mM MgCl2, 420 mM NaCl, 0.5 mM DTT, 0.2 mM EDTA, 0.2 mM PMSF, 0.2 mM leupeptin, 0.2 mM pepstatin A, 0.1 mM Na3VO4) was added to the nuclei, and incubated 30 min at 4°C with rocking. The samples were centrifuged at 10,000 × g for 15 min at 4°C and the supernatants containing nuclear proteins were harvested.
Preparation of whole cell extracts
Cells were washed with ice-cold PBS and scraped into 1 ml of PBS. The cells were pelleted by centrifugation and lysed in whole cell extract lysis buffer (50 mM Tris-HCl, pH 7.9, 150 mM NaCl, 1.5 mM MgCl2, 1% Triton X-100, 10% glycerol, 1.0 mM EDTA, 1 mM Na3VO4, 1 mM PMSF, 0.2 mM leupeptin, 0.2 mM pepstatin A) for 30 min on ice. The lysates were centrifuged at 10,000 × g for 10 min at 4°C and supernatants were isolated.
The protein concentration was measured by Bradford assay (Bio-Rad, Hercules, CA). EMSA was performed as previously described (1), except that the distal κB site of the murine NOS2 promoter 5′-TAG GGG GAT TTT CCC CTC-3′ was used as probe at a quantity of 50,000 cpm per sample. The Abs used for supershift were goat polyclonal anti-p65, rabbit polyclonal anti-RelB, and rabbit polyclonal anti-p50 (all obtained from Santa Cruz Biotechnology, Santa Cruz, CA).
IPs were performed on 1 mg of whole cell extract using 1 μg of rabbit polyclonal anti-IKKαβ (H470) or mouse monoclonal anti-NIK (A-12) (both Santa Cruz Biotechnology). Normal rabbit IgG was used as negative control. IPs were allowed to proceed for 1 h at 4°C with rotation before addition of 50 μg of a 50% slurry of protein A-agarose (Santa Cruz Biotechnology). The mixture was left overnight under the same conditions. Immunoprecipitates were washed six times with whole cell extract lysis buffer before analysis by Western blotting.
Western blotting was performed as previously described (3). The following Abs and concentrations were used: mouse monoclonal anti-glycoprotein D (gD) (Virusys, North Berwick, ME), 1/10,000; mouse monoclonal anti-IκBα (H-4), 1/200; mouse monoclonal anti-p-IκBα (B-9) 1/100; rabbit polyclonal anti-IKKαβ (H470), 1/200; mouse monoclonal anti-NIK (A-12), 1/100; rabbit polyclonal anti-MEKK1 (43-Y), 1/200; rabbit polyclonal anti-TAK1 (M579), 1/100; goat polyclonal anti-Cot (N-17), 1/200; goat polyclonal anti-MEKK3 (D-17), 1/200; rabbit polyclonal anti-p38 (H-147), 1/100; rabbit polyclonal anti-calcium/calmodulin-dependent protein kinase II (M-176), 1/200 (all Santa Cruz Biotechnology); mouse monoclonal anti-β-actin (AC-15), 1/5000; mouse monoclonal anti-FLAG-M2, 10 μg/ml (both Sigma-Aldrich); rabbit polyclonal anti-phosphoserine (Zymed Laboratories, San Francisco, CA), 2 μg/ml; rabbit polyclonal anti-phospho-mitogen-activated protein kinase kinase (MKK) 3/6, 1/2000; rabbit polyclonal anti-phospho-MKK4, 1/1000 (both Cell Signaling Technology, Beverly, MA); HRP-conjugated goat polyclonal anti-mouse IgG, 1/2000; HRP-conjugated goat polyclonal anti-rabbit IgG, 1/2000 (both BD Transduction Laboratories, Lexington, KY).
The chemical inhibitors, concentrations, and solvents were: 4-hydroxy-3-methoxyacetophenone (apocynin; Fluka, Buchs, Switzerland), 100 μM, H2O; CGP371572O; diphenyleneiodonium chloride (DPI; Sigma-Aldrich), 10 μM, ethanol; EGTA tetra-(acetoxymethyl-ester) (EGTA-AM; Molecular Probes, Eugene, OR), 100 μM, DMSO; KN93 (Seikagaku Kogyo, Tokyo, Japan), 5 μM, H2O; N-acetyl-l-cysteine (NAC; Sigma-Aldrich), 45 mM, PBS; phosphonoacetic acid (PAA; Sigma-Aldrich), 100 μg/ml, H2O; pyrollidine dithiocarbamate (PDTC; Sigma-Aldrich), 25 μM, PBS; rotenone (Sigma-Aldrich), 2.5 μg/ml, ethanol; ruthenium red (RR; Sigma-Aldrich), 20 μM, PBS; wortmannin (Sigma-Aldrich), 100 nM, DMSO. None of the solvents alone affected HSV-induced NF-κB-dependent gene expression in the concentrations used in this study (data not shown).
The DNA plasmids used for transfections encoded dominant-negative mutants of IKKβ (22), NIK (8), MEKK1 (provided by J. A. Didonato, The Cleveland Clinic Foundation, Cleveland, OH; Ref.13), p38 (H. Holtmann, Medical School Hannover, Hannover, Germany; Ref.23), NAK (M. Nakanishi, Nagoya City University Medical School, Nagoya, Japan; Ref.16), TAK1 (K. Matsumoto, University of Tokushima, Tokushima, Japan; Ref.14), Cot (W. C. Greene, University of California, San Francisco, CA; Ref.19), MEKK3 (B. Su, University of Texas, Houston, TX; Ref.15), and IκBα (1). pcDNA3 was used as empty vector control. For transfection, the cells were seeded at a density of 2.5 × 106 cells per 25 cm2 plate and left for 24 h. The cells were transfected using LipofectAMINE (Life Technologies). Briefly, 7.5 μg of DNA and 20 μl of LipofectAMINE was mixed in a volume of 200 μl of serum-free DMEM. After 20 min of incubation at room temperature, 1 ml of serum-free medium was added to the mixture. The cells were washed once with 5 ml of preheated serum-free medium, and the DNA-LipofectAMINE solution was gently added to the cells. The plates were gently rocked every 30 min for 3 h, at which point 9 ml of preheated medium supplemented with 11% FCS was added. The cells were incubated 24 h before application of selection with G418 (300 μg/ml). Every 7 days, half of the growth medium was exchanged with new preheated selection medium. Surviving colonies were pooled to avoid single-clone abnormalities and cells were grown three to five passages in DMEM supplemented with 10% FCS in the presence of selection before being used for experiments.
Detection of intracellular ROIs
Cells were treated with 4 μM dihydroethidium (Sigma-Aldrich) for 45 min, harvested, and washed twice with PBS. The cells were subsequently suspended in PBS, and ROIs were measured using a flow cytometer with an excitation emission of 605 nm.
NF-κB is required for induction of proinflammatory mediators during HSV infection
The production of proinflammatory cytokines during an HSV infection in murine macrophages has been studied previously in our laboratory (1, 2, 3). As expected, HSV-1 or HSV-2 infection in the murine macrophage cell line J774A.1, as well as in primary murine peritoneal cells, induced production of RANTES, IL-6, and IL-12 p40 (Fig. 1⇓). To determine whether activation of NF-κB was essential for this proinflammatory response, we generated a cell line, J774A.1-dnIκBα, stably transfected with a nondegradable version of the inhibitory subunit IκBα. Indeed, the dominant-negative IκBα mutant strongly inhibited production of RANTES, IL-6, and IL-12 p40 during HSV infection (Fig. 1⇓). These data demonstrated that NF-κB activation was required for HSV-induced expression of these proinflammatory mediators.
Activation of HSV immediate-early (IE) or early (E) genes is essential for nuclear translocation of p50/p65 NF-κB complexes
To elucidate the mechanisms of NF-κB activation during HSV infection, we examined the kinetics of the NF-κB complex formation in addition to the IκB degradation in response to HSV-1 infection (Fig. 2⇓). Weak NF-κB binding to DNA was detectable in mock-infected cells, but increased substantially at 2 h postinfection, after which DNA binding decreased by 4 h and returned to baseline levels after 6 h of infection (Fig. 2⇓A). Supporting the data on the DNA-binding activity of NF-κB, IκBα phosphorylation as detected by Western blotting was maximal 1.5 h postinfection and preceding nuclear translocation of NF-κB (Fig. 2⇓B). When the same extracts were examined for levels of total IκBα, we were unable to demonstrate degradation of IκBα. However, we did observe the well-described poststimulatory superinduction of IκBα (24). This finding suggests that degradation of only a small pool of total cellular IκBα is responsible for the observed NF-κB activation. Supershifts using Abs against p50, p65, and c-Rel, respectively, identified anti-p50 and anti-p65 but not anti-c-Rel in the DNA-binding complex after 2 h of HSV infection (Fig. 2⇓C). Importantly, the identification of p65 in the DNA-binding complexes indicated that the NF-κB complex activated during HSV infection was in possession of transactivating potential (5).
Finally, to further characterize the viral mechanism, whereby HSV triggers signaling to NF-κB, HSV was irradiated with UV light, which destroys the viral genome and thus renders the virus unable to produce first IE and subsequently E and late viral proteins (3). As seen in Fig. 2⇑D, NF-κB activation was abrogated, when UV-irradiated HSV was used, indicating the requirement for a functional viral genome for induction of NF-κB DNA-binding activity. However, viral late genes appeared to be dispensable for NF-κB activation, because the presence of PAA, a specific inhibitor of the viral DNA polymerase the activity of which precedes expression of late genes, had no impact on NF-κB activation by HSV (Fig. 2⇑E).
Taken together, these results demonstrated that HSV infection resulted in IκBα phosphorylation and subsequent activation of a transcriptionally active p50/p65 complex within 2 h of infection. The mechanisms of NF-κB activation seemed to require viral IE, or E genes, whereas viral late genes were dispensable.
HSV-induced RANTES production is abolished in cell lines expressing dominant-negative mutants of IKKβ, TAK1, MEKK1, or NIK
IKK appears to be the central kinase, upon which most NF-κB-inducing signals converge (7, 8, 9, 10, 11, 12). Thus a growing number of upstream kinases regulating IKK activity have been identified. To investigate which of these kinases are important in HSV-induced NF-κB activation, we generated cell lines stably transfected with dominant-negative mutants of IKKβ or several potential upstream kinases. All cell lines obtained displayed high levels of ectopic protein expression (Fig. 3⇓A). Because an anti-NAK Ab was not available to us, we were unable to compare the expression of NAK between the J774-dnNAK and J774-pcDNA cell lines. For the remaining cell lines, the ectopic protein expression surpassed expression of the endogenous protein several-fold.
The transfected cell lines were initially tested for HSV-induced production of RANTES. As seen in Fig. 3⇑B, HSV infection induced a 20-fold induction of RANTES secretion in mock-transfected cells. Similar results were obtained with cell lines expressing dominant-negative mutants of p38, MEKK3, NAK, and Cot, or after inhibition of the Akt pathway by wortmannin, demonstrating that these kinases were dispensable for HSV-induced RANTES production. In contrast, the presence of dominant-negative mutants of either, IKKβ, TAK1, MEKK1, or NIK prevented induction of RANTES during HSV infection.
NF-κB activation during HSV infection is inhibited by dominant-negative mutants of IKKβ, TAK1, MEKK1, or NIK
The observation that some of the dominant-negative cell lines displayed an impaired ability to produce RANTES prompted us to explore whether they were also unable to activate NF-κB following HSV infection. NF-κB DNA-binding activity induced 2 h postinfection was analyzed by EMSA as shown in Fig. 4⇓A. Whereas NF-κB activation was strongly induced by HSV in mock-transfected cells, no increase in NF-κB DNA-binding activity was detected in response to HSV infection in cell lines expressing dominant-negative mutants of either, IKKβ, TAK1, MEKK1, or NIK. This suggested that each of these kinases was involved in the signaling pathway mediating NF-κB activation. As these results were obtained by overexpressing the dominant-negative mutants, we wanted to assure that the kinases investigated were actually present in the parental cells. By Western blotting with Abs against IKK, TAK1, MEKK1, and NIK we confirmed the presence of these kinases in whole cell extracts from the murine macrophage cell line J774A.1. IKKα, IKKβ, MEKK1, and TAK1 were easily detectable, requiring only short times of exposure of the blots (Fig. 4⇓B), whereas NIK was barely detectable even after very long times of exposure (data not shown). However, when Western blotting was performed on anti-NIK immunoprecipitates, we were also able to detect NIK protein in J774A.1 cells (Fig. 4⇓B). The detection of only low amounts of NIK in the cell lysates is in agreement with previous findings from other laboratories (25).
MEKK1 and NIK interact physically with the IKK complex
To further study the involvement of TAK1, NIK, and MEKK1 in NF-κB activation, we investigated whether any of these kinases might interact with the IKK complex. To address this question, we immunoprecipitated the endogenous IKK complex in cell extracts from cell lines expressing kinase inactive mutants of TAK1, NIK, or MEKK1 followed by Western blotting for TAK1, NIK, or MEKK1, respectively (Fig. 4⇑C). Whereas MEKK1 (the low m.w. form) and NIK coprecipitated with IKK, we were unable to detect TAK1 in immunoprecipitates of IKK. These results demonstrated that under the given experimental conditions, MEKK1 and NIK associated physically with the IKK complex.
IKK, TAK1, and MEKK1 are activated during HSV infection in macrophages
To establish the involvement of the kinases identified above in HSV-induced NF-κB activation in macrophages, we wanted to examine whether these kinases were activated during HSV infection. Because IKK is the only kinase known to specifically phosphorylate IκBα at serines 32 and 36, our finding in Fig. 2⇑B that HSV infection triggered this phosphorylation strongly suggested that IKK was activated. To analyze for in vivo kinase activity of MEKK1 and TAK1, we next examined the phosphorylation status of MKK4 and MKK3/6, substrates for MEKK1 and TAK1, respectively (26, 27). As seen in Fig. 4⇑D, we found that HSV-2 infection augmented the level of phosphorylation of both MKK4 and MKK3/6.
As for NIK, it has been difficult to detect stimulus-dependent NIK activation in response to most stimuli examined in the literature and, furthermore, cytoplasmic protein levels of NIK are low (25). To the best of our knowledge, only H2O2 has been shown to stimulate NIK phosphorylation (28). Therefore, we examined whether HSV infection affected the activation status of NIK by immunoblotting anti-NIK immunoprecipitates with antiphosphoserine. Although we were able to immunoprecipitate NIK, no serine phosphorylation could be detected.
NF-κB activation during HSV infection is dependent on intracellular calcium possibly originating from mitochondria
We next wanted to identify cellular components upstream of IKK, TAK1, MEKK1, and NIK in the HSV-induced pathway. Because the intracellular calcium concentration is known to be of importance in many intracellular signaling events, we decided to focus on this molecule. An increase in intracellular calcium levels can occur by two different mechanisms, i.e., either by influx of extracellular calcium through the plasma membrane or by calcium release from intracellular stores, such as the endoplasmic reticulum (ER) or mitochondria (29, 30).
To establish whether intracellular calcium is important in HSV-induced NF-κB activation, cells were incubated in the presence of EGTA-AM, an esterified form of the calcium chelator, that penetrates the plasma membrane and chelates intracellular but not extracellular calcium. The presence of EGTA-AM was able to completely inhibit NF-κB DNA-binding activity 2 h after HSV-1 or HSV-2 infection (Fig. 5⇓A). Moreover, and in agreement with the data described in Fig. 5⇓A, the presence of EGTA-AM also resulted in an almost complete inhibition of HSV-induced RANTES production (Fig. 5⇓B).
To explore the origin of HSV-induced intracellular calcium, we incubated cells with either CGP or RR, which are inhibitors of the mitochondrial sodium/calcium exchanger and mitochondrial calcium uptake and release, respectively (31, 32). As shown in Fig. 5⇑C, each of these inhibitors was able to abolish HSV-induced NF-κB DNA-binding activity, although a slight increase was observed during HSV-2, but not HSV-1, infection in the presence of CGP. To confirm this result, we also investigated RANTES production performing ELISA on culture supernatants. This demonstrated that CGP and RR inhibited RANTES production during HSV infection (Fig. 5⇑D). To address the question of whether mitochondria were the sole source of calcium release in HSV-infected macrophages, we blocked calcium release from the ER using the ryanodine receptor inhibitor dantrolene. This compound reduced HSV-induced RANTES expression by ∼30% (Fig. 5⇑F), whereas only a marginal decline in NF-κB DNA-binding activity was detected (Fig. 5⇑E). Taken together, these results indicated that intracellular calcium originating primarily from mitochondria, with possibly a minor contribution from the ER, was an essential component in the signal transduction pathway induced by HSV infection and leading to activation of NF-κB.
Generation of ROIs in mitochondria is a prerequisite for NF-κB activation during HSV infection
The experiments described above revealed that intracellular calcium was necessary for HSV-induced NF-κB activation and further suggested that HSV, by an as yet unknown mechanism, induced calcium release by targeting mitochondria and possibly the ER. We hypothesized that oxidative stress and the generation of ROIs in mitochondria may be involved. To test this, we first examined whether HSV infection triggered production of ROIs in macrophages. The cells were treated with mock virus preparation or infected with HSV-2 for 1.5 h and then incubated with dihydroethidium. This compound is oxidized by superoxide anions to ethidium, which intercalates with DNA and emits fluorescence at 605 nm. As seen in Fig. 6⇓A, infection of macrophages with HSV-2 for 1.5 h stimulated the generation of ROIs, hence preceding the activation of NF-κB. To examine whether ROIs contributed to activation of NF-κB, we treated HSV-infected J774A.1 cells with the antioxidants PDTC and NAC and examined the nuclear NF-κB DNA-binding activity. As shown in Fig. 6⇓, B and C, removing oxidants from the intracellular milieu with either of these inhibitors completely prevented NF-κB activation and RANTES production by HSV.
To identify the origin of ROIs produced during HSV infection, we targeted enzymes responsible for ROI formation. Apocynin is an inhibitor of cytosolic NADPH oxidase, DPI is a general inhibitor of various ROI generating systems in the cell, and rotenone is described as a specific inhibitor of mitochondrial O2− formation by NADPH reductase (29). Fig. 6⇑D shows that the presence of DPI or rotenone completely abolished HSV-induced NF-κB activation, in contrast to apocynin, which had no effect on the DNA-binding activity of NF-κB. These data suggested that mitochondrial NADPH reductase and possibly other ROI-forming enzymes were activated during an HSV infection. The level of RANTES in the culture supernatants supported these conclusions (Fig. 6⇑E). Together with our finding that mitochondrial calcium release may be involved, the results described in this study pointed to an important role for mitochondria in HSV-induced NF-κB activation.
Calcium acts downstream of oxidative stress in the NF-κB activation pathway in macrophages through a pathway independent of calcium/calmodulin-dependent protein kinases
We wanted to challenge our hypothesis that intracellular calcium acts downstream of ROIs in the signaling pathway. To this end, we treated cells with H2O2, simulating mitochondrially produced ROIs, in the absence or presence of EGTA-AM. As shown in Fig. 7⇓A, H2O2-induced activation of NF-κB was abolished when cytosolic calcium was bound to EGTA-AM, thus demonstrating that intracellular calcium was required for the signal induced by ROIs to proceed to NF-κB.
In an attempt to identify the downstream target of calcium, probably acting upstream of TAK1, NIK, and MEKK1, we examined how inhibition of calcium/calmodulin-dependent protein kinases by KN93 affected HSV-induced NF-κB activation and RANTES expression. Calcium/calmodulin-dependent protein kinase II has previously been shown to participate in NF-κB activation in T lymphocytes and to activate Akt, a well-described IKK-regulating kinase (33, 34). By Western blotting we found that calcium/calmodulin-dependent protein kinase II was expressed in J774A.1 cells and that kinase levels were not affected by virus infection (Fig. 7⇑B). To examine whether KN93 was active in J774A.1 cells, we tested whether this compound was capable of inhibiting UTP-mediated potentiation of LPS-induced nitrite production, as reported by others (35). Indeed KN93 did abolish the effect of UTP on LPS-driven production of nitrite (data not shown). However, even when using high doses of KN93, this inhibitor did not affect the ability of HSV to activate NF-κB and stimulate NF-κB-dependent gene transcription (Fig. 7⇑, C and D), thus excluding calcium/calmodulin-dependent protein kinases from playing an essential role in this pathway.
Effect of chemical inhibitors on virus replication and LPS-induced activation of NF-κB
One major concern with respect to inhibitor studies is the specificities of the compounds. To address this problem, we examined how the inhibitors used in our studies affected cell viability, virus replication, and NF-κB activation by LPS, a classical NF-κB activator. Staining of the cells with trypan blue, showed that between ∼80 and 95% of the cells excluded the dye as a sign of viability after 20 h of treatment with the different inhibitors, as compared to ∼90% in control cells (data not shown). To monitor virus replication, we performed Western blotting on whole cell extracts from 14 h-infected macrophages using a mAb against the viral late gene gD. As seen in Fig. 8⇓A, most chemical inhibitors left virus replication unaffected. Only the specific herpesvirus DNA polymerase inhibitor PAA and the two antioxidants PDTC and NAC reduced gD expression. Finally, as for NF-κB activation by LPS, the antioxidants PDTC, NAC, and to a minor extent DPI, reduced NF-κB activation, whereas the remaining inhibitors did not affect the response (Fig. 8⇓B). The potent inhibitory activity of PDTC and NAC is in agreement with the literature (20). These results demonstrate that the inhibitors used did not compromise cell viability within the time frame of the experiments, and did not prevent HSV from replicate to the stage of late gene expression. Finally, the majority of the inhibitors did not affect NF-κB activation globally, because LPS remained fully capable of activating NF-κB in the presence of most of the inhibitors.
Macrophages are tissue cells present in the organism at most portals of entry, and hence are likely to encounter infectious agents including viruses, at the very early stages of infection, as well as at later stages where monocyte-derived macrophages are recruited to the site of infection. It has been demonstrated that macrophages are important for restriction of HSV infections in vivo through their ability to directly restrict virus replication and to promote an antiviral proinflammatory response (36, 37).
NF-κB is an important player in the proinflammatory host response to infections. We have previously shown that inducible gene expression of many cytokines and chemokines in virus-infected macrophages requires NF-κB (1, 2, 3). Various different mechanisms for activation of NF-κB by viruses have been described (4). These include 1) interaction between viral glycoproteins and cellular receptors (38), 2) accumulation of viral dsRNA (39), 3) overload of viral proteins in the ER (40), and 4) interaction between virus-encoded proteins and the NF-κB-activating pathway (10). In the present study we have investigated the mechanisms by which NF-κB is activated during an HSV infection in macrophages. We demonstrate that HSV-induced NF-κB activation is dependent on viral IE and/or E proteins and proceeds through IKK and the upstream kinases TAK1, MEKK1, and possibly NIK. Furthermore, we present data that suggest mitochondrial generation of ROIs and intracellular calcium are essential components in this signaling pathway. These data suggest the model shown in Fig. 9⇓.
Our finding in this study that IKK is essential for HSV-induced NF-κB activation is in agreement with a recent study by Amici et al. (12) who showed NF-κB DNA-binding activity and IKK activation in the early phase of HSV infection. In contrast to our data, these authors reported a sustained activation of NF-κB in epithelial cells. This difference may be explained by the fact that HSV replicates poorly in macrophages (41). The observation that IKK is essential is not surprising, because most NF-κB-activating stimuli known to date appear to signal through this kinase (7, 8, 9, 10, 11, 12). Therefore, what distinguishes different signaling pathways is the involvement of different upstream mediators. In our studies, we identified three different MAP3Ks-TAK1, MEKK1, and NIK-for which we observed inhibition of HSV-induced NF-κB activation in the presence of dominant-negative mutants that competitively inhibited the kinase activity. In support of these findings, we were able to detect increased phosphorylation of MKK4 and MKK3/6, substrates for MEKK1 and TAK1, respectively. Although MKK4 and MKK3/6 are not phosphorylated exclusively by MEKK1 and TAK1, respectively, these results further support that MEKK1 and TAK1 are involved in HSV-induced NF-κB activation. However, we cannot formally exclude the involvement of other upstream kinases.
The role of MEKK1 in NF-κB activation is controversial. Whereas MEKK1 has been found to participate in IKK activation in response to TNF-α in 293 cells, MEKK1-deficient murine fibroblasts exhibits intact NF-κB activation in response to TNF-α and IL-1 (6). Another kinase we identified as being essential for NF-κB activation by HSV was NIK. Although we did not detect virus-induced phosphorylation of NIK, this does not preclude a role of this kinase in HSV-induced NF-κB activation. First, NIK is present in the cell in only low amounts and, therefore, an increase in phosphorylation may not be easily detectable. Second, NIK may be regulated by other mechanisms. NIK is present in the IKK complex under certain conditions and is the kinase activating IKK in response to a number of stimuli, including TNF-α, IL-1, and CD95 (42). However, no consensus has been reached as to its precise mechanism of action in the signaling cascade. NIK may exert its major role in transactivation of NF-κB in the nucleus rather than in the kinase pathway leading to IκBα phosphorylation and degradation (43). NIK itself can be activated by the proto-oncogene Cot in CD3/CD28 signaling or by the kinase TAK1 in IL-1 and TNF-α signaling pathways (19). Because we also identified TAK1 as an essential player, this kinase might act upstream of NIK during HSV-induced NF-κB activation. This is supported by our finding that NIK as well as MEKK1 could be coimmunoprecipitated with IKK, whereas TAK1 could not. This scenario is supported by a recent report by Shuto et al. (17) showing that activation of NF-κB by Haemophilus influenzae/Toll-like receptor 2 proceeds through TAK1, NIK, and IKK in epithelial cells. Whether TAK1 is also upstream of MEKK1 and can activate this kinase remains to be investigated.
The finding of both MEKK1 and NIK as essential components is surprising, because MEKK1 and NIK are thought to act in separate signaling pathways. However, it is possible that both MEKK1 and NIK need to be present for signaling to occur. For example, it may be required that both kinases are associated with the IKK complex for signaling to proceed. This hypothesis is supported by our present finding, and previous observations by others (13, 44), that MEKK1 and NIK can physically interact with the IKK complex. Although it is difficult to draw any conclusions as to the physiological relevance of these protein associations from in vitro studies, the interactions observed might play a role in the mechanism of IKK activation by MEKK1 and NIK. Moreover, it has been previously suggested that MEKK1 and NIK may synergistically regulate IKK activity (13), emphasizing the importance of these two kinases upstream of IKK in signaling to NF-κB.
There are several pieces of evidence in the literature that implicate intracellular calcium in NF-κB activation by oxidants and ER stress (29, 45). For instance it has been shown that the NS5A protein of hepatitis C virus alters intracellular calcium levels and induces oxidative stress, which eventually results in activation of STAT3 and NF-κB (32). These transcription factors can also be activated by mitochondrially associated hepatitis B virus X protein (46). Although the latter work did not address the question of whether calcium is implicated in this signaling pathway, it was subsequently shown that hepatitis B virus X protein increases cytosolic calcium mediated by mitochondrial calcium channels, a process resulting in activation of the calcium-dependent proline-rich tyrosine kinase-2, Pyk2 (31). These recent reports, in addition to our data, may begin to elucidate a common mechanism involving signaling by oxidative stress and intracellular calcium, which may be used by the cell in response to several different viruses. Based on our finding that NF-κB activation was abrogated by both CGP and RR and is slightly reduced following treatment with dantrolene, we suggest that HSV IE or E proteins target primarily mitochondria, and possibly the ER, and induce calcium release. These two organelles have recently been demonstrated to cross-talk with respect to intracellular calcium homeostasis and have tightly interconnected functions (47). However, virus-induced calcium flux from mitochondria remains to be formally demonstrated.
Our data imply a link between cytosolic calcium and the kinases TAK1, NIK, and MEKK1. We hypothesized that one or more calcium-dependent kinases might be involved. For example, it has been demonstrated that calcium/calmodulin-dependent protein kinase II mediates TCR/CD3- and phorbol ester-induced activation of IKK and NF-κB (33), and that this kinase is regulated by oxidation-sensitive mechanisms (34). However, experiments using KN93, an inhibitor of calcium/calmodulin-dependent protein kinases, did not affect HSV-induced NF-κB activation. In addition, we have previously demonstrated that inhibition of protein kinase C did not prevent HSV-induced IL-6 expression in macrophages (1). Thus, how intracellular calcium may activate IKK and upstream kinases remains to be determined and is a subject of current research in the laboratory.
Mitochondria are key organelles which generate cellular energy and control apoptosis by releasing death-promoting proteins, most notably cytochrome c, into the cytoplasm (48). In addition, it is the principal organelle in which ROIs are generated in response to cellular stress, including viral infection (49). Our observation that rotenone inhibited NF-κB activation by HSV suggests that mitochondrial stress by oxidants plays the major role in mobilization of calcium and subsequent activation of NF-κB. Nevertheless, our finding that DPI also inhibited NF-κB activation leaves open the possibility of additional sources of ROIs. The significance of mitochondria in HSV-induced signaling is, however, further supported by our finding that apocynin did not prevent HSV-induced NF-κB activation. In addition, we found that in the presence of EGTA-AM, H2O2-induced activation of NF-κB was abrogated, thus demonstrating that calcium signaling is an essential step downstream of ROI production in signaling to HSV.
Altogether, the data presented in this study suggest that oxidative stress in mitochondria and mobilization of calcium in virus-infected macrophages trigger subsequent activation of kinases regulating IKK, eventually leading to IκB phosphorylation and NF-κB activation. The results thus suggest that mitochondria, which have long been recognized to play a central role in apoptosis, are also capable of sensing cellular stress and initiating a proinflammatory response during a virus infection in macrophages.
We thank Drs. J. A. Didonato, H. Holtmann, M. Nakanishi, K. Matsimuto, W.C. Greene, and B. Su for generous donations of DNA constructs.
↵1 This work was supported by grants from the Dagmar Marshalls Fond, the Novo Nordisk Foundation, and the Carlsberg Foundation. T.H.M. and J.M. were supported by fellowships from the Faculty of Health Sciences, University of Aarhus.
↵2 Address correspondence and reprint requests to Dr. Trine H. Mogensen, Department of Medical Microbiology and Immunology, University of Aarhus, The Bartholin Building, DK-8000 Aarhus C, Denmark. E-mail address:
↵3 Abbreviations used in this paper: IKK, IκB kinase; NIK, NF-κB-inducing kinase; MEKK, mitogen-activated kinase/extracellular signal-regulated kinase kinase; MAP3K, mitogen-activated protein kinase kinase kinase; TAK, TGF-β-activated kinase; NAK, NF-κB-activating kinase; ROI, reactive oxygen intermediate; IP, immunoprecipitation; gD, glycoprotein D; MKK, mitogen-activated protein kinase kinase; DPI, diphenyleneiodonium chloride; EGTA-AM, EGTA tetra-(acetoxymethyl-ester); NAC, N-acetyl-l-cysteine; PDTC, pyrollidine dithiocarbamate; RR, ruthenium red; PAA, phosphonoacetic acid; IE, immediate early; E, early; ER, endoplasmic reticulum; HBx, hepatitis B virus X; PC, peritoneal cell; MOI, multiplicity of infection.
- Received September 19, 2003.
- Accepted April 7, 2003.
- Copyright © 2003 by The American Association of Immunologists