HIV infects the key players of the immune system including the CD4+ T cells and macrophages. The viral gene sequences are integrated into the host genome for subsequent expression of viral proteins for replication, assembly and release of the virus . To enhance its own survival, HIV gradually depletes immune responsiveness of the host leading to an immunodeficient state . Consequently, coinfections with other non-HIV pathogens including viruses, parasites, fungi and bacteria are common in AIDS patients. These opportunistic infections influence the course of disease progression, decrease survival of the patients and increase the risk of HIV transmission . For example, these pathogens may have beneficial effects on HIV dissemination by enhancing retrovirus replication via Toll-like receptor (TLR)-dependent or pathogen-induced cytokine signaling pathways, or both .
During Gram-negative bacteria infection, lipopolysaccharide (LPS), a bacterial cell wall component, is recognized by Toll-like receptor 4 (TLR4) to induce signaling cascades for triggering innate immune responses. TLR4 recruits a series of adaptor proteins including MyD88, IRAK4 and IRAK1 and subsequently transduces the signal to TRAF6-associated signaling complex for activating the I kappa B kinase (IKK) complex . The IKK complex phosphorylates IκBα leading to the degradation of IκBα. NFκB p65, which is constitutively inhibited by its binding to IκBα, is subsequently released and translocated from the cytoplasm into the nucleus to induce the expression of genes including TNF-α, IL-6 and IFN-β [4–7]. Apart from the NFκB signaling pathway, TLR4 activates the mitogen-activated protein kinases (MAPKs) including ERK1/2 and p38 MAPK for the induction of cytokines including IL-6 and TNF-α [4,6,8,9]. These cytokines induced by LPS play different and specific roles in combating the invading Gram-negative bacteria. For example, IL-6 stimulates the B-cell proliferation leading to the production of antibacterial antibodies . IFN-β, in addition to its well known antiviral activity, induces inducible nitric oxide synthase (iNOS) expression to enhance NO synthesis for the clearance of the Gram-negative bacteria in mouse models . Furthermore, IFN-β synergizes with TNF-α and IL-1β for inducing the indoleamine-2,3-dioxygenase expression to inhibit bacterial growth .
Tat, the transactivating protein of HIV, is expressed within the virus-infected cells and released extracellularly . In the infected cells, Tat transactivates the long terminal repeat (LTR) region of the integrated HIV genome to enhance the retrovirus replication . Additionally, Tat is released extracellularly from the HIV-1 infected cells  and circulates in the sera of HIV-infected patients with concentrations ranging from 0.2 to 4 nmol/l . However, the local concentration of Tat has been suggested to be higher than these figures . Tat, whether produced intracellularly or taken up from an extracellular source, dysregulates cytokine responses of the targeted cells. For example, it induces cytokines such as IL-10, IL-6 and TNF-α in monocytes/macrophages [1,15–21]. These cytokines play different roles in the progression of AIDS including activation of HIV-1 replication [22–27], induction of cytotoxicity of T cells , promotion of the development of HIV-associated B-cell lymphomas  and suppression of Th1 cytokine induction resulting in impaired T-cell signaling [1,20,22,23]. However, the role of extracellular Tat in modulating the cytokine and interferon responses during infection by opportunistic pathogens in HIV-infected patients remains to be investigated. Here, by using the endotoxin-induced signaling pathway as an in-vitro model, we investigated the effects of Tat on the immune responses triggered by Gram-negative bacteria.
Materials and methods
Recombinant HIV-1 Tat protein and lipopolysaccharide
The recombinant HIV-1 Tat protein (amino acid 1–86) was purchased from Advanced BioScience Laboratories, Kensington, Maryland, United States and was reconstituted in PBS with 1 mg/ml BSA. Its endotoxin level was less than 0.0025 EU/mg as determined by Pyrochrome Chromogenic Kit (Associates of Cape Cod, Falmouth, Massachusetts, USA), and its biological activity was validated as described . LPS extracted from Escherichia coli serotype O26:B6 was purchased from Sigma-Aldrich, St Louis, Missouri, United States.
Isolation and culture of primary human CD14+ blood monocytes and macrophages
Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coat of healthy blood donors from the Hong Kong Red Cross Blood Transfusion Service by Ficoll-Paque (GE Healthcare Medical Systems, Piscataway, New Jersey, USA) density gradient centrifugation [19,29]. Human primary blood monocytes (PBMos) were then separated from the PBMCs by magnetic cell sorting (MACS) using anti-CD14 antibody-coated MicroBeads following the manufacturer's instruction (Miltenyi Biotec, Bergisch Gladbach, Germany). Isolated PBMos, shown to be more than 90% CD14 positive as assayed by flow cytometry (data not shown), were cultured in Roswell Park Memorial Institute (RPMI) medium 1640 (Invitrogen, Carlsbad, California, USA) with 5% heat-inactivated autologous plasma (RPMI-AP). Human primary blood macrophages (PBMacs) were obtained by the adherence method  and cultured for 14 days in RPMI-AP.
RNA extraction and reverse transcription-polymerase chain reaction
Total RNA, extracted from PBMos/PBMacs (0.5 × 106 cells) by TRIzol Reagent (Invitrogen), was treated with DNase I (Roche Applied Science, Basel, Switzerland) and reverse transcribed into cDNA by using SuperScript II system (Invitrogen). Expression level of IκBα, TLR4 and glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was assayed by PCR as in our previous report . The primer sets for IκBα, TLR4 and GAPDH were: forward, 5′-AGAAGGAGCGGCTACTGGAC-3′, reverse, 5′-TCATGGATGATGGCCAAGT-3′; forward, 5′-TGGATACGTTTCCTTATAAG-3′, reverse, 5′-GAAATGGAGGCACCCCTTC; and forward, 5′-ACCACAGTCCATGCCATCAC-3′, reverse, 5′-TCCACCACCCTGTTGCTGTA-3′, respectively.
Quantitative reverse transcription-PCR
DNase I (Roche Applied Science)-treated total RNA was reverse transcribed into cDNA by using the TaqMan reverse transcription reagent kit (Applied Biosystems, Foster City, California, USA). Quantitative PCR was performed in triplicates for each RNA sample by using the TaqMan gene-specific assays-on-demand reagent kits (Applied Biosystems). The comparative cycle number to threshold (CT) method was used for analyzing mRNA induction levels [29,30]. The mRNA levels of the target genes were normalized by reference genes, 18S rRNA for PBMos and GAPDH for PBMacs. The fold induction of mRNA expression of the treated cells was compared with that of mock-treated cells.
Enzyme-linked immunosorbent assay
The supernatant samples of the PBMo cultures were obtained at the indicated time of treatment. The levels of the cytokine proteins were measured by the specific enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, Minnesota, USA).
The IFN-β bioassay was performed as previously described with minor modifications [31,32]. Briefly, supernatants of the PBMac cultures were collected at 6 h after LPS addition. The supernatant samples or 400 pg/ml recombinant IFN-β (PBL, Piscataway, New Jersey, USA) were mixed with control rabbit immunoglobulin fraction (Dako, Glostrup, Denmark) or rabbit anti-IFN-β-neutralizing antibodies (Abcam, Cambridge, UK) for 30 min of incubation and added into T98G cells (0.1 × 106 cells). After 24-h incubation, the supernatant samples were removed. The adherent T98G cells were washed with PBS and were infected by encephalomyocarditis virus (EMCV) (108 titer/ml) suspended in 2% fetal bovine serum medium. After 24-h infection, the virus-induced cytopathic effects were visualized by light microscopy after staining the cells with 0.1% crystal violet dissolved in 5% ethanol.
PBMos (1 × 106 cells) were stained with phycoerythrin-conjugated anti-TLR4 antibody (eBioscience, San Diego, California, USA) or phycoerythrin-conjugated IgG (eBioscience) and fixed in 1% paraformaldehyde (PFA). They were analyzed by a flow cytometer (Elite; Beckman Coulter, Fullerton, California, USA). Positive events refer to the fluorescent levels when compared to the cells treated with an IgG control.
As in our previous reports [29,30], cytoplasmic and nuclear proteins were extracted from PBMos/PBMacs (2 × 106 cells) and analyzed by western blots using specific primary antibodies from Cell Signaling Technology (Danvers, Massachusetts, USA) for recognizing ERK1/2, phospho-ERK1/2, p38 MAPK or phospho-p38 MAPK from Santa Cruz Biotechnology (Santa Cruz, California, USA) for recognizing IκBα or actin from Millipore (Billerica, Massachusetts, USA) for recognizing IRAK1 and from Calbiochem (San Diego, California, USA) for recognizing NFκB p65 and subsequently using the respective secondary antibodies conjugated with horseradish peroxidase (BD Bioscience, San Jose, California, USA).
Immunocytochemistry for assaying nuclear factor-κB localization
PBMacs were fixed with 4% PFA and permeabilized with 0.25% Triton-X 100. Cells were stained with anti-NFκB p65 primary antibodies (Santa Cruz Biotechnology) followed by staining with rhodamine-conjugated secondary antibodies (Millipore). Cell nuclei were stained with 4,6-diamidine-2-phenylindole dihydrochloride (DAPI). Immunofluorescence of the stained cells was quantified by Cellomics ArrayScan HCS VTI Reader (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The nuclear translocation of NFκB p65 was determined by Nuclear Translocation BioApplications from Cellomics and expressed as nucleus/cytoplasm intensity ratio. Higher intensity ratio indicated a higher level of nuclear localization of NFκB p65. Representative images were captured using a 40× objective lens by immunofluorescence microscopy.
Experimental results were analyzed by two-tailed and paired Student's t-test.
To investigate the potential effects of Tat on the LPS-induced IFN-β expression, PBMos were preincubated with Tat prior to LPS treatment. Although Tat by itself did not induce IFN-β transcription, it suppressed the LPS-induced IFN-β mRNA expression in a dose-dependent manner (Fig. 1a). These suppressive effects of Tat were more effective with increasing length of Tat preincubation time from 1 to 4 h (Fig. 1b) and sustained in longer Tat pretreatment time periods of 8 and 24 h (data not shown). Similarly, the elevation of IFN-β mRNA levels by 10 and 100 ng/ml of LPS treatment decreased significantly after Tat pretreatment in PBMacs (Fig. 1c). The antiviral activity elicited by LPS-induced IFN-β was determined by IFN-β bioassay. IFN-β was released in the supernatant from the LPS-treated cells as demonstrated by the antiviral activity induced (Fig. 1d, panel [v] versus panel [i]) and its neutralization by anti-IFN-β antibodies (Fig. 1d, panels [v] and [vi]). However, the antiviral activity of the supernatant from the PBMacs pretreated with Tat before LPS treatment (Fig. 1d, panel [vii]) was decreased in comparison to the supernatant from cells with LPS treatment alone (Fig. 1d, panel [v]). The effectiveness of Tat (Fig. 1d, panel [vii]) and the anti-IFN-β antibodies (Fig. 1d, panel [vi]) in suppressing the LPS-induced antiviral activities was comparable. These suggest that Tat inhibits the LPS-induced production of IFN-β specifically. As controls, the anti-IFN-β antibodies were demonstrated to completely neutralize the antiviral activity of recombinant IFN-β (Fig. 1d, panels [ix] and [x]). As additional controls, direct addition of Tat or LPS to the T98G cells was shown to have no protection from EMCV-induced cytotoxicity, indicating that Tat or LPS cannot induce any antiviral proteins in T98G cells (Fig. 1d, panels [xi] and [xii]). Taken together, the results demonstrated that Tat inhibited the LPS-induced expression of IFN-β resulting in a significant decrease of IFN-β-induced antiviral activity.
To examine the effects of Tat on LPS-induced TNF-α and IL-6, PBMos were pretreated with different doses of Tat prior to the LPS treatment. Pretreatment of Tat led to a dramatic dose-dependent enhancement of the LPS-induction of IL-6 mRNA levels, whereas treatment with Tat or LPS alone induced lower levels of IL-6 mRNA (Fig. 2a). Besides, Tat pretreatment for 1–4 h greatly enhanced the LPS-induced IL-6 mRNA transcription in a time-dependent manner, whereas Tat or LPS alone only induced a lower level of IL-6 mRNA synthesis (Fig. 2b). These enhancing effects of Tat were also found in cells pretreated with Tat for a longer time of 8 and 24 h (data not shown). Furthermore, Tat pretreatment for 4 h significantly enhanced the LPS-induced release of IL-6 protein both at 6 and 24 h after LPS treatment as assayed by ELISA (Fig. 2c). Similarly, Tat pretreatment also significantly enhanced the LPS-induction of IL-6 mRNA in PBMacs (Fig. 2d). In contrast, Tat pretreatment had no significant effect on the LPS-upregulation of TNF-α both in PBMos and PBMacs (Fig. 2e and f). These results suggest that Tat significantly enhanced the LPS-induction of IL-6 production but not the TNF-α production in PBMos/PBMacs.
In light of Tat impairment of the LPS-stimulated response, the effect of Tat on TLR4 expression was examined. The levels of TLR4 mRNA remained unchanged with Tat pretreatment over the range of 1–20 nmol/l (Fig. 3a). Furthermore, the surface expression level of TLR4 on the cells treated with Tat for 4 h was found to be comparable to the cells treated with mock reagents (Fig. 3b). This suggests that the Tat impairment of LPS-induced cytokine responses was not due to the viral protein's effects on TLR4 mRNA and its surface protein expression.
To explore the effects of Tat on the LPS-induced activation of MAPKs, cells were pretreated with Tat for 4 h prior to LPS addition. As shown, LPS induced phosphorylation of ERK1/2 and p38 MAPK after 10 and 20 min of stimulation (Fig. 3c, lanes 6 and 7 versus lanes 1–3). Intriguingly, the level of LPS-stimulated ERK1/2 phosphorylation decreased with Tat pretreatment for 4 h (Fig. 3c, lanes 8 and 9 versus lanes 6 and 7), while ERK1/2 phosphorylation remained at the basal level with the Tat pretreatment (Fig. 3c, lanes 4 and 5). By contrast, the LPS-activated phosphorylation of p38 MAPK was not affected by the Tat pretreatment (Fig. 3c, lanes 8 and 9 versus lanes 6 and 7). The results suggest Tat selectively inhibited the LPS-stimulated activation of ERK1/2 but not the activity of p38 MAPK.
To examine the effects of Tat on other downstream pathways of TLR4, we investigated whether Tat regulates IRAK1 activation. We showed IRAK1 was degraded after LPS stimulation for 20 min (Fig. 4a, lane 10). The levels of IRAK1 degradation by LPS with or without Tat preincubation for 4 h were comparable (Fig. 4a, lane 13 versus 10). This suggests that Tat did not affect the LPS-activated IRAK1 degradation.
Next, the effects of Tat on the IκBα degradation were examined. Although LPS induced rapid IκBα degradation starting at 5 min after stimulation, Tat pretreatment suppressed this process (Fig. 4b, lanes 11–13 versus lanes 8–10). This effect may be partially explained by the results showing that Tat alone induced the IκBα protein expression (Fig. 4b, lanes 5–7). To verify this, the levels of IκBα protein expression were monitored over the course of Tat treatment. Tat initially induced IκBα degradation from 30 to 60 min after stimulation (Fig. 4c, lanes 8 and 9). The IκBα protein level started to rise with significant increased expression at 4 h of the Tat treatment (Fig. 4c, lane 11). When followed for an extended period of time over 24 h, there was a sustained induction of IκBα protein by the Tat treatment (Fig. 4d, lanes 3 and 4). These suggest though Tat initially induced IκBα degradation, it induced IκBα protein at later time points.
To elucidate whether Tat affects the NFκB p65 activation, translocation of NFκB p65 was examined by western analysis. Although Tat induced low levels of NFκB p65 nuclear translocation (Fig. 5a, lane 3), LPS induced much higher levels of this translocation with the peak level at 10 min after stimulation (Fig. 5a, lanes 4–6). However, Tat pretreatment inhibited this nuclear translocation and retained NFκB p65 in the cytosol throughout the LPS stimulation for 20 min, as demonstrated by a decrease of the NFκB p65 protein levels in the nucleus but an increase of the protein levels in the cytosol in comparison to the levels induced by LPS (Fig. 5a, lanes 7–9 versus lanes 4–6). To verify these results, NFκB p65 nuclear translocation was assayed by quantitative immunfluoresence by using Cellomics ArrayScan HCS VTI Reader. As shown in Fig. 5b, whereas mock-treated and Tat-treated cells showed higher levels of NFκB p65 localization in the cytoplasm (Fig. 5b, panel [i] to [vi]), LPS stimulated significant increases of NFκB p65 nuclear translocation (Fig. 5b, panels [vii]–[ix]). In contrast, with Tat pretreatment, this LPS-induced translocation of NFκB p65 was dramatically decreased in the nucleus but increased in the cytoplasm (Fig. 5b, panels [x]–[xii] versus panels [vii]–[ix]). Consistent with these results, as shown by immunofluorescence quantification of these images, while LPS induced NFκB p65 nuclear translocalization with an elevation of the nuclear/cytoplasmic intensity ratio, Tat suppressed this NFκB p65 nuclear translocation with a significant decrease of the ratio (Fig. 5c). These results suggest that Tat suppressed the LPS-induced NFκB p65 activation via the retention of NFκB p65 in the cytosol.
HIV dysregulation of cytokines' and chemokines' systems has been suggested to play a role in enhancing HIV replication and its associated HIV pathogenesis [20,33]. For example, elevation of TNF-α expression participates in the HIV-induced cytotoxicity . In addition, IL-6 induction can dysregulate the proliferation of B cells leading to the development of B-cell lymphomas . High levels of CCL2 in HIV-infected individuals have been suggested to contribute to the HIV-associated dementia . In addition, IL-6, TNF-α and CCL2 can enhance HIV replication [22–24,34]. In contrast, C-C chemokines (including CCL3, CCL4 and CCL5) and one of the C-X-C chemokines (CXCL12) can inhibit HIV infection by blocking the HIV coreceptors, namely CCR5 and CXCR4 . Furthermore, as IFN-β can abrogate the infection and replication of HIV , the inability of HIV-infected cells to mount IFN-β induction [36–40] allows the retrovirus to evade the cellular antiviral effects. Abnormal inductions of these cytokines including TNF-α, IL-6 and IL-10 have been demonstrated to be partly regulated by extracellular HIV-1 Tat [16,18–21]. In this report, we demonstrated the role of Tat in dysregulating the LPS-activated interferon and cytokine responses, thus providing a cellular model to investigate the interaction of HIV with Gram-negative bacteria.
In a report using mouse macrophages as a model, it was shown that Tat did not impair the LPS-induction of nitrite . As HIV is a human virus, it is pertinent to further investigate the effects of Tat on LPS-induced cytokine response to signaling pathways. Our report demonstrates that Tat suppressed the LPS-induced IFN-β expression and its associated antiviral activity in human monocytes/macrophages (Fig. 1). Our results are consistent with a previous study showing that stably expressed Tat is incapable of inducing IFN-β expression in human immature dendritic cells , thus partly accounting for the HIV inability to induce IFN-β [36–40]. Concomitantly, with the same dosage of Tat for the inhibition of IFN-β expression, Tat was able to enhance the LPS-induced IL-6 production (Fig. 2). As previous studies including ours have reported that Tat or LPS alone can induce IL-6 expression [6,19], our results further elucidated the enhancing effect of Tat on the LPS-induction of IL-6.
Our results showed that Tat had no effect on the LPS-activation of p38 MAPK but was potent in inhibiting the ERK1/2 phosphorylation (Fig. 3c) and NFκB activation by blocking the NFκB p65 translocation to the nucleus (Fig. 5). Previous report has demonstrated that upon LPS stimulation, NFκB p65 and ERK1/2 are both activated by IKK-β via the specific degradation of IκBα and TPL2, respectively . However, IKK-β does not regulate the LPS-activation of p38 MAPK . Hence, our results suggest that Tat may inhibit IKK-β leading to the specific inhibition on activation of both ERK1/2 and NFκB p65 but not p38 MAPK. Furthermore, consistent with previous reports [19,44], our results showed that extracellular Tat activates NFκB via IκBα degradation at an early time of treatment in vitro. However, we demonstrated that prolonged treatment with Tat suppresses NFκB activation in PBMos/PBMacs (Fig. 5) without interfering with the TLR4 expression and the downstream IRAK1 activation (Figs 3a, 3b and 4a). This suppression may have been the result of Tat inhibition on the IκBα degradation (Fig. 4b), thus leading to the retention of NFκB in the cytosol by IκBα (Fig. 5). This IκBα degradation inhibition resulted from the sustained elevated protein levels of IκBα caused by Tat (Fig. 4c and d). It is possible that this increased IκBα protein level may be partly due to the Tat inhibition on the IKK-β activity as discussed above.
It is well documented that LPS-activation of p38 MAPK and ERK1/2 can induce the production of TNFα and IL-6 [6,8,9]. Our results showed that Tat enhanced the LPS-induction of IL-6 but had no significant effect on the TNF-α expression (Fig. 2). This did not correlate with the results of Tat effects on these kinases' activities (Fig. 3c). Nevertheless, p38 MAPK can activate a number of specific downstream kinases, for example, MK2 that regulates the IL-6 expression by modulating its mRNA stability . Hence, it is possible that Tat may regulate the downstream targets of p38 MAPK leading to upregulation of IL-6 expression. Moreover, IL-6 expression can be regulated by other downstream kinases or transcription factors including C/EBP-β . As a previous study has shown that Tat interacts with C/EBP-β and binds to the IL-6 leader RNA for inducing IL-6 expression , it is conceivable that Tat may enhance the LPS-induction of IL-6 via these mechanisms.
As shown in Fig. 1c, the inhibitory effect of Tat on the LPS-induction of IFN-β was less effective at a higher concentration of LPS (100 ng/ml). This implied that Tat may only inhibit selected but not all branches of the pathways that lead to the LPS-induction of IFN-β. As NFκB activation has been shown to be a major player in regulating IFN-β expression [4,46], our data supported that one of these Tat-inhibited pathways is the NFκB signaling. Tat may inhibit this signaling by inducing high levels of IκBα proteins for the cytosolic retention of NFκB p65. Apart from NFκB, c-Jun, ATF-2, IRF3, IRF7, NFκB and CBP/p300 have been suggested to form an enhanceosome to regulate the transcription of IFN-β by interacting with the cytokine promoter [47–49]. It is plausible that Tat may inhibit one or more pathways, which lead to specific inhibition on the activity of some of the above transcription factors but not the others. For example, IRF3 is a crucial mediator for inducing IFN-β expression in the TLR4 and TLR3 pathways triggered by LPS and the double-stranded RNA of viruses, respectively. [4,5]. It has been demonstrated that HIV-1 accessory proteins, Vif and Vpr, contributed to the ubiquitin-mediated degradation of IRF3 leading to the inhibition of IFN-β transcription in HIV proviral DNA-transfected cells . Therefore, it remains to be investigated whether Tat may also impair the IRF3-dependent induction of IFN-β by LPS and viruses via similar mechanisms, thus delineating whether Tat can suppress the host antiviral responses at multiple levels of the IFN-induction pathway.
Recent clinical studies have suggested that LPS plays a role in HIV pathogenesis via a process called microbial translocation [50–53]. This translocation may lead to chronic systemic immune activation and bacterial sepsis in AIDS patients [50,54–56]. This postulate has been supported by the evidence that the plasma level of LPS is increased in HIV-infected individuals (ranging between 25 and 500 pg/ml), when compared with the uninfected counterparts [50–53]. However, the level of LPS in vivo in the cellular microenvironment sites may be much higher than these plasma levels in the HIV-infected individuals. Consequently, the elevated plasma LPS levels have been found to be associated with the hyperactivation of T cells, which may partly contribute to the decreased CD4+ T-cell counts in HIV-infected patients [50–52]. In addition, monocytes/macrophages are suggested to be hyperinduced by the circulating LPSs. These processes may partly account for the pathogenesis of AIDS including the HIV-associated dementia . Therefore, microbial translocation may play a major role in the progression of AIDS.
Despite these clinical findings, the in-vitro detailed interactions between HIV and Gram-negative bacteria in the infected host have not been well defined. Previous report showed that LPS from the bacteria could induce HIV transcription via the TLR4-dependent activation of NFκB, AP-1, C/EBP-β, cAMP response element binding (CREB) protein and Sp1, as well as via the autocrine/paracrine effects of bacteria-induction of TNF-α [3,57]. However, LPS can inhibit HIV infection by induction of IFN-β in vitro, which in turn inhibits transcription of the retrovirus [35,58,59]. Moreover, it has been found that LPS-induced cytokines including TNF-α and IL-6 are either unchanged, enhanced or suppressed in the HIV-infected cells [60–64]. Our findings contribute to the understanding of the underlying mechanisms in causing these paradoxical effects. Our results support the idea that HIV may evade from the antiviral effects induced by bacteria [35,58,59] to enhance its own survival by Tat suppression on the LPS-induction of IFN-β. It may further promote its own replication and dissemination of the infection by Tat enhancement on the LPS-induction of IL-6 during the bacterial infection. Hence, HIV may take advantage of the host response and utilize the differential effects of different cytokines for promoting its dissemination and adversely influencing the progression of AIDS.
This study was supported in part by the Hong Kong Research Grants Council (HKU7594/06M) and the Hong Kong Research Fund for the Control of Infectious Diseases (06060612). H.C.H.Y. is the recipient of a postgraduate studentship from the University of Hong Kong. We would like to acknowledge the Genome Research Centre and University Development Fund, The University of Hong Kong, for the support.
The data were presented in part in the 7th Joint Conference of the International Society for Interferon and Cytokine Research and International Cytokine Society – Cytokines 2008, October 2008, Montreal, Canada (abstract no. 500).
H.C.H.Y., J.C.B.L. and J.S.H.L. performed experiments; H.C.H.Y., J.C.B.L., J.S.H.L. and A.S.Y.L. analyzed results. H.C.H.Y., J.C.B.L. and A.S.Y.L. designed the research and wrote the article.
There was no conflict of interests.
1. Levy JA. HIV and the pathogenesis of AIDS. 2nd ed. Washington, DC: ASM Press; 1998.
2. Helweg-Larsen J, Benfield T, Lundgren J. Opportunistic infections. Dordrecht: Kluwer Academic; 2000.
3. Bafica A, Scanga CA, Schito M, Chaussabel D, Sher A. Influence of coinfecting pathogens on HIV expression: evidence for a role of Toll-like receptors. J Immunol 2004; 172:7229–7234.
4. O'Neill LA, Bowie AG. The family of five: TIR-domain-containing adaptors in Toll-like receptor signalling. Nat Rev Immunol 2007; 7:353–364.
5. Fitzgerald KA, Rowe DC, Barnes BJ, Caffrey DR, Visintin A, Latz E, et al
. LPS-TLR4 signaling to IRF-3/7 and NF-kappaB involves the toll adapters TRAM and TRIF. J Exp Med 2003; 198:1043–1055.
6. Guha M, Mackman N. LPS induction of gene expression in human monocytes. Cell Signal 2001; 13:85–94.
7. Ghosh S, May MJ, Kopp EB. NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol 1998; 16:225–260.
8. Matsuzawa A, Saegusa K, Noguchi T, Sadamitsu C, Nishitoh H, Nagai S, et al
. ROS-dependent activation of the TRAF6-ASK1-p38 pathway is selectively required for TLR4-mediated innate immunity. Nat Immunol 2005; 6:587–592.
9. Huang Q, Yang J, Lin Y, Walker C, Cheng J, Liu ZG, Su B. Differential regulation of interleukin 1 receptor and Toll-like receptor signaling by MEKK3. Nat Immunol 2004; 5:98–103.
10. Oppenheim JJ, Feldmann M, Durum SK. Cytokine reference: a compendium of cytokines
and other mediators of host defense. San Diego: Academic Press; 2001.
11. Decker T, Muller M, Stockinger S. The yin and yang of type I interferon
activity in bacterial infection. Nat Rev Immunol 2005; 5:675–687.
12. Westendorp MO, Frank R, Ochsenbauer C, Stricker K, Dhein J, Walczak H, et al
. Sensitization of T cells to CD95-mediated apoptosis by HIV-1 Tat
and gp120. Nature 1995; 375:497–500.
13. Rice AP, Mathews MB. Transcriptional but not translational regulation of HIV-1 by the tat
gene product. Nature 1988; 332:551–553.
14. Xiao H, Neuveut C, Tiffany HL, Benkirane M, Rich EA, Murphy PM, Jeang KT. Selective CXCR4 antagonism by Tat
: implications for in vivo expansion of coreceptor use by HIV-1. Proc Natl Acad Sci U S A 2000; 97:11466–11471.
15. Ambrosino C, Ruocco MR, Chen X, Mallardo M, Baudi F, Trematerra S, et al
. HIV-1 Tat
induces the expression of the interleukin-6 (IL6) gene by binding to the IL6 leader RNA and by interacting with CAAT enhancer-binding protein beta (NF-IL6) transcription factors. J Biol Chem 1997; 272:14883–14892.
16. Badou A, Bennasser Y, Moreau M, Leclerc C, Benkirane M, Bahraoui E. Tat
protein of human immunodeficiency virus type 1 induces interleukin-10 in human peripheral blood monocytes: implication of protein kinase C-dependent pathway. J Virol 2000; 74:10551–10562.
17. Buonaguro L, Barillari G, Chang HK, Bohan CA, Kao V, Morgan R, et al
. Effects of the human immunodeficiency virus type 1 Tat
protein on the expression of inflammatory cytokines
. J Virol 1992; 66:7159–7167.
18. Chen P, Mayne M, Power C, Nath A. The Tat
protein of HIV-1 induces tumor necrosis factor-alpha production. Implications for HIV-1-associated neurological diseases. J Biol Chem 1997; 272:22385–22388.
19. Li JC, Lee DC, Cheung BK, Lau AS. Mechanisms for HIV Tat
upregulation of IL-10 and other cytokine expression: kinase signaling and PKR-mediated immune response. FEBS Lett 2005; 579:3055–3062.
20. Shearer GM. HIV-induced immunopathogenesis. Immunity 1998; 9:587–593.
21. Zauli G, Furlini G, Re MC, Milani D, Capitani S, La Placa M. Human immunodeficiency virus type 1 (HIV-1) tat
-protein stimulates the production of interleukin-6 (IL-6) by peripheral blood monocytes. New Microbiol 1993; 16:115–120.
22. Duh EJ, Maury WJ, Folks TM, Fauci AS, Rabson AB. Tumor necrosis factor alpha activates human immunodeficiency virus type 1 through induction of nuclear factor binding to the NF-kappa B sites in the long terminal repeat. Proc Natl Acad Sci U S A 1989; 86:5974–5978.
23. Folks TM, Clouse KA, Justement J, Rabson A, Duh E, Kehrl JH, Fauci AS. Tumor necrosis factor alpha induces expression of human immunodeficiency virus in a chronically infected T-cell clone. Proc Natl Acad Sci U S A 1989; 86:2365–2368.
24. Poli G, Bressler P, Kinter A, Duh E, Timmer WC, Rabson A, et al
. Interleukin 6 induces human immunodeficiency virus expression in infected monocytic cells alone and in synergy with tumor necrosis factor alpha by transcriptional and posttranscriptional mechanisms. J Exp Med 1990; 172:151–158.
25. Poli G, Kinter AL, Fauci AS. Interleukin 1 induces expression of the human immunodeficiency virus alone and in synergy with interleukin 6 in chronically infected U1 cells: inhibition of inductive effects by the interleukin 1 receptor antagonist. Proc Natl Acad Sci U S A 1994; 91:108–112.
26. Naif H, Ho-Shon M, Chang J, Cunningham AL. Molecular mechanisms of IL-4 effect on HIV expression in promonocytic cell lines and primary human monocytes. J Leukoc Biol 1994; 56:335–339.
27. Griffin GE, Leung K, Folks TM, Kunkel S, Nabel GJ. Induction of NF-kappa B during monocyte differentiation is associated with activation of HIV-gene expression. Res Virol 1991; 142:233–238.
28. Westendorp MO, Shatrov VA, Schulze-Osthoff K, Frank R, Kraft M, Los M, et al
. HIV-1 Tat
potentiates TNF-induced NF-kappa B activation and cytotoxicity by altering the cellular redox state. EMBO J 1995; 14:546–554.
29. Cheung BK, Lee DC, Li JC, Lau YL, Lau AS. A role for double-stranded RNA-activated protein kinase PKR in mycobacterium-induced cytokine expression. J Immunol 2005; 175:7218–7225.
30. Lee DC, Cheung CY, Law AH, Mok CK, Peiris M, Lau AS. p38 mitogen-activated protein kinase-dependent hyperinduction of tumor necrosis factor alpha expression in response to avian influenza virus H5N1. J Virol 2005; 79:10147–10154.
31. Lau AS, Der SD, Read SE, Williams BR. Regulation of tumor necrosis factor receptor expression by acid-labile interferon
-alpha from AIDS sera. AIDS Res Hum Retroviruses 1991; 7:545–552.
32. Lau AS, Hannigan GE, Freedman MH, Williams BR. Regulation of interferon
receptor expression in human blood lymphocytes in vitro and during interferon
therapy. J Clin Invest 1986; 77:1632–1638.
33. Kedzierska K, Crowe SM, Turville S, Cunningham AL. The influence of cytokines
, chemokines and their receptors on HIV-1 replication in monocytes and macrophages. Rev Med Virol 2003; 13:39–56.
34. Ansari AW, Heiken H, Moenkemeyer M, Schmidt RE. Dichotomous effects of C-C chemokines in HIV-1 pathogenesis. Immunol Lett 2007; 110:1–5.
35. Kornbluth RS, Oh PS, Munis JR, Cleveland PH, Richman DD. Interferons and bacterial lipopolysaccharide protect macrophages from productive infection by human immunodeficiency virus in vitro. J Exp Med 1989; 169:1137–1151.
36. Goldfeld AE, Birch-Limberger K, Schooley RT, Walker BD. HIV-1 infection does not induce tumor necrosis factor-alpha or interferon
-beta gene transcription. J Acquir Immune Defic Syndr 1991; 4:41–47.
37. Gendelman HE, Friedman RM, Joe S, Baca LM, Turpin JA, Dveksler G, et al
. A selective defect of interferon
alpha production in human immunodeficiency virus-infected monocytes. J Exp Med 1990; 172:1433–1442.
38. Okumura A, Alce T, Lubyova B, Ezelle H, Strebel K, Pitha PM. HIV-1 accessory proteins VPR and Vif modulate antiviral response by targeting IRF-3 for degradation. Virology 2008; 373:85–97.
39. Woelk CH, Ottones F, Plotkin CR, Du P, Royer CD, Rought SE, et al
gene expression following HIV type 1 infection of monocyte-derived macrophages. AIDS Res Hum Retroviruses 2004; 20:1210–1222.
40. Vahey MT, Nau ME, Jagodzinski LL, Yalley-Ogunro J, Taubman M, Michael NL, Lewis MG. Impact of viral infection on the gene expression profiles of proliferating normal human peripheral blood mononuclear cells infected with HIV type 1 RF. AIDS Res Hum Retroviruses 2002; 18:179–192.
41. Barton CH, Biggs TE, Mee TR, Mann DA. The human immunodeficiency virus type 1 regulatory protein Tat
-induced iNos activity in a murine macrophage cell line. J Gen Virol 1996; 77(Pt 8):1643–1647.
42. Izmailova E, Bertley FM, Huang Q, Makori N, Miller CJ, Young RA, Aldovini A. HIV-1 Tat
reprograms immature dendritic cells to express chemoattractants for activated T cells and macrophages. Nat Med 2003; 9:191–197.
43. Waterfield M, Jin W, Reiley W, Zhang M, Sun SC. IkappaB kinase is an essential component of the Tpl2 signaling pathway. Mol Cell Biol 2004; 24:6040–6048.
44. Blazquez MV, Macho A, Ortiz C, Lucena C, Lopez-Cabrera M, Sanchez-Madrid F, Munoz E. Extracellular HIV type 1 Tat
protein induces CD69 expression through NF-kappaB activation: possible correlation with cell surface Tat
-binding proteins. AIDS Res Hum Retroviruses 1999; 15:1209–1218.
45. Neininger A, Kontoyiannis D, Kotlyarov A, Winzen R, Eckert R, Volk HD, et al
. MK2 targets AU-rich elements and regulates biosynthesis of tumor necrosis factor and interleukin-6 independently at different posttranscriptional levels. J Biol Chem 2002; 277:3065–3068.
46. Algarte M, Nguyen H, Heylbroeck C, Lin R, Hiscott J. IkappaB-mediated inhibition of virus-induced beta interferon
transcription. J Virol 1999; 73:2694–2702.
47. Panne D, Maniatis T, Harrison SC. An atomic model of the interferon
-beta enhanceosome. Cell 2007; 129:1111–1123.
48. Merika M, Williams AJ, Chen G, Collins T, Thanos D. Recruitment of CBP/p300 by the IFN beta enhanceosome is required for synergistic activation of transcription. Mol Cell 1998; 1:277–287.
49. Wang H, Garcia CA, Rehani K, Cekic C, Alard P, Kinane DF, et al
. IFN-beta production by TLR4-stimulated innate immune cells is negatively regulated by GSK3-beta. J Immunol 2008; 181:6797–6802.
50. Brenchley JM, Price DA, Schacker TW, Asher TE, Silvestri G, Rao S, et al
. Microbial translocation is a cause of systemic immune activation in chronic HIV infection. Nat Med 2006; 12:1365–1371.
51. Anselmi A, Vendrame D, Rampon O, Giaquinto C, Zanchetta M, De Rossi A. Immune reconstitution in human immunodeficiency virus type 1-infected children with different virological responses to antiretroviral therapy. Clin Exp Immunol 2007; 150:442–450.
52. Hunt PW, Brenchley J, Sinclair E, McCune JM, Roland M, Page-Shafer K, et al
. Relationship between T cell activation and CD4+ T cell count in HIV-seropositive individuals with undetectable plasma HIV RNA levels in the absence of therapy. J Infect Dis 2008; 197:126–133.
53. Ancuta P, Kamat A, Kunstman KJ, Kim EY, Autissier P, Wurcel A, et al
. Microbial translocation is associated with increased monocyte activation and dementia in AIDS patients. PLoS ONE 2008; 3:e2516.
54. Farizo KM, Buehler JW, Chamberland ME, Whyte BM, Froelicher ES, Hopkins SG, et al
. Spectrum of disease in persons with human immunodeficiency virus infection in the United States. JAMA 1992; 267:1798–1805.
55. Proctor RA. Bacterial sepsis in patients with acquired immunodeficiency syndrome. Crit Care Med 2001; 29:683–684.
56. Rosenberg AL, Seneff MG, Atiyeh L, Wagner R, Bojanowski L, Zimmerman JE. The importance of bacterial sepsis in intensive care unit patients with acquired immunodeficiency syndrome: implications for future care in the age of increasing antiretroviral resistance. Crit Care Med 2001; 29:548–556.
57. Equils O, Faure E, Thomas L, Bulut Y, Trushin S, Arditi M. Bacterial lipopolysaccharide activates HIV long terminal repeat through Toll-like receptor 4. J Immunol 2001; 166:2342–2347.
58. Honda Y, Rogers L, Nakata K, Zhao BY, Pine R, Nakai Y, et al
. Type I interferon
induces inhibitory 16-kD CCAAT/enhancer binding protein (C/EBP)beta, repressing the HIV-1 long terminal repeat in macrophages: pulmonary tuberculosis alters C/EBP expression, enhancing HIV-1 replication. J Exp Med 1998; 188:1255–1265.
59. Liu X, Mosoian A, Li-Yun Chang T, Zerhouni-Layachi B, Snyder A, Jarvis GA, Klotman ME. Gonococcal lipooligosaccharide suppresses HIV infection in human primary macrophages through induction of innate immunity. J Infect Dis 2006; 194:751–759.
60. Gan HX, Ruef C, Hall BF, Tobin E, Remold HG, Mellors JW. Interleukin-6 expression in primary macrophages infected with human immunodeficiency virus-1 (HIV-1). AIDS Res Hum Retroviruses 1991; 7:671–679.
61. Molina JM, Schindler R, Ferriani R, Sakaguchi M, Vannier E, Dinarello CA, Groopman JE. Production of cytokines
by peripheral blood monocytes/macrophages
infected with human immunodeficiency virus type 1 (HIV-1). J Infect Dis 1990; 161:888–893.
62. Patel NR, Zhu J, Tachado SD, Zhang J, Wan Z, Saukkonen J, Koziel H. HIV impairs TNF-alpha mediated macrophage apoptotic response to Mycobacterium tuberculosis. J Immunol 2007; 179:6973–6980.
63. Tachado SD, Zhang J, Zhu J, Patel N, Koziel H. HIV impairs TNF-alpha release in response to Toll-like receptor 4 stimulation in human macrophages in vitro. Am J Respir Cell Mol Biol 2005; 33:610–621.
64. Tsai WP, Hirose K, Nara PL, Kuang YD, Conley S, Li BQ, et al
. Decrease in cytokine production by HIV-infected macrophages in response to LPS-mediated activation. Lymphokine Cytokine Res 1991; 10:421–429.