HIV-1 Nef Inhibits Lipopolysaccharide-induced IL-12p40 Expression by Inhibiting JNK-activated NFκB in Human Monocytic Cells*

Impaired cellular immunity caused by decreased production of Th1-type cytokines, including interleukin-12 (IL-12) is a major feature of HIV-1-associated immunodeficiency and acquired immunodeficiency syndrome. IL-12p40, an inducible subunit shared between IL-12 and IL-23, plays a critical role in the development of cellular immunity, and its production is significantly decreased during HIV infection. The mechanism by which HIV induces loss of IL-12p40 production remains poorly understood. We have previously shown that lipopolysaccharide (LPS)-induced IL-12p40 production in monocytic cells is regulated by NFκB and AP-1 transcription factors through the activation of two distinct upstream signaling pathways, namely the c-Jun-N-terminal kinase (JNK) and the calmodulin-dependent protein kinase-II-activated pathways. Herein, we show that intracellular nef expressed through transduction of primary monocytes and promonocytic THP-1 cells with retroviral-mediated nef gene inhibited LPS-induced IL-12p40 transcription by inhibiting the JNK mitogen-activated protein kinases without affecting the calmodulin-dependent protein kinase-II-activated pathway. In addition, nef inhibited JNK-activated NFκB without affecting the AP-1 activity. Overall, our results suggest for the first time that intracellular nef inhibited LPS-activated JNK, which may cause inhibition of IL-12p40 expression in human monocytic cells by selectively inhibiting NFκB activity.

HIV 5 infection results in a progressive loss of general and HIV-specific cellular immunity by inhibiting the production of Th1-type cytokines such as IL-12 (1)(2)(3)(4)(5). IL-12 acts as a bridge between innate and adaptive immune responses and plays a critical role in the immunopathogenesis of various diseases, including HIV infection, inflammation, and autoimmune disorders (3,4,6,7). It promotes Th1-type cell-mediated immune responses by inducing interferon-␥ from NK and T cells and enhances their cytotoxicity (7,8). IL-12 is produced by monocytic and dendritic cells and B cells (7,8). It is a 70-kDa heterodimer composed of p35 and p40 subunits that are disulfidelinked together to form biologically active IL-12 (9,10). The p35 and p40 subunits are encoded by two distinct and differentially regulated genes: the p40 gene is tightly regulated at transcriptional level and detected only in IL-12-producing cells, whereas the p35 gene is constitutively expressed in various cell types (9,10). IL-12p40, therefore, constitutes an indicator for IL-12 production. Moreover, IL-12p40 subunit is also shared by another Th1 cytokine IL-23, which makes it highly significant for determination of cell-mediated immune response (6).
The signaling pathways involved in the regulation of IL-12p40 synthesis in monocytic cells following LPS stimulation have been investigated. Multiple transcription factors, including NFB, Ets-2, AP-1, and C/EBP, PU.1, and interferon-␥ regulatory factors and their complexes have been suggested to regulate IL-12p40 transcription in LPS-stimulated murine and human monocytic cells (7,(11)(12)(13)(14). We and others have demonstrated that c-Jun-N-terminal kinase (JNK) plays a key role in the regulation of IL-12p40 production in LPS-stimulated human monocytic cells (13,15). Recently, we have also shown that LPS-induced IL-12p40 production is regulated by another distinct pathway, the calmodulin/CaM-activated protein kinase (CaMK-II)-activated phosphatidylinositol-3-kinase pathway (16). Interestingly, both pathways regulated IL-12p40 production through the NFB and AP-1 transcription factors (13,16).
Monocytic cells play a key role in HIV pathogenesis and serve as long-term reservoirs in chronically infected patients (2,17). IL-12 and in particular IL-12p40 production is impaired in HIV-infected patients and in monocytic cells infected in vitro with HIV (3)(4)(5). Active cellular infection and HIV replication inhibited IL-12p40 transcription and its synthesis in monocytic cells (5). We and others have shown that IL-12 production is decreased in HIV-infected patients and treatment with antiretroviral therapy enhanced IL-12 production (3-5, 18 -22). Furthermore, exogenous addition of IL-12 enhanced IL-2 production, cell proliferation, and the development of cell-mediated cytotoxicity of HIV antigen-stimulated PBMCs from HIVinfected individuals (3,4,18,19,(22)(23)(24).
To understand the mechanism underlying the loss of cellmediated immune response during HIV infection and development of AIDS, it is imperative to investigate the signaling pathways responsible for the loss of Th1 cytokines IL-12 and IL-23 and in particular the inducible IL-12p40 subunit shared between these two cytokines. At present, little is known regarding the regulation and expression of IL-12p40 in monocytic cells following HIV infection. There is evidence to suggest that HIV regulatory protein, nef, inhibits IL-12 synthesis. IL-12p40 production was shown to be suppressed in lymph nodes of macaques infected with simian immunodeficiency virus compared with those infected with the non-pathogenic nef-deleted strain, SIVmac239⌬ nef (25). However, the exact role of nef and the mechanism involved in the inhibition of IL-12p40 production in monocytic cells are not known. Nef is a 27-kDa myristoylated protein expressed early in HIV infection (26). In addition to the well known down-regulation of the cell surface CD4 and MHC-I receptors (27,28), nef uniquely can interact with a number of signaling molecules through its myristoyl moiety, leading to the dysregulation of host immune responses (29 -34). In this study, we show for the first time that intracellular expression of HIV-nef by retroviral infection of primary monocytes and promonocytic THP-1 cells and following stable transfection of THP-1 cells with HIV-nef gene resulted in the inhibition of LPS-induced IL-12p40 production. Studies conducted to understand the underlying mechanism revealed that nef inhibited IL-12p40 transcription by selectively inhibiting LPS-activated JNK without affecting the calcium signaling pathway. Moreover, nef inhibited JNK-activated NFB without affecting the activity of AP-1 transcription factor.
Isolation of Monocytes-Monocytes were purified from PBMCs by negative selection as described earlier (16,35). Briefly, PBMCs were incubated with magnetic polystyrene Dynabeads (Dynal Biotech, Oslo, Norway) coated with anti-CD2 (T cells) and anti-CD19 (B cells) antibodies for 30 min on ice for depletion of T and B cells, respectively. Cells were further incubated for 2 h at 37°C to eliminate nonadherent cells. The monocytes obtained contained Ͻ1% T and B cells as determined by flow cytometry. Some experiments were confirmed by using monocytes isolated by automacs negative selection (Miltenyi Biotech Inc., Auburn, CA). Briefly, PBMCs were washed twice in phosphate-buffered saline containing 2% EDTA followed by incubation with automacs FcR blocking reagent along with biotin antibody mixture for 10 min at 4°C. Following incubation, cells were treated with anti-biotin microbeads for 15 min at 4°C. Cells were then washed once and subjected to automacs negative selection separation as per the manufacturer's instructions. Cell populations thus obtained contained more than 90% CD14ϩ monocytes.
Production of nef Retroviruses-The retroviral vector pSR␣-MSVtkneo containing nef gene (pSR-␣-Nef) derived from HIV-1 SF2 strain (GenBank TM accession number K02007, nucleotides 8504 -9573) was a gift from Dr. T. Smithgall (University of Pittsburgh) (33). The control retroviruses were prepared by cleaving nef fragment by using Klenow Fragment (New England Biolabs). To produce high titer nef retrovirus stocks, PT67 cells (1.5 ϫ 10 6 ) were transduced with 4 g of nef retrovirus along with 12 g of FuGENE 6 (Roche Applied Science) in 2 ml of Iscove's modified Dulbecco's medium-fetal calf serum (10%) containing 4 mg/ml polybrene. After 24 h, the culture medium was replaced with fresh Iscove's modified Dulbecco's medium, and cells were selected by 300 g/ml G418 (Invitrogen). Seven days post-transfection, genomic DNA was analyzed to determine nef gene integration by PCR, and total proteins were analyzed for nef expression by immunoblotting with anti-nef monoclonal antibody, EH-1 (NIH AIDS Research and Reference Reagent Program, Rockville, MD). The neftransfected PT67 cells were grown to collect nef virus-containing supernatants followed by their titration by employing NIH 3T3 cells according to the manufacturer's protocol (BD Bioscience Clontech).
Infection of Monocytic Cells with nef Retrovirus and Measurement of IL-12p40 by ELISA-Briefly, cells were cultured in 3 ml of virus-containing supernatant collected from packaging PT67 cells for 24 h and infected a second time under identical conditions for another 24 h followed by stimulation with LPS for various times. Cells were harvested for protein and RNA extraction. The supernatants were collected for measurement of IL-12p40 production by ELISA (R & D Systems) as described earlier (13,16).
Stable Transfection of THP-1 Cells with pcDNA-Nef-The full-length HIV-1-nef gene plasmid derived from pclonsnefSN (NIH AIDS Research and Reference Reagent) was subcloned into the pcDNA3.1zeoϩ expression vector (Invitrogen) at the EcoRI restriction site. THP-1 cells were transfected with pcDNA3.1zeoϩ containing nef gene (pcDNA-Nef) by Lipofectamine reagent as per the manufacturer's instructions (Invitrogen). Briefly, 2 g of the pcDNA-Nef and 8 l of Lipofectamine were incubated with 200 l of Opti-MEM-I Reduced Serum Medium (Invitrogen) for 30 min at room temperature to allow formation of DNA-liposome complexes. The resulting complexes were added to the cell culture for 48 h following which the medium was replaced with fresh Iscove's modified Dulbecco's medium containing 200 g/ml of Zeocin. After 7 days, nef-mRNA and proteins were measured by RT-PCR and Western blotting, respectively.
For determination of equal infectivity of monocytic cells with nef retroviruses, RT-PCR analysis was performed for the neomycin gene present in the pSR␣ retroviruses containing nef gene. The primer sequence for the neomycin gene is as follows: Neo: sense, 5Ј-AGA GGC TAT TCG GCT ATG ACT G-3Ј; antisense, 5Ј-TTC GTC CAG ATC ATC CTG ATC-3Ј. The conditions for PCR amplification were essentially as described above for IL-12p40 (13,16).
Calcium Influx-Calcium influx was measured as described earlier (16,37). Briefly, cells were washed and suspended in Buffer A (RPMI 1640 containing 20 mM HEPES, pH 7.4) containing 1 mM Fluo-3/AM (Molecular Probes, Eugene, OR). The reaction was stopped by adding an equal volume of Buffer B (Buffer A containing 5% fetal calf serum, pH 7.4) followed by incubation for 15 min at 37°C. The cells were washed, and intracellular Ca 2ϩ levels were measured by FACScan flow cytometer (BD Biosciences) equipped with CellQuest software, version 3.2.1fl.
Western Blotting-Briefly, total proteins were subjected to Western immunoblotting as described earlier (13,16). All immunoblots were visualized by ECL (Santa Cruz Biotechnology, Santa Cruz, CA). The densitometry analysis was preformed by CHEMIGENIUS2 XE Bio Imaging System (PerkinElmer Life Sciences).
Transient Transfection with IL-12p40 Promoter-luciferase Reporter and Luciferase Assay-A series of truncated hIL-12p40 promoter fragments (Ϫ880 to ϩ108) were generated by PCR and subcloned into the NheI/NcoI sites of pGL3B luciferase reporter plasmid as described earlier (13,16). To generate mutations in key transcription factor binding sites, site-directed mutagenesis was performed by PCR using mutagenic primers. All the sequences were confirmed by the Biotechnology Research Institute, University of Ottawa. THP-1 cells were transiently transfected by FuGENE6 reagent (Roche Diagnostics) as described earlier (13,16). Briefly, THP-1 cells infected with pSR␣-Nef retroviruses were transfected with 1 g of IL-12p40 promoter/luciferase constructs and 0.5 g of the pSV-␤-galactosidase (Promega, Madison, WI) by employing FuGENE6 reagent as described earlier (13,16). After 24 h, cells were stimulated with LPS for another 24 h following which cells were assayed for luciferase and ␤-galactosidase activity by using respective assay kits (Promega). IL-12p40 promoter luciferase activity was normalized by measuring ␤-galactosidase values.
Electrophoretic Mobility Shift Assay-Electrophoretic mobility shift assays were performed as described earlier (13,16). Briefly, cells were infected twice with pSR␣-Nef for 24 h each and stimulated with LPS for various times. The nuclear proteins (5 g) were mixed with 32 P-labeled NFB or AP-1 oligonucleotide probes for 20 min, and the resulting complexes were separated on a 5% nondenaturing gel. The oligonucleotide probe sequences corresponding to the NFB and AP-1 binding sites in the IL-12p40 promoter have been described (13). To determine the specificity of NFB/AP-1 binding, parallel electrophoretic mobility shift assay reactions were performed by using a 200-fold excess of unlabeled specific and nonspecific oligonucleotides. Supershift experiments were performed by using mouse anti-NFB p50 and p65, anti-c-Fos, and anti-c-Jun antibodies (Santa Cruz Biotechnology).
Statistical Analysis-Means were compared by two-tailed Student's paired t test. The results are expressed as mean Ϯ S.D.

Intracellular nef Inhibits LPS-induced IL-12p40 Expression in
Human Monocytic Cells-To determine the effect of intracellular HIV-nef on IL-12p40 expression in monocytic cells, HIVnef retroviruses were generated to infect monocytic cells because of their ability to transduce gene with high efficiency. HIV-nef retroviruses were generated by transfecting amphotrophic packaging cell line PT67 with either pSR␣-Nef retrovirus (pSR␣-Nef) or the retrovirus containing the empty vector pSR␣. The integration of pSR␣-Nef in PT67 cells was verified by subjecting the genomic DNA to PCR and intracellular expression of nef protein was confirmed by Western blotting (Fig. 1, A  and B). The supernatant from a 3-day culture of PT67 cells stably transfected with pSR␣-Nef was used as a source of nefcontaining retroviruses. The supernatants were assayed for determination of viral titers by infecting NIH 3T3 cells. Monocytes and THP-1cells were infected with either pSR␣-Nef or pSR␣ at a multiplicity of infection of 1 for 24 h. To obtain maximal viral infectivity, cells were infected again for another 24 h. THP-1 cells and monocytes infected with pSR␣-Nef expressed nef as determined by PCR and Western blotting (Fig. 1, A and  B). Moreover, monocytes and THP-1 cells infected with pSR␣-Nef did not induce morphological changes, and this level of infectivity was optimal for influencing IL-12p40 expression (data not shown).
To determine whether intracellular nef affects LPS-induced IL-12p40 expression, THP-1 cells and monocytes were infected with nef or pSR␣ retroviruses at a multiplicity of infection of 1 followed by stimulation with LPS for another 24 h. LPS-induced IL-12p40 production was significantly reduced by ϳ80% in pSR␣-Nef-infected monocytes and THP-1 cells compared with the pSR␣-infected cells (p Ͻ 0.001, Fig. 1C). To determine if intracellular nef inhibits IL-12p40 transcription, total RNA prepared from LPS-stimulated and pSR␣-Nefor pSR␣-infected monocytes and THP-1 cells was analyzed for IL-12p40 mRNA by semi-quantitative RT-PCR. LPS-induced IL-12p40 mRNA expression was significantly inhibited in pSR␣-Nef-infected THP-1 cells and monocytes compared with the pSR␣-infected cells (Fig. 1D). The specificity of the inhibitory effect of nef on IL-12p40 production was confirmed by showing that intracellular nef did not inhibit LPSinduced IL-10 production. THP-1 cells infected with nef retroviruses produced 114 Ϯ 102 pg/ml IL-10 as compared with the 142 Ϯ 110 pg/ml in cells infected with control viruses following LPS stimulation. These results confirmed our prior observations that recombinant nef did not affect LPS-induced IL-10 production (38). Equal infectivity of primary monocytes and THP-1 cells with nef retroviruses was shown by performing RT-PCR analysis on the same samples for the neomycin gene present in both nef and pSR␣ retroviruses (Fig. 1D, lower panel).
We also determined that transduction of THP-1 cells with another gene such as gag by retroviruses did not affect IL-12p40 production. Infection of THP-1 cells with gag retroviruses resulted in the expression of gag p55, p41, and p24 proteins as determined by Western blot analysis by using anti-gag antibodies received from the NIH reagent and reference laboratory (data not shown). Furthermore, infection of THP-1 cells with gag retroviruses did not affect the production of IL-12p40 in response to stimulation with LPS (2817 Ϯ 130 pg/ml in gaginfected cells versus 2967 Ϯ 120 pg/ml of IL-12p40 in control virus infected cells). Inhibition of LPS-induced IL-12p40 expression by HIV-nef in monocytic cells was confirmed by stable transfection of THP-1 cells with a nef plasmid from another HIV strain (pcDNA-Nef). The expression of nef in THP-1 cells stably transfected with pcDNA-Nef was confirmed by PCR as well as immunoblotting (Fig. 1, A and B, right panels). To determine the effect of intracellular nef on IL-12p40 production, THP-1 cells stably transfected with pcDNA-Nef were stimulated with LPS followed by determination of IL-12p40 mRNA and protein expression. Similar to the results obtained with pSR␣-Nef-infected cells, LPS-induced IL-12p40 expression was significantly inhibited in pcDNA-Nef-transfected THP-1 cells compared with the cells transfected with the control plasmid (p Ͻ 0.001) as determined by ELISA and semi-quantitative RT-PCR analysis (Fig. 1, C and D, right panels).

FIGURE 1. Intracellular nef inhibits LPS-induced IL-12p40 expression in human monocytic cells.
A, Nef is expressed in pSR␣-Nef-transfected PT67 cells, and pSR␣-Nef-infected monocytes and THP-1 cells. Left panel: genomic DNA obtained from PT67 cells 7 days after transduction and from monocytes, and THP-1 cells 2 days after infection with either pSR␣ or pSR␣-Nef retroviruses were subjected to PCR for HIV-nef using primers to amplify a 237-bp fragment. Right panel: genomic DNA obtained from THP-1 cells stably transfected with pcDNA-Nef plasmid was subjected to PCR for HIV-nef using primers to amplify a 639-bp fragment. B, left panel: total proteins obtained from PT67 cells 7 days after transduction and from monocytes, and THP-1 cells 2 days after infection with either pSR␣ or pSR␣-Nef retroviruses were analyzed by Western blotting using monoclonal anti-nef antibodies. Right panel: total proteins obtained from THP-1 cells stably transfected with pcDNA-Nef plasmid were analyzed by Western blotting using monoclonal anti-nef antibodies. C, Nef inhibits LPS-induced IL-12p40 production in monocytes and THP-1 cells. 2 ϫ 10 6 monocytes (left panel) and THP-1 cells (middle panel) were infected with either pSR␣-Nef or control virus at a multiplicity of infection of 1 at 37°C for 24 h and infected again for a second time for 24 h following which cells were stimulated with LPS (1 g/ml) for another 24 h. THP-1 cells stably transfected with pcDNA-Nef or a control plasmid were also stimulated with LPS for 24 h. The culture supernatants were assayed for IL-12p40 production by ELISA. Right panel: THP-1 cells (1 ϫ 10 6 /ml) were infected with equal amounts of nef-deleted and wild-type HIV (100 pg/ml p24) containing ⌬nef and wild-type HIV-1 NL4 -3 clones, respectively. After 2 days, cells were stimulated with LPS (1 g/ml) for another 24 h followed by measurement of IL-12p40 production by ELISA. The results shown are mean Ϯ S.D. of three independent experiments. D, monocytes (left panel) and THP-1 cells (middle panel) infected with either pSR␣ or pSR␣-Nef retroviruses described as above were stimulated with LPS (1 g/ml) for another 4 h. THP-1 cells stably transfected with pcDNA-Nef or a control plasmid were also stimulated with LPS for 4 h (right panel). Total RNA was isolated and subjected to RT-PCR for IL-12p40 and ␤-actin as a control. Equal levels of infectivity were determined by performing RT-PCR analysis for the neomycin gene present in pSR␣ and pSR␣-Nef retroviruses. The results shown in A, B, and D are representative of three independent experiments.
The inhibitory effect of nef on IL-12p40 expression was also confirmed by infecting THP-1 cells with equal amounts (100 pg/ml p24) of nef-deleted (⌬nef-HIV-1 pNL4 -3 ) and wild-type (HIV-1 pNL4 -3 ) HIV molecular clones. Two days after infection, cells were stimulated with LPS for another 24 h followed by measurement of IL-12p40 production by ELISA. Infection with wild type HIV significantly inhibited IL-12p40 production compared with the cells infected with ⌬nef-HIV-1 pNL4 -3 viruses (Fig. 1C, right panel).
Intracellular nef Down-regulates LPS-induced IL-12p40 Production by Selectively Inhibiting JNK MAPK-We have previously shown that LPS-induced IL-12p40 production in human monocytic cells is regulated by two distinct signaling pathways namely the JNK MAPK and the CaMK-II pathways (13,16). HIV-1-nef is also known to interfere with a number of signaling molecules, including MAPKs (30 -32). Hence, it is possible that nef inhibits LPS-induced IL-12p40 expression through the inhibition of either JNK or CaMK-II alone or JNK and CaMK-II together. Therefore, to ensure that the activities of JNK and Ca 2ϩ /CaM/CaMK-II are not influenced by retroviruses, we first determined that LPS-induced IL-12p40 expression is regulated by JNK as well as CaMK-II in control pSR␣-infected THP-1 cells and monocytes by employing specific pharmacological inhibitors. Prior to LPS stimulation, cells were treated for 2 h with various concentrations of either JNK inhibitor SP600125 (39), calcium chelating agent EGTA, SKF96365, an inhibitor of receptor-mediated calcium entry (40), calmodulin antagonist W-7, or CaMK-II inhibitor KN-93 (41) followed by analysis for IL-12p40 production by ELISA. As expected, JNK ( Fig. 2A) and all calcium signaling inhibitors (Fig. 2B)  To determine if HIV-nef affects LPS-activated JNK and/or calcium signaling, monocytes and THP-1 cells were infected with either pSR␣-Nef or pSR␣ retroviruses followed by LPS stimulation for 0 -120 min. The pSR␣-infected THP-1 cells and monocytes exhibited 3-to 5-fold and 6-to 8-fold increases in JNK phosphorylation, respectively, within 30 min post-LPS stimulation. In contrast, LPS stimulation of pSR␣-Nef-infected THP-1 cells and monocytes failed to significantly induce JNK phosphorylation at any time post-stimulation (Fig. 3A). The ERK and p38 MAPKs have also been shown to regulate IL-12p40 production in some cell types (42,43). Therefore, we determined if nef interfered with p38 and ERK activation following LPS stimulation. However, following stripping of the same blots, LPS induced similar levels of ERK (Fig. 3B) and p38

MAPKs phosphorylation (data not shown) in both pSR␣-and pSR␣-Nef-infected THP-1 cells and monocytes.
Stress-activated protein kinase/extra-cellular signal-regulated kinase 1 (SEK1) is a dual kinase immediate upstream of JNK, which phosphorylates JNK and p38 on threonine 183 and tyrosine 185 residues (44). To further understand the role of intracellular nef, we determined if infection with pSR␣-nef retroviruses down-regulated SEK1 activity. LPS stimulation did not induce SEK1 activation in pSR␣-nef-infected monocytes and THP-l cells in contrast to the 6-to 8-fold increased phosphorylation observed in pSR␣-infected cells (Fig. 4). These results suggest that intracellular nef down-regulated LPS-induced IL-12p40 production by inhibiting the JNK pathway in primary monocytes and THP-1 cells.

Intracellular nef-mediated Inhibition of LPS-induced IL-12p40 Expression Does Not Involve Calcium Signaling and CaMK-II
Activity-Subsequently we investigated if intracellular nef inhibits LPS-induced IL-12p40 production by inhibiting the calcium influx, and/or CaM/CaMK-II activity (16). Since the function of CaMK-II is calcium-dependent, we first determined the effect of nef retrovirus infection on calcium influx by flow cytometry using Fluo-3 as a calcium binding dye. LPS induced an increase in the levels of intracellular calcium at 12 min poststimulation in primary monocytes and THP-1 cells infected with pSR␣-nef retroviruses that was similar to that observed in pSR␣-infected cells (Fig. 5A). Furthermore, EGTA inhibited LPS-induced calcium influx to the basal levels in uninfected, pSR␣-and pSR␣-Nef-infected THP-1 cells (Fig. 5A) and monocytes (data not shown). Similar levels of LPS-induced CaMK-II phosphorylation were observed in both monocytes and THP-1 cells infected with either nef or control retroviruses (Fig. 5B). Because intracellular nef did not affect LPS-induced calcium influx or CaMK-II activity, the results suggest that nef inhibited IL-12p40 expression by down-regulating the LPS-activated JNK pathway independent of the calcium/CaMK-II pathway in human monocytic cells. Fig. 6A) in human monocytic cells (13,16), we determined if intracellular nef inhibits LPS-induced IL-12p40 transcription by inhibiting the promoter activity of NFB, AP-1 or both transcription factors. To confirm our earlier observations and to ensure that infection with control retroviruses did not impair IL-12p40 promoter activity, THP-1 cells infected with the pSR␣ retroviruses were transfected with a series of IL-12p40 promoter 5Ј-deletion mutants linked with the luciferase reporter plasmid, pGL3B, for 24 h followed by stimulation with LPS. Subsequent analysis of luciferase activity revealed that transfection with the full-length Ϫ880 to ϩ118 promoter construct induced significant luciferase activity compared with the cells transfected with the control vector (Fig. 6B). In contrast, transfection with the vectors containing mutant NFB in the presence of the PU.1 binding sequence (Ϫ128, pIL-12p40.PU.1, and NFBm), abrogated luciferase activity (Fig.  6B). Transfection with vectors containing wild-type AP-1 and mutant NFB sequences (Ϫ232, pIL-12p40.AP-1, Ets-2, PU.1, and NFBm) still exhibited an increased luciferase activity after LPS stimulation. However, when both AP-1 and NFB sites were mutated (Ϫ232, pIL-12p40.AP-1m, Ets-2, PU.1, and NFBm), LPS-induced luciferase activity was significantly decreased (Fig. 6B). These results suggest that both AP-1 and NFB can drive LPS-induced IL-12p40 transcription in THP-1 cells infected with the control retroviruses.

Intracellular nef Inhibits IL-12p40 Promoter Activity in LPSstimulated THP-1 Cells-Because LPS-induced IL-12p40 is regulated by NFB and AP-1 transcription factors (sequence shown in
To investigate if intracellular nef inhibited LPS-induced IL-12p40 transcription by inhibiting NFB and/or AP-1 activity, THP-1 cells infected with pSR␣-Nef were transiently transfected with either the full-length IL-12p40 promoter construct (Ϫ880/ϩ118 bp), NFB containing plasmid (Ϫ116, pIL-12p40.NFB) or the vectors containing wild-type AP-1 and mutant NFB sites (Ϫ232, pIL-12p40.AP-1, Ets-2, PU.1, and NFBm). LPS-induced luciferase activity was significantly reduced in nef-infected cells transfected with full-length IL-12p40 promoter construct and NFB containing Ϫ116, pIL-12p40.NFB plasmids compared with the cells infected with control virus (Fig. 6C). LPS stimulation induced a significant ϳ3-fold increase in luciferase activity in pSR␣-infected cells transfected with a plasmid containing wild-type AP-1 and mutant NFB sites (Ϫ232, pIL-12p40.AP-1, Ets-2, PU.1, and NFBm), although this increase in luciferase activity was low compared with the cells transfected with plasmids containing wild-type NFB sites. On the other hand, LPS-induced luciferase activity was not significantly reduced in pSR␣-Nef-infected cells transfected with the vectors containing wild-type AP-1 and mutant NFB sites compared with the cells infected with the control pSR␣ viruses (Fig. 6C). Similar results were obtained by cotransfecting THP-1 cells with the pcDNA-Nefand the NFB-containing IL-12p40 promoter constructs (data not shown). These results suggest that intracellular nef inhibited LPS-induced IL-12p40 promoter activity primarily by inhibiting NFB activity.
Nef Inhibits IL-12p40 Transcription through the Selective Suppression of NFB in LPS-stimulated THP-1 Cells-The involvement of NFB in nef-mediated down-regulation of IL-12p40 transcription was further elucidated by determining the binding of NFB and AP-1 transcription factors to their respective binding sites in IL-12p40 promoter by the gel shift assays. THP-1 cells infected with either control or pSR␣-Nef retroviruses were stimulated with LPS for 0 -4 h followed by analysis of NFB and AP-1 binding to their binding sites using the ␥-32 P-labeled oligonucleotide probes containing NFB or AP-1 sequences corresponding to the IL-12p40 promoter. LPS stimulation of uninfected THP-1 cells or THP-1 cells infected with pSR␣ retrovirus significantly enhanced the binding of NFB to its binding site at 30 -45 min post-LPS stimulation. However, in contrast to the cells infected with the control retroviruses, binding of NFB to its binding site was not induced in cells infected with pSR␣-Nef retroviruses at any time following LPS stimulation (Fig. 7A, upper and lower panels). The specificity of NFB binding was demonstrated by competition with specific and nonspecific oligonucleotides and by supershift analysis with anti-NFB p50 and p65 antibodies (Fig. 7A, lower  panel).
To understand the effect of pSR␣-Nef on AP-1 induction, LPS stimulation of uninfected THP-1 cells or THP-1 cells infected with pSR␣ retroviruses significantly enhanced the binding of AP-1 to its binding site at 30 -45 min post-LPS stimulation. However, in contrast to NFB, THP-1 cells infected with the nef retroviruses exhibited significant binding of AP-1 to its binding site at a level similar to that observed in cells infected with the control retroviruses following LPS stimulation (Fig. 7B). The specificity of AP-1 binding was demon- the control retroviruses. 2 ϫ 10 6 THP-1 cells were infected twice with pSR␣ retroviruses for 24 h followed by transient transfection with 1 g of various IL-12p40 promoter/ luciferase constructs containing either wild-type, mutant NFB or AP-1 binding sequences, or control reporter vector plus 0.5 g of ␤-galactosidase, an internal control, for 24 h. Cells were harvested following LPS (1 g/ml) stimulation for another 24 h and lysed for analysis of luciferase activity. C, loss of IL-12p40 promoter activity by pSR␣-Nef through NFB binding motifs. THP-1 cells (2 ϫ 10 6 ) were infected with pSR␣-Nef twice for 24 h each following which cells were transiently transfected with 1 g of IL-12p40 promoter/ luciferase constructs containing either AP-1 or NFB binding motif plus 0.5 g of ␤-galactosidase for 24 h. Cells were harvested following LPS (1 g/ml) stimulation for another 24 h and lysed for analysis of luciferase activity. Luciferase activities were normalized with the baseline activity of control vector and the ␤-galactosidase activity. Results shown in B and C are represented as a mean Ϯ S.D. of three independent experiments. strated by competition with specific and nonspecific oligonucleotides (Fig. 7B). These results suggest that intracellular nef selectively inhibits the binding of NFB without affecting the binding of AP-1.

DISCUSSION
Deficiency in IL-12 production by monocytes/macrophages after infection has been identified as a potential factor responsible for impaired innate and cellular immune responses observed in AIDS patients (1,3,4). However, the mechanism by which HIV-1 infection inhibits IL-12 production remains unknown. Herein, we show for the first time that intracellularly expressed nef in primary monocytes and THP-1 cells resulted in the inhibition of LPS-induced IL-12p40 expression. Moreover, intracellular nef inhibits IL-12p40 expression through the selective inhibition of JNK-activated NFB pathway in these cells.
HIV regulatory proteins tat, nef, and vpr are known to modulate production of cytokines, including IL-12 (25,(45)(46)(47). Variable effects of tat and nef on IL-12 expression in B cells and monocytes have been observed possibly due to different cell types and the models used to deliver these genes/gene products into the cells. For example, tat was shown to suppress IL-12 production in human PBMCs and enhance its production in dendritic cells (45,48). We have previously shown that tat did not affect IL-12p40 production in either unstimulated or LPSstimulated monocytic cells (Ref. 49 and data not shown). On the other hand, HIV vpr protein was shown to inhibit IL-12 production by down-regulating IL-12p35 subunit without affecting the synthesis of IL-12p40 subunit (47). Similarly, there are reports that nef protein did not affect IL-12 expression in human PBMCs and U937 cells (45,50). However, introduction of nef through adenoviruses and recombinant nef enhanced IL-12 production in immature dendritic cells (46). Conversely, there is evidence for the inhibitory role for nef in IL-12p40 production in monocytic cells. IL-12p40 production was suppressed in lymph nodes of macaques infected with simian immunodeficiency virus compared with those infected with the corresponding non-pathogenic nef-deleted strain (25).
HIV-nef has been shown to alter cytokine expression (51) by modulating the activation of host cell signaling molecules (29). For example, intracellular nef down-regulated the expression of Th1 cytokines, including interferon-␥ (51). Recombinant myristoylated HIV-nef enhanced the release of interferon-␤ in monocytes/macrophages through the activation of MAPKs, IB kinase and IRF-3 (52). HIV-nef was also shown to up-regulate CCL2/MCP-1 expression through calmodulin activation (53). In contrast, HIV-nef was shown to inhibit macrophagecolony stimulating factor receptor signaling through Hck activation in monocytes/macrophages (54). Our results suggest that intracellular nef interferes with the LPS-activated JNK pathway to inhibit IL-12p40 production. LPS mediates its effects through the CD14/TLR4 receptor complex involving the activation of tyrosine and serine/threonine kinases, including protein kinase C, MAPKs, phosphatidylinositol 3-kinase, and the calcium pathway (55). We have previously shown a critical role for JNK and the upstream SEK1 in IL-12p40 regulation by LPS-activated monocytic cells (13). Because nef-expressing cells exhibited specifically down-regulation of the JNK pathway without any effect on p38 and ERK MAPKs following LPS stimulation, it is reasonable to conclude that CD14 and the LPS-activated downstream signaling molecules, including Myd88 and the interleukin-1 receptor-associated kinase remained unaffected following nef expression.
JNK plays a critical role in Th1/Th2 cell differentiation, cytokines production, and cell survival (56 -58). It contains three members, JNK1, -2, and -3. JNK3 is selectively expressed in FIGURE 7. Nef inhibits LPS-induced NFB binding to IL-12p40 promoter in THP-1 cells. A, upper panel: inhibition of NFB binding activity by pSR␣-Nef. THP-1 cells (5 ϫ 10 6 ) were infected with either pSR␣-Nef or control virus twice followed by stimulation with LPS for 0 -120 min. Nuclear extracts (5 g) were incubated for 1 h with 32 P-labeled oligonucleotide probes corresponding to the NFB binding sequences derived from the IL-12p40 promoter. The binding activity of NF-B is shown in parental uninfected THP-1 cells, control virus-infected, and pSR␣-Nef-infected THP-1 cells following LPS stimulation. The lower panel shows the specificity of NF-B binding in pSR␣-infected cells. The nuclear proteins (5 g) obtained from LPS-stimulated cells (30 min) were incubated with 32 P-labeled oligonucleotide probes corresponding to the NFB sequences located in the IL-12p40 promoter. The specificity of NFB binding was determined by incubating nuclear proteins with 100-to 200-fold excess of unlabeled specific (CC) and nonspecific (NS) oligonucleotides for 30 min before addition of labeled probes. The supershift analysis was performed by treating the nuclear proteins with oligonucleotide probes in the presence or the absence of anti-p50 or anti-p65 NFB antibodies. The supershifted bands are indicated. The inhibitory effect of nef on NFB binding activity is shown in the last two lanes. B, the AP-1 binding activity is not influenced by pSR␣-Nef. The nuclear extracts obtained as above were incubated with 32 Plabeled oligonucleotides probes corresponding to the AP-1 binding sequences derived from the IL-12p40 promoter. The binding activity for AP-1 in parental THP-1 cells, control pSR␣, and pSR␣-Nef-infected THP-1 cells is shown in the left panel. The right panel shows specificity of AP-1 binding by incubating nuclear extracts with specific (CC) and nonspecific (NS) unlabeled oligonucleotide probes as described above. The experiments shown in A and B are representatives of three different experiments.
neuronal and cardiac tissues and associated with neuron cell apoptosis, whereas JNK1/2 are highly inducible in monocytic and T cells (56,57,59). It is not clear whether JNK1 or JNK2 regulate LPS-induced IL-12p40 expression. Our results suggest that intracellular nef inhibited LPS-activated both JNK1 and JNK2 isoforms in monocytes and THP-1 cells. JNK is activated by SEK1, a dual kinase upstream of JNK MAPKs (44). The results of this study suggest that inhibition of LPS-activated SEK1 may be responsible for the inhibition of JNK activation and subsequent IL-12p40 transcription in both monocytes and THP-1 cells.
JNK is a serine/threonine kinase that activates various transcription factors, including AP1 and NFB (56). Our results revealed that intracellular nef selectively inhibited NFB without affecting AP-1 activation. It is not clear if nef inhibited NFB by inhibiting NFB-JNK interactions as JNK has been shown to influence NFB pathway by regulating IB␣ activation (60). Because nef has been shown to modulate several signaling molecules, including IB kinase (33,34,52), it is possible that nef may inhibit binding of LPS-activated NFB to the IL-12p40 promoter by directly affecting the components of the NFB pathway. However, it remains to be determined if nef inhibits NFB independent of its interactions with the upstream JNK/SEK1 kinases. JNK can also activate AP-1 mainly via phosphorylation of the c-Jun component (56). However, AP-1 can also be activated independent of the JNK MAPKs (61,62). Because nef did not inhibit binding of AP-1 to the IL-12p40 promoter, it is likely that infection of monocytic cells with retroviruses in the present study activated AP-1 independent of the JNK pathway.
We have previously shown that LPS-induced IL-12p40 production is regulated by two distinct and independent signaling pathways namely the CaM/CaMK-II-and the JNK-activated pathways. Interestingly, both pathways regulate IL-12p40 transcription through the NFB and AP-1 transcription factors (13,16). At present, the precise role of NFB and AP-1 in IL-12p40 transcription is not clear; however, a potential cooperation between AP-1 and NFB may be required for LPS-induced IL-12p40 transcription in monocytic cells. This hypothesis is supported by studies showing interactions of c-Jun-containing complexes with NFB proteins p50/p65 (60). The observations that nef inhibited IL-12p40 transcription in the presence of functional AP-1 suggests that AP-1 in the presence of dysfunctional NFB may not be able to drive LPS-induced IL-12p40 transcription further suggesting a potential cooperation between the two factors. Besides signal transduction pathways, molecular mechanisms controlling gene expression involve chromatin remodeling and DNA methylation (63). In addition to the inhibition of JNK and NFB, intracellular nef may inhibit IL-12p40 transcription by altering the status of promoter methylation, and chromatin remodeling. Further studies are necessary to elucidate the mechanisms underlying nef-mediated inhibition of IL-12p40 transcription.
Nef is believed to promote viral pathogenicity by altering signaling pathways in infected cells through interactions with several signaling proteins, including Src tyrosine kinases (Hck, Lck, Lyn, Fyn, and c-Src) (33,34), serine/threonine kinases (p21-activated protein kinase) (64), isoform of protein kinase C (65), Vav (66), and MAPKs (30,32). These interactions are mediated by a highly conserved proline repeat motif (PXXP) present in the nef proteins, which interact directly with the Src homology domain 3 region (67). The precise mechanism by which intracellular nef inhibits JNK/SEK1 phosphorylation and NFB activation leading to impaired LPS-induced IL-12p40 transcription is not clear. Nef may impair JNK phosphorylation by direct interactions with upstream signaling molecules through the PXXP motif (64 -67).
NFB plays a key role in the development of innate and adaptive immunity and is involved in the pathogenesis of a number of diseases, including cancer, infectious diseases, and inflammatory disorders (68). The biological significance of JNK-mediated activation of NFB signaling has been documented in cytokine production (IL-6, tumor necrosis factor-␣, and MIP-1␣) and susceptibility to bacterial and viral infection in animal models (60,69). In addition, NFB is a molecular basis for the aberrant growth and cytokine gene expression observed in AIDS (70). Therefore, the inhibition of JNK-mediated NFB activation by intracellular nef may not only downregulate IL-12 production but may also be implicated broadly in the down-regulation of Th1 cytokines, Th1 responses, and development of immunodeficiency.