CpG Protects Human Monocytic Cells against HIV-Vpr–Induced Apoptosis by Cellular Inhibitor of Apoptosis-2 through the Calcium-Activated JNK Pathway in a TLR9-Independent Manner

Monocytic cells represent a key target of HIV and play a crucial role in disease progression (1). Monocytes, monocyte-derived macrophages (MDMs), and tissue macrophages can be productively infected by HIV (2, 3). Persistently infected monocytic cells serve as a major reservoir of HIV in lymphoid tissues at all stages of disease and represent a key challenge to eradicating HIV infection. It is generally believed that monocytic cells, unlike T cells, survive HIV replication without major signs of HIV-induced cytopathic effects (1–3). However, we and others have shown that monocytic cells from HIV-infected patients and HIV-negative individuals when infected in vitro can undergo apoptosis (4, 5). Moreover, MDMs were shown to undergo apoptosis following in vitro infection with HIV, which was mediated by downregulation of Akt-1 and the FOXO3a transcription factor (6, 7). It appears that monocytic cells can undergo apoptosis but may escape HIV cytopathic effects as a result of certain factors reducing their sensitivity to apoptosis (8). For example, nerve growth factor was shown to act as an autocrine survival factor that rescued monocytic cells from the cytopathic effects of HIV (9), whereas IL-13 decreased their spontaneous apoptosis from HIV-infected patients (10). However, the mechanism(s) underlying the development of resistance to HIV-induced apoptosis in monocytic cells are poorly understood and may result in viral persistence.

Viral protein R (Vpr), a 14-kDa 96-aa multifunctional regulatory protein of HIV, can induce cell-cycle arrest at the G₂/M phase of the cell cycle (11) and promotes apoptosis in T cells (12), monocytes (13, 14), and neuronal cells (15). Vpr-induced cell-cycle arrest has been suggested to create a favorable environment for maximal virus production (12). Vpr is also secreted from HIV-infected cells and has been detected in the serum and cerebrospinal fluid of HIV-infected patients at levels similar to those of the HIV-p24 Ag (16). Notably, extracellular Vpr can also be efficiently taken up by cells, and its transport appears to be independent of cellular receptors or ionic gradients (11, 17). Moreover, circulating Vpr is biologically active, as it has been shown to induce virion production from latently infected cells and apoptosis of uninfected bystander cells (16, 18).

Multiple functions of Vpr have been attributed to its different domains. Mapping studies performed on isolated mitochondria revealed that the N-terminal 1–51 aa of Vpr, Vpr(1–51), are vital for virion incorporation and nuclear localization, whereas the C-terminal 52–96 aa, Vpr(52–96), induce cell-cycle arrest and apoptosis (11, 17, 19, 20). Vpr can cause apoptosis either upon infection with Vpr-expressing HIV isolates or following exposure of cells to the purified protein (14, 15, 21). We and others have shown that this apoptotic effect is mimicked by Vpr(52–96) but not by the Vpr(1–51) moiety (13, 22). Moreover, Vpr(52–96)-induced apoptosis in monocytic cells occurred through the mitochondrial pathway and requires JNK MAPK activation (13).

Activation of monocytic cells by microbial components has been shown to confer antiapoptotic survival signals (23, 24). Recently, elevated levels of microbial products, as a consequence of their translocation from the gut, were found in the serum of persons with chronic HIV infection, and this was linked to systemic immune activation and immunodeficiency (25, 26). Moreover, immune responses to several coinfections, like those causing endotoxemia and tuberculosis, have been considered to be a crucial factor in HIV pathogenesis and disease progression (27, 28). We hypothesized that monocyte activation by such microbial products interacting with their corresponding TLRs may confer antiapoptotic survival signals against HIV-induced cytopathic effects in these cells. Our results show for the first time, to our knowledge, that among various TLR agonists examined, pretreatment with TLR9 ligand CpG DNA afforded maximum protection in monocytic cells against HIV-Vpr–induced apoptosis. Subsequently, we investigated the mechanism underlying CpG-mediated protection against HIV-induced cytopathic effects in monocytic cells by using Vpr(52–96) peptide as a model apoptosis-causing agent. Our results further suggest that this resistance to apoptosis was mediated through TLR9-independent signaling via induction of the cellular inhibitor of apoptosis (c-IAP)-2 gene and involved the selective activation of calmodulin (CaM)/CaM-dependent protein kinase-II (CaMK-II) and JNK MAPK.

PBMCs were isolated by density gradient centrifugation over Ficoll-Hypaque (Pharmacia Biotech, Piscataway, NJ). PBMCs thus obtained were subjected to AutoMACS negative selection (Miltenyi Biotec, Auburn, CA) as per manufacturer’s instructions. THP-1 cells, a promonocytic cell line derived from an acute monocytic leukemia patient, were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in IMDM-10 (Sigma-Aldrich, St. Louis, MO) supplemented with 10% FBS (Invitrogen, Grand Island, NY), 100 U/ml penicillin, and 100 μg/ml gentamicin (all from Sigma-Aldrich). Second mitochondrial activator of caspases (SMAC) mimetic AEG-730 was a gift from Dr. Korneluk (Apoptosis Research Centre, Ottawa, ON, Canada). Lipoteichoic acid (LTA), imiquimod, LPS (Sigma-Aldrich), CpG-B oligodeoxynucleotide (ODN) 2006 (Hycult Biotech, Plymouth Meeting, PA), Lyovec Escherichia coli DNA, and GpC control ODN (Invitrogen) were purchased. The following signaling inhibitors were used: chloroquine and EGTA (Sigma-Aldrich); 2-APB, W-7 hydrochloride, KN-93, SKF-96365 hydrochloride, PD98059, Ly294002, SB203580 (Calbiochem, San Diego, CA) (29, 30); FK-506 (AG Scientific, San Diego, CA) (31); and SP600125 (Enzo Life Sciences) (13). All other chemicals used for electrophoresis and immunoblot analysis were obtained from Sigma-Aldrich.

For generation of MDMs, monocytes were isolated by the adherence method. Briefly, PBMCs were resuspended in serum-free medium (5 × 10⁶/ml) and cultured in 12-well polystyrene plates (BD Biosciences, Mississauga, ON, Canada) for 3 h to adhere to the plate. The nonadherent cells were washed off, and adherent cells were cultured for another 6 d in IMDM-10 supplemented with 10 ng/ml M-CSF (R&D Systems, Minneapolis, MN).

The Vpr(52–96) peptide was synthesized by automated solid-phase synthesis and purified by reverse-phase HPLC (>95%) (Invitrogen). The amino acid sequence of Vpr peptide Vpr(52–96) is ⁵²DTWAGVEAI IRILQQLLFI HFRIGCRHSR IGVTRQRRAR NGASRS⁹⁶. The mutant Vpr peptide with three arginine to alanine mutations at sites R73, R77, and R80 and indicated in bold letters in the above sequence was synthesized (Genemed Synthesis). Cells were cultured for 12 h in serum-free media before treatment with Vpr peptides, as described previously (13, 21).

Apoptotic cells exhibiting sub-G₀ DNA content were identified and analyzed by flow cytometry using propidium iodide (PI) staining of permeabilized cells, as described previously (13). Briefly, cells (1.0 × 10⁶/ml) were washed twice with PBS containing 1% FBS, fixed with methanol for 15 min at 4°C, and treated with 1 μg/ml RNAse A (Roche Applied Science, Laval, QC, Canada), followed by staining with 50 μg/ml PI (Sigma-Aldrich) at 4°C for 1 h. The DNA content was then analyzed by flow cytometry (BD FACS Canto equipped with BD FACS Diva software v5.0.3; BD Biosciences). Apoptosis was also measured by staining cells (1.0 × 10⁶/ml) with FITC-labeled Annexin V (Molecular Probes, Eugene, OR) for 15 min at room temperature in the dark followed by flow cytometry and data analysis using Win-MDI version 2.8 software (J. Trotter, The Scripps Research Institute, San Diego, CA).

Briefly, total proteins from cell lysates were subjected to SDS-PAGE followed by transfer onto polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA). The membranes were probed with Abs specific for c-IAP-1, c-IAP-2, XIAP, Bcl2, CaMK-II, phospho-p38, phospho-Akt, Akt, Mcl-1, Bax, and GAPDH (all from Cell Signaling Technology, Danvers, MA), phospho–CaMK-II (Stressgen Bioreagents, Victoria, BC, Canada), phospho-JNK, JNK, phospho-ERK, ERK, and p38 (Santa Cruz Biotechnology, Santa Cruz, CA), followed by donkey anti-rabbit secondary polyclonal Abs conjugated to HRP (Amersham Bioscience, Montreal, QC, Canada). All immunoblots were visualized by ECL (Amersham Bioscience) as described previously.

THP-1 cells were transiently transfected with either dominant-negative (DN)–CaMK-II or empty vector plasmid using transfection reagent FuGENE6 (Roche Applied Science), as described previously (32). For transfection, 5 μg test plasmid was incubated for 30 min at room temperature with 10 μl FuGENE6 in 100 μl OPTI-MEM1 (Invitrogen) medium to allow formation of DNA–liposome complexes. These complexes were then added to the cell suspension (1.0 × 10⁶/ml) for 24 h followed by stimulation with CpG for 12 h. The cells were treated with Vpr(52–96) overnight, and the percentage of apoptotic cells was determined by staining with Annexin V and flow cytometry.

The small interfering RNAs (siRNAs) against c-IAP-2, JNK-1, JNK-2, and CaMK-IIγ (Santa Cruz Biotechnology) were used to inhibit CpG-induced c-IAP-2 induction. Briefly, the control nonsilencing siRNA (Qiagen, Mississauga, ON, Canada) and c-IAP-2 siRNA were incubated with 3 μl FuGENE6 (Roche Applied Science) at a 1:3 ratio (micrograms/microliters) in 100 μl serum-free medium for 30 min at room temperature before adding to THP-1 cells (0.25 × 10⁶/0.5 ml). After 5 h of transfection, cells were transferred into complete medium, stimulated with CpG (5 μM) for 48 h, and treated with 1.5 μM Vpr(52–96) for another 24 h, followed by the measurement of apoptosis using Annexin V staining and flow cytometry.

The nonsilencing siRNA or JNK-1 and JNK-2 siRNA was used to transfect THP-1 cells (0.25 × 10⁶/0.5 ml) using TransMessenger (Qiagen) transfection reagent at a ratio of 1:5 (micrograms/microliters) as per the manufacturer’s protocol. Similarly, the control nonsilencing siRNA (Qiagen) and CaMK-IIγ siRNA were transfected using FuGENE6 (Roche Applied Science) transfection reagent at a 1:3 ratio (micrograms/microliters). After 5 h of transfection, cells were transferred into complete medium and incubated for 24–48 h followed by stimulation with CpG (5 μM) for 12 h and treated with 1.5 μM Vpr(52–96) overnight followed by measurement of apoptotic cells by Annexin V staining and flow cytometry. Cell pellets were collected prior to Vpr treatment to perform immunoblotting to detect c-IAP-2 expression.

Briefly, cells were washed with PBS and plated on poly-l-lysine–coated (Sigma-Aldrich) coverslips in 12-well plates for 20 min at 37°C. The adhered cells were fixed (4% paraformaldehyde in PBS for 30 min at 37°C) and quenched (10 min in 50 mM NH₄Cl in PBS). This was followed by permeabilization in 0.1% Triton in PBS (10 min) and incubation with FITC-conjugated monoclonal TLR9–specific Ab and FITC-conjugated mouse IgG_2a isotype control Ab (both from Hycult Biotech) overnight at 4°C. Thereafter, the coverslips were mounted using Prolong Gold Antifade reagent with DAPI nuclear stain (Invitrogen). Confocal fluorescent images were obtained using a Zeiss LSM510 confocal scan head mounted on a Zeiss Axiovert 200M (Carl Zeiss) on an inverted-base microscope with a ×63 objective. Images were analyzed by Zeiss software (Carl Zeiss) and ImageJ (National Institutes of Health freeware).

Data sets were graphed and analyzed by the two-tailed Student t test using Microsoft Excel software (Microsoft). Apoptosis results were normalized against the percentage Annexin V-positive cells in unstimulated samples. The protective effect of TLR ligands was calculated as percent apoptosis relative to the Vpr-induced apoptosis.

Blood was obtained from healthy volunteers after approval of the protocol by the ethics review committee of the Ottawa Hospital (Ottawa, ON, Canada). A written informed consent was obtained from the study participants.

We have previously reported that Vpr(52–96) induced apoptosis in THP-1 cells and primary human monocytes (13). In addition, Vpr-containing retroviruses have been shown to induce apoptosis in these cells (14, 33). To precisely delineate the signaling pathways involved in Vpr(52–96)-induced apoptosis, we used synthetic Vpr peptides, as these are free of contaminating bacterial products present in recombinant Vpr proteins and certain nonspecific or undefined factors present in retroviral supernatants that may be capable of nonspecifically and/or transiently activating various signaling pathways. Moreover, Vpr(52–96) peptide mimics the apoptotic activity of full-length Vpr peptide (13, 33). Even though Vpr(52–96) induced significant apoptosis in undifferentiated primary monocytes and THP-1 cells (Fig. 1A, 1B), differentiated MDMs displayed a marked resistance to Vpr(52–96)-induced apoptosis (Fig. 1C). Three Vpr arginine residues at sites 73, 77, and 80 have been shown to be essential for Vpr-mediated mitochondrial permeabilization and apoptosis (19, 34). The control mutant Vpr(52–96) peptide with arginine to alanine mutations at sites 73, 77, and 80 did not induce cell death either in MDMs or THP-1 cells (Fig. 1C, 1D).

Despite the apoptogenic affect of Vpr at high concentrations, low concentrations of Vpr have been shown to protect CD4⁺ T cells against apoptotic agents like cyclohexamide, TNF-α, and sorbitol (35, 36). To determine if low concentrations of Vpr protected monocytic cells against Vpr-mediated cytopathic effects, we sequentially treated monocytic cells with two low, nonapoptogenic concentrations of Vpr(52–96) at intervals of 24 h in THP-1 cells and 2 h in primary monocytes followed by measurement of apoptosis. It was observed that cells that received two treatments with low doses of Vpr(52–96) exhibited significantly higher apoptosis compared with cells receiving single dose of Vpr (Fig. 2). These results suggest that low concentrations of Vpr(52–96) instead of conferring protection enhanced apoptosis upon subsequent exposures to a low nonapoptotic dose of Vpr(52–96). Therefore, low nonapoptotic concentrations of Vpr present in the serum of HIV-infected individuals (16) may be apoptotic for primary monocytes and thus may have biological relevance as an apoptosis-inducing agent under in vivo conditions.

Recently, translocation of various microbial products, like LPS, from the gut has been implicated in causing systemic immune activation, TLR stimulation (25, 26), and upregulation of programmed death receptor-1 on monocytes (37) in HIV-infected individuals. Therefore, we hypothesized that stimulation with various TLR ligands may protect monocytic cells against Vpr(52–96)-induced apoptosis. To determine the effect of TLR ligands on Vpr(52–96)-induced apoptosis, THP-1 cells and monocytes were stimulated initially with LTA, LPS, imiquimod, and CpG, the ligands for TLR2, TLR4, TLR7, and TLR9, respectively, followed by treatment with Vpr(52–96) peptide and analysis for apoptosis. Pretreatment with LTA decreased apoptosis by 60% in THP-1 cells (Fig. 3A) and monocytes (data not shown), whereas LPS pretreatment exhibited a modest protection in both cell types (Fig. 3A and data not shown). In contrast, imiquimod did not induce protection against Vpr(52–96)-induced apoptosis in both cell types (Fig. 3A and data not shown). Interestingly, pretreatment with TLR9 agonists, synthetic CpG DNA, and E. coli DNA at concentrations recommended by the manufacturer reduced apoptosis by >80 and 50%, respectively, in THP-1 cells in a dose-dependent manner (Fig. 3A–C). Similar results were obtained in primary monocytes following CpG and E. coli DNA stimulation (Fig. 3D, 3E). Notably, CpG failed to induce protection if cells were treated with Vpr(52–96) prior to CpG stimulation (Fig. 3F).

Cell survival is regulated by distinct antiapoptotic genes in different cell types (38). To identify antiapoptotic gene(s) involved in CpG-induced protection against Vpr(52–96)-mediated apoptosis, we first analyzed the induction of pro-/antiapoptotic genes c-IAP-1, c-IAP-2, XIAP, Bcl2, Mcl1, and Bax in CpG-stimulated THP-1 cells and primary monocytes. Our results show that both CpG and E. coli DNA upregulated c-IAP-2 expression in THP-1 cells in a dose-dependent manner. Similar results were obtained in primary monocytes following CpG treatment as well. However, no significant change was observed in the expression of c-IAP-1, XIAP, Bcl2, Mcl1, or Bax genes in either cell type following CpG treatment (Fig. 4A). In accordance with our above results showing that pretreatment with Vpr(52–96) abrogated CpG-induced resistance against Vpr(52–96)-mediated apoptosis (Fig. 3F), monocytes did not exhibit c-IAP-2 induction in response to CpG if they had been pretreated with Vpr(52–96) (Fig. 4B).

Because CpG selectively induced c-IAP-2 expression, we hypothesized that CpG-induced protection may be regulated by c-IAP-2. Therefore, THP-1 cells were transfected with siRNA against c-IAP-2 prior to stimulation with CpG. c-IAP-2–specific siRNA significantly inhibited CpG-induced c-IAP-2 expression compared with the cells transfected with nonsilencing control siRNA (Fig. 5A). Significantly, prior stimulation with CpG failed to inhibit Vpr(52–96)-induced apoptosis in cells transfected with c-IAP-2 siRNA compared with the cells transfected with control siRNA (Fig. 5B). These results suggest that the protective effect of CpG against Vpr(52–96)-mediated apoptosis may be regulated via c-IAP-2 induction in monocytic cells.

To confirm the role of CpG-induced c-IAP-2 in conferring protection against Vpr(52–96)-mediated apoptosis, we used SMAC mimetic AEG-730. These are synthetically produced novel small molecules that mimic the activity of cellular SMAC and target IAPs for rapid degradation (39). THP-1 cells and primary monocytes were treated with SMAC mimetic and CpG. Consistent with the results observed with c-IAP-2 siRNA, stimulation with CpG failed to inhibit Vpr(52–96)-induced apoptosis in THP-1 cells or primary monocytes treated with SMAC mimetic (Fig. 5C, 5D). Treatment with SMAC mimetic alone did not cause apoptosis in either cell type (data not shown). Significantly, treatment with SMAC mimetic also inhibited CpG-induced c-IAP-2 expression (Fig. 5E). These results combined with the siRNA data support a protective role for c-IAP-2 in CpG-induced resistance to apoptosis caused by Vpr(52–96) in human monocytic cells.

The c-IAP-2 gene has been shown to be regulated by the ERK and p38 MAPKs in human colon epithelial cells and by the PI3K pathway in human breast cancer MCF-7 cells (40, 41). To determine the role of MAPK and PI3K pathways in CpG-induced protection against Vpr(52–96)-induced apoptosis, specific pharmacological inhibitors of these pathways were used. First, the biological activity of SB203580, SP600125, PD98059, and Ly294002, the specific inhibitors for p38, JNK, ERK, and Akt, respectively, were confirmed for their ability to inhibit phosphorylation of corresponding kinases in response to CpG stimulation (Fig. 6A). Thereafter, THP-1 cells were treated with SP600125, PD98059, SB203580, and Ly294002 before stimulation with CpG followed by treatment with Vpr(52–96) and analysis for apoptosis. Interestingly, pretreatment with JNK inhibitor SP600125 significantly prevented protection afforded by CpG in THP-1 cells. In contrast, inhibitors for p38, ERK, or PI3K pathways did not affect CpG-mediated protection in these cells (Fig. 6B, top panel). Similar results were obtained in primary monocytes with respect to JNK inhibition (Fig. 6B, bottom panel). In accordance with the protection results, pretreatment of THP-1 cells and primary monocytes with SP600125 prevented CpG-induced c-IAP-2 expression, whereas inhibiting p38, ERK, or Akt phosphorylation did not affect c-IAP-2 induction (Fig. 6C).

The involvement of JNK in CpG-induced protection was further confirmed by transfecting THP-1 cells with either control nonsilencing siRNA or JNK siRNAs for 48 h and stimulating with CpG for 12 h followed by Vpr(52–96) treatment. Consistent with the JNK inhibitor studies, cells transfected with JNK siRNAs displayed a marked lack of protection against Vpr(52–96)-induced apoptosis compared with control siRNA-transfected cells (Fig. 7A). Moreover, cells transfected with JNK siRNA failed to upregulate CpG-induced c-IAP-2 expression compared with control siRNA-transfected cells (Fig. 7B).

CaM/CaMK-II pathway has previously been demonstrated to regulate response to TLR ligands in monocytic cells (32, 42). Therefore, we hypothesized that in addition to the JNK pathway, CpG-induced protection against Vpr-mediated apoptosis may also be regulated by the calcium signaling pathway in monocytic cells. Consistent with our hypothesis, CpG treatment was found to cause rapid calcium influx in THP-1 cells that was inhibited upon addition of calcium chelator EGTA in THP-1 cells (data not shown). The biological activity of various pharmacological inhibitors of the calcium signaling pathway, namely SKF, W-7, EGTA, and KN-93, was confirmed by their ability to inhibit CpG-induced CaMK-II phosphorylation in a dose-dependent manner in THP-1 cells (Fig. 8A). Elevated cytoplasmic calcium concentrations occur in response to stimuli that activate voltage or ligand-gated calcium channels in the plasma membrane or following the release of calcium mainly from the endoplasmic reticulum (ER) (43). Interestingly, treatment with calcium chelator EGTA prior to stimulation with CpG reversed the CpG-mediated protection against Vpr(52–96)-induced apoptosis in both THP-1 cells and primary monocytes (Fig. 8B). The role of receptor-mediated entry of extracellular Ca²⁺ was studied by using SKF-96365 (29). To determine whether calcium release from ER regulates CpG-induced resistance to Vpr-mediated apoptosis, we used 2-APB, which inhibits the release of calcium from ER by blocking inositol 1,4,5-triphosphate receptor-gated channels (30). Unlike 2-APB, SKF-96365 treatment prior to stimulation with CpG reversed the CpG-mediated protection against Vpr(52–96)-induced apoptosis in both cell types (Fig. 8B and data not shown). Calmodulin, a major calcium receptor, is present in both cytoplasmic and nuclear compartments. The calcium/CaM complex regulates several downstream targets including protein kinases and phosphatases. One major family of calcium/CaM effectors is CaMK, which includes multifunctional kinases CaMK-II and calcineurin (44, 45). The role of CaM, CaMK-II, and calcineurin was determined by using their inhibitors W-7, KN-93, and FK-506, respectively. Unlike FK506, pretreatment with both W-7 and KN93 significantly reversed the CpG-mediated protection against Vpr(52–96)-induced apoptosis in both cell types (Fig. 8B and data not shown). Notably, 24 h treatment of THP-1 cells and primary monocytes with the highest concentrations of all of the inhibitors used in this study did not cause significant apoptosis (data not shown). These inhibitor studies suggest that protection induced by CpG is mediated via influx of extracellular calcium and CaM/CaMK-II activation, whereas ER calcium stores and calcineurin do not play a significant role in CpG-induced protection from apoptosis caused by Vpr(52–96) treatment. In addition, pretreatment with EGTA, SKF, KN-93, and W-7 also prevented CpG-induced expression of c-IAP-2 in THP-1 cells and monocytes (Fig. 8C), suggesting that c-IAP-2 induced by CpG was instrumental in protecting cells from apoptosis caused by Vpr(52–96).

The role of CaMK-II was confirmed by transfecting THP-1 cells with CaMK-II–specific siRNA as well as a DN–CaMK-II construct followed by stimulation with CpG and subsequent determination of c-IAP-2 expression and Vpr(52–96)-induced apoptosis. Transfection with both CaMK-II siRNA and DN–CaMK-II construct significantly inhibited both the c-IAP-2 expression induced by CpG (Fig. 9A, 9C) and the protective effects of CpG (Fig. 9B, 9D) compared with the cells transfected with control siRNA or control vector. These results suggest that CpG-induced protection against Vpr(52–96)-mediated apoptosis is regulated, at least in part, by c-IAP-2 expression through the activation of JNK and CaMK-II in human monocytic cells.

The above results suggest that CpG-induced c-IAP-2 expression was regulated by both activation of JNK and calcium pathways. Therefore, it was interesting to determine if these two pathways cross-talked and regulated each other. For this, we inhibited either calcium signaling using EGTA or JNK activation by SP600125 followed by stimulation with CpG. Blocking calcium signaling not only inhibited phosphorylation of CaMK-II but also prevented JNK activation (Fig. 10A). In contrast, blocking JNK activation did not prevent CaMK-II activation (Fig. 10B), suggesting that JNK activation takes place downstream of CaMK-II phosphorylation in response to CpG stimulation.

Human monocytes have been reported to express low levels of TLR9 mRNA (46). Keeping in view the above-mentioned results, we determined if CpG-mediated protection from Vpr(52–96)-induced apoptosis is indeed mediated through TLR9 signaling. For this, we first demonstrated that THP-1 cells and monocytes expressed TLR9 (Fig. 11A). Chloroquine has been shown to inhibit endocytic maturation and subsequent TLR9 activation (47). Therefore, to further confirm the involvement of TLR9 signaling, THP-1 cells and monocytes were treated with chloroquine followed by CpG stimulation and Vpr(52–96) treatment. Cells pretreated with chloroquine at concentrations of 25 μM failed to significantly reverse CpG-induced protection (Fig. 11B), suggesting that CpG, under our experimental conditions, may exert its effects through TLR9-independent mechanisms (48–51). In support of this, we found that stimulation with non-TLR9–activating GpC control ODNs prior to Vpr(52–96) treatment was equally capable of inhibiting Vpr(52–96)-induced apoptosis (Fig. 11C). Thus, our results suggest that CpG-induced protection in human monocytes from Vpr(52–96)-mediated apoptosis may be regulated by the inhibitor of apoptosis c-IAP-2 through the calcium-activated JNK pathway but in a TLR9-independent manner (Fig. 12).

Recent developments indicating translocation of microbes or their components from the gut into the circulation during chronic HIV infection suggest a critical role for TLR ligands in HIV pathogenesis. In fact, high levels of bacterial LPS and DNA have been found in the plasma of persons infected with HIV. Moreover, LPS levels in plasma have also been correlated with indices of T cell activation, IFN-α plasma levels, and CD4⁺ T cell restoration following antiretroviral therapy (25, 26). Besides, chronic HIV-1 infection was associated with increased TLR expression and immune responsiveness (52). Because LPS and CpG DNA have been implicated in enhanced cell survival (8, 24, 53), we determined whether monocytic cells’ stimulation by various TLR agonists can modulate their survival in response to apoptogenic Vpr(52–96) peptide. In this study, we show for the first time, to our knowledge, that TLR2, TLR4, and TLR9 agonists induce resistance to Vpr(52–96)-mediated apoptosis, with CpG inducing maximum resistance in primary human monocytes and THP-1 cells. Furthermore, this protection is mediated by c-IAP-2 induction through the calcium/CaMK-II–activated JNK pathway in a TLR9-independent manner.

Synthetic/bacterial DNA rich in unmethylated CpG motifs has previously been shown to enhance cell survival by preventing spontaneous apoptosis in mouse B cells and dendritic cells (DCs) via PI3K signaling and upregulation of antiapoptotic Bcl2, BclXl, c-IAP-1, and c-IAP-2 genes (24, 54). CpG DNA is a classical TLR9 ligand (55) and has been shown to induce potent Th1 responses, resulting in secretion of IL-12 and IFN-γ via a MyD88-dependent signaling pathway involving MyD88, IL-1R–associated kinases, and TNFR-associated factor-6 (56, 57). In the recent past, a growing body of evidence suggests that DNA can also be recognized by the host cells independent of TLR9, leading to inflammatory events such as activation of a set of genes encoding type I IFNs, chemokines, histocompatibility complex, costimulatory molecules, transcription factors STAT1 and IFN regulatory factor, and protein kinase R (48–51, 58, 59). The precise mechanism by which CpG exerts its effects via TLR9-independent pathways is not well understood. Several TLR9-independent CpG signaling cascades have been suggested, including activation of two Src family kinases, Hck and Lyn (51), activation of transcription factor IFN regulatory factor-3 and IFN-β promoter stimulator-1 through the signaling pathway requiring the kinases TANK-binding kinase-1 and inducible κB kinase-i (60), DNA-dependent protein kinase-induced Akt activation in murine macrophages (61), and inhibition of Smad protein-regulated signaling in human osteoblasts (48).

Calcium (Ca²⁺), a pervasive intracellular second messenger, plays a key role in mediating the transcription of several cellular genes (44, 62). In general, binding of a ligand to its receptor induces influx of extracellular Ca²⁺ and/or release of intracellular Ca²⁺ stores from the ER. Many of the effects of Ca²⁺ are mediated via CaM. CaM is a highly conserved 17-kD protein associated either with membrane, cytoplasm, or nucleus. It binds up to four calcium ions and undergoes a conformational change that renders it active to bind its target proteins with high affinity. CaMK-II is one of the key targets for CaM (43). It is activated by increases in intracellular calcium levels and essential for the translation of calcium signals to enable gene transcription. CaMK-II can phosphorylate several downstream effectors including transcription factors involved in diverse cellular functions such as T cell activation and cytokine production (43, 44).

Ca²⁺ influx in various cell types including monocytic cells in response to TLR4 ligands has been shown (62, 63). However, very little is known about the involvement of Ca²⁺ signaling and its major downstream kinases, CaMKs, in CpG-mediated activation of cellular genes. In the current study, we provide evidence that CpG can trigger elevation of intracellular Ca²⁺ by inducing influx of extracellular Ca²⁺ and activation of CaMK-II in monocytic cells. These observations clearly suggest that calcium signaling and in particular CaMK-II activation may serve as a key pathway in TLR9-independent CpG signaling, leading to the development of resistance to apoptosis in human monocytic cells.

JNK, a serine/threonine kinase, plays a critical role in cell survival, as it has been shown to be both pro- and antiapoptotic depending upon the cell type and the apoptosis-inducing agent (64). For example, thymocytes from JNK null mice were shown to suffer from increased Fas/CD95- and CD3-mediated apoptosis (65). In contrast, jnk1^−/− and jnk2^−/− murine embryonic fibroblasts were found to be resistant to UV-induced apoptosis (66). In this study, we report an antiapoptotic role of JNK in CpG-induced protection against apoptosis caused by Vpr(52–96) in human monocytic cells via upregulation of c-IAP-2. In accordance with previous studies showing cross interaction between MAPK and Ca⁺ signaling (44), our results demonstrate that CaM/CaMK-II activation is a prerequisite for JNK phosphorylation in response to TLR9-independent CpG signaling.

The IAP genes are key regulators of apoptosis and overexpressed in many cancer cells (38). There are now eight mammalian homologs of IAPs: XIAP (hILP), NAIP, c-IAP-1 (HIAP2), c-IAP-2 (HIAP1), livin (ML-IAP/KIAP), survivin, Ts-IAP (hILP2), and Apollon (Bruce) (38, 67, 68). The results of this study suggest that CpG selectively upregulates the expression of c-IAP-2. Moreover, inhibition of c-IAP-2 expression by CaM/CaMK-II or JNK antagonists reversed CpG-mediated protection against Vpr(52–96)-induced apoptosis. The expression of other antiapoptotic gene products such as c-IAP-1, XIAP, and Bcl2 was not upregulated in our studies, suggesting these genes may not play a significant role in development of resistance to Vpr-induced apoptosis in monocytic cells following CpG stimulation. However, determination of the precise role of these genes in the development of resistance to Vpr-induced apoptosis needs further investigation.

Vpr has been shown to induce apoptosis by directly affecting the mitochondrial permeability transition pore complex and specifically acting on the mitochondrial adenine nucleotide translocator, a component of the permeability transition pore complex. This event enhances permeability of the outer mitochondrial membrane with consequent release of apoptogenic factors such as apoptosis-inducing factor and cytochrome C (22, 33). Cytochrome C interacts with Apaf-1 and procaspase 9 to create an apoptosome, the caspase activation complex that causes activation of other caspases, resulting in apoptosis (69). The precise mechanism by which c-IAP-2 prevents Vpr-induced apoptosis remains to be investigated.

We and others have used relatively high concentrations of Vpr as apoptosis-inducing agents compared with the physiologically relevant low concentrations present in the plasma of HIV-infected individuals (16). Moreover, low concentrations of Vpr have been suggested to be protective rather than apoptosis-inducing agents in T cells (35, 36). Our results clearly suggest that undifferentiated primary monocytes and THP-1 cells, when exposed sequentially to low nonapoptogenic concentrations of Vpr, undergo enhanced apoptosis. Therefore, primary monocytes exposed persistently to low concentrations of Vpr present in serum/lymph nodes of HIV-infected individuals under in vivo conditions may in fact undergo apoptosis. However, these cells may be protected from the apoptogenic effects of Vpr following differentiation in tissues or exposure to various microbial products present in the circulation of HIV-infected individuals. In addition, interesting parallels can be drawn between resistance of MDMs and CpG-stimulated monocytes to Vpr(52–96)-driven apoptosis. Because CpG has previously been reported to induce macrophage-like characteristics in monocytes, such as increased expression of costimulatory and Ag-presenting molecules (70), it is possible that CpG induces resistance against Vpr-mediated apoptosis by propelling undifferentiated monocytes into a more mature and macrophage-like state of differentiation. The molecular mechanism responsible for the development of resistance to Vpr-induced apoptosis is not clear. It is possible that the differentiation process or prior stimulation with microbial products may interfere with the process of Vpr internalization and/or its interaction with adenine nucleotide translocator and subsequent release of apoptotic factors such as apoptosis-inducing factor and cytochrome C.

Highly purified monocytes depleted of plasmacytoid DCs do not express TLR9 or express low levels of TLR9 and do not respond to TLR9 agonists unless reconstituted with the plasmacytoid DCs (46). However, other reports indicate that monocytes isolated by adherence method or positive selection respond to CpG and express TLR9 (71–75), suggesting that the expression of TLR9 may depend on the method of isolation, cell activation, and the presence of nonmonocytic cells in the preparation. We have shown that both THP-1 cells and monocytes do express TLR9 and respond to CpG, although the monocytes used in this study may contain nonmonocytic cells (2–5%), including plasmacytoid DCs. Our results also suggest that TLR9-independent mechanisms may be operative in CpG-induced protection from Vpr(52–96)-mediated apoptosis, as chloroquine was unable to prevent CpG-induced protection. Concordantly, GpC control ODNs consistently prevented Vpr(52–96)-induced apoptosis, whereas TLR9 antagonist ODNs failed to inhibit CpG-induced protection (data not shown), further suggesting the role of TLR9-independent mechanisms. These observations are in accordance with several studies that describe TLR9-independent CpG signaling in neutrophils, DCs, mouse embryonic stem cells, macrophages, and THP-1 cells (48–51, 58, 59).

In summary, this is the first study, to our knowledge, that demonstrates a CpG/bacterial DNA-dependent mechanism for the resistance of human monocytic cells to an HIV accessory protein, Vpr. Moreover, TLR9-independent CpG-mediated resistance to Vpr-induced apoptosis occurred via expression of c-IAP-2 regulated by activation of CaM/CaMK-II and JNK signaling pathways (Fig. 12). Therefore, it is reasonable to speculate that monocytic cells from HIV-infected patients may have a reduced propensity to undergo apoptosis at least in part due to the presence of microbial products, including LPS and DNA present in chronically infected HIV patients (25) with various opportunistic infections. Keeping in view our results implicating c-IAP-2 in CpG-induced survival of human monocytic cells against Vpr-induced apoptosis, strategies based on suppression of c-IAP-2 (76, 77) induction by agents known to inhibit CaM/CaMK-II/JNK signaling pathways may prove helpful in controlling HIV reservoir formation.

We thank Dr. M. Kozlowski for critically reading the manuscript.

The authors have no financial conflicts of interest.