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Keywords:

  • CC-chemokines;
  • HTLV-2;
  • innate immunity;
  • NF-κB pathway;
  • Tax2

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Retroviral co-infections with human immunodeficiency virus type-1 (HIV-1) and human T cell leukaemia virus type 1 (HTLV-1) or type 2 (HTLV-2) are prevalent in many areas worldwide. It has been observed that HIV-1/HTLV-2 co-infections are associated with slower rates of CD4+ T cell decline and delayed progression to AIDS. This immunological benefit has been linked to the ability of Tax2, the transcriptional activating protein of HTLV-2, to induce the expression of macrophage inflammatory protein (MIP)-1α/CCL3, MIP-1β/CCL4 and regulated upon activation normal T cell expressed and secreted (RANTES)/CCL5 and to down-regulate the expression of the CCR5 co-receptor in peripheral blood mononuclear cells (PBMCs). This study aimed to assess the role of Tax2-mediated activation of the nuclear factor kappa B (NF-κB) signalling pathway on the production of the anti-viral CC-chemokines MIP-1α, MIP-1β and RANTES. Recombinant Tax1 and Tax2 proteins, or proteins expressed via adenoviral vectors used to infect cells, were tested for their ability to activate the NF-κB pathway in cultured PBMCs in the presence or absence of NF-κB pathway inhibitors. Results showed a significant release of MIP-1α, MIP-1β and RANTES by PBMCs after the activation of p65/RelA and p50. The secretion of these CC-chemokines was significantly reduced (P < 0·05) by canonical NF-κB signalling inhibitors. In conclusion, Tax2 protein may promote innate anti-viral immune responses through the activation of the canonical NF-κB pathway.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

The human T cell leukaemia viruses types 1 and 2 (HTLV-1, HTLV-2) infect approximately 15–25 million individuals worldwide [1]. Both viruses have similar biological properties, genomic structures and tropism for immune cells, and they establish lifelong infection in their hosts with rare expression of clinical disease [2]. The neurological disorder HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) [3, 4], adult T cell leukaemia (ATL) [5, 6] and inflammatory diseases [7-9] have been reported in 3–5% of individuals infected with HTLV-1. In contrast, HTLV-2 has not been linked clearly to any disease, although long-term carriers of HTLV-2 have subtle alterations in their immunological phenotypes [10]. Due to common modes of retroviral transmission [11], co-infections with human immunodeficiency virus type 1 (HIV-1) and HTLV-1 or HIV-1 and HTLV-2 are prevalent in many metropolitan areas in the United States and worldwide (reviewed recently in [12]). In the absence of therapy, HIV-1 results in massive depletion of CD4+ T cells, with development of severe immunodeficiency and death from opportunistic infections. Interestingly, cohort studies have shown immunological benefits among HIV-1/HTLV-2 co-infected patients compared to the HIV-1 mono-infected population, including: (i) delayed rate of decrease in CD4+ T cell count, (ii) induction of clonal expansion of both CD4+ and CD8+ T cells, (iii) lower levels of HIV-1 plasma RNA, (iv) delay in progression to AIDS and (v) significantly slower progression to death [13, 14]. In addition, the HTLV-2 tax/rex mRNA levels were found to be increased in the HIV-1/HTLV-2 co-infected population [15] and high HTLV-2 proviral loads correlated with long-term non-progression to AIDS [14].

Tax1 and Tax2, the regulatory proteins of HTLV-1 and HTLV-2, activate viral and host cellular gene transcription and are essential for viral replication; in addition they have considerable effects on the level of clinical disease expression [16-18]. Tax1 induces multiple functions in the host cells (e.g. modulation of cell cycle checkpoint, interference with DNA repair, induction of cellular senescence, inhibition of apoptosis) and interacts with numerous cellular proteins regulating the activation of multiple signalling pathways [e.g. cyclic adenosine monophosphate (AMP)-responsive element-binding protein (CREB), serum response factor (SRF), mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), activator protein 1 (AP1), transforming growth factor (TGF)-β, nuclear factor (NF)-κB], whereas Tax2 has only been identified to interact with proteins involved mainly in the NF-κB canonical pathway [19]. The canonical and non-canonical NF-κB activation pathways have distinct regulatory functions. In the canonical pathway, the NF-κB/Rel family of transcription factors exist in the cytoplasm bound and inhibited by IκB proteins. Cellular stimulation by a variety of inducers (e.g. cytokines, mitogens, free radicals, Tax1, Tax2) results in phosphorylation, polyubiquitination and proteosomal degradation of IκB allowing translocation of the active dimer p65/RelA-p50 to the nucleus inducing the transcription of target genes (chemokines, cytokines and adhesion molecules) promoting cell survival, immune regulation and inflammatory responses [18, 20]. In the non-canonical pathway, p100/RelB complexes are inactive in the cytoplasm. Signalling through a subset of tumour necrosis factor (TNF) receptors (e.g. LTβR, CD40, BR3) phosphorylates IKKα complexes which, in turn, activate p100 leading to its ubiquitination and proteosomal processing to p52. The transcriptionally competent p52/RelB complexes translocate to the nucleus and induce target gene expression that regulates the development of lymphoid organs and the adaptive immune responses [18, 20]. Tax1 and Tax2 mediate activation of key cellular pathways involved in cytokine and chemokine production via the NF-κB pathway [20], but the ability of Tax2 to induce cytokine gene expression have been reported to be lower than Tax1 [21]. The NF-κB pathway is constitutively activated in HTLV-1-infected cells due to the persistent dissociation of IκB from the NF-κB/IκB complex induced by Tax1 [22]. Tax1 has been shown to deregulate both canonical and non-canonical NF-κB pathways by interacting with and activating several factors including the RelA and IκB kinase complex, as well as mediating p100 processing and p52 nuclear translocation [19]. In contrast, Tax2 protein does not contain NF-κB2 domain, does not bind p100, and therefore does not induce its processing to the active p52 subunit [19, 20]. Tax1, but not Tax2, has been found to have a co-operative role with the non-canonical NF-κB pathway to mediate T cell transformation and leukaemogenesis [23].

Recently our group reported that extracellular Tax1 and Tax2 proteins induce the expression of macrophage inflammatory protein (MIP)-1α/CCL3, MIP-1β/CCL4 and regulated upon activation normal T cell expressed and secreted (RANTES)/CCL5 from peripheral blood mononuclear cells (PBMCs) and monocyte-derived macrophages (MDMs) [24, 25] with the concomitant down-regulation of CCR5, the HIV-1 co-receptor [24]. Additionally Tax1 and Tax2 expressed via adenoviral vectors delivered into MDMs also induced the secretion of CC-chemokines [25]. CC-chemokines have been correlated with innate resistance to HIV-1 infection, decreased viral loads in individuals already infected and protection against disease progression to AIDS [26]. We have hypothesized that Tax2 has the potential to alter innate host immune responses and may be capable of modifying HIV-1 pathogenesis in HIV-1/HTLV-2 co-infected individuals. In this study we aimed to investigate whether or not Tax2 could induce the expression of CC-chemokines in cultured PBMCs through the canonical NF-κB signalling pathway. The effect of potent inhibitors of the canonical NF-κB signalling was examined to determine whether CC-chemokine production is dependent upon this pathway.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Human peripheral blood mononuclear cells (PBMCs), cell lines and reagents

Blood samples from three HIV-1 and HTLV-1/-2 seronegative donors were obtained following informed consent using a protocol that was approved by the Institutional Review Board for Human Investigation of the Milwaukee Veterans Affairs, Research Service Committee. Whole blood was collected in CPT/Vacutainer BD tubes (BD Biosciences, San Jose, CA, USA) and PBMCs were obtained following the manufacturer's instructions. Phorbol 12-myristate 13-acetate (PMA at 50 ng/ml; Sigma, St Louis, MO, USA) and phytohaemagglutinin (PHA at 5 μg/ml; Sigma) were used to stimulate PBMCs. The NF-κB inhibitor pyrrolidine dithiocarbamate was used to pretreat PBMCs (PDTC at 30 μM; Sigma). Antibodies specific for phospho-p65/RelA (Ser536) were from Cell Signaling Technology and fluorescein isothiocyanate (FITC)-labelled goat anti-rabbit immunoglobulin (Ig)G (H + L), F(ab′)2 was obtained from KPL Inc. (Gaithersburg, MD, USA).

The HTLV-2-infected human T cell line (known as MoT, ATCC CRL-8086) expresses Tax2 and mature HTLV-2 viral particles and exhibits constitutive activation of NF-κB [27]. MoT cells, used as positive control, were grown in complete RPMI medium [RPMI medium supplemented with 10% fetal bovine serum (FBS), 2·05 mM L-glutamine, 1% penicillin/streptomycin (P/S v/v), 1% sodium pyruvate (v/v)] and cultured in a humidified incubator at 37°C with 5% CO2. Jurkat cells (human T cell lymphoblast-like cell line; American Type Culture Collection TIB-152) were used as negative control and grown in complete Iscove's modified Eagle's medium (IMDM) (IMDM supplemented with 10% FBS and P/S and L-glutamine).

Recombinant proteins and recombinant vectors

Recombinant Tax1 and Tax2A (subtype A) proteins were purified as described recently in detail [24, 25]. Truncated Tax2A/NH2term-His tagged sequence aa 1–198 (Tax2A/1–198) (MRGSHHHHHHGS AHFPGFGQSL LYGYPVYVFG DCVQADWCPV SGGLCSTRLH RHALLATCPE HQL TWDPIDG RVVSSPLQYL IPRLPSFPTQ RTSRTLKVLT PPTTPVSPKV PPAFFQSMRK HTPYRNGCLE PTLGDQLPSL AFPEPGLRPQ NIYTTWGKTV VCLYLYQLSP PMTWPLIPHV IFCHPRQLGA FLTKVPLKRL EELLYKM LDLQPSLIS) and truncated Tax2A/COOHterm-His tagged sequence aa 135–331 (Tax2A/135–331) (MRGSHHHHHHGS EPGLRPQNIY TTWGKTVVCL YLYQLSPPMT WPLIPHVIFC HPRQLGAFLT KVPLKRLEEL LYKMFLHTGT VIVLPEDDLP TTMFQPVRAP CIQTAWCTGL LPYHSILTTP GLIWTFNDGS PMISGPCPKA GQPSLVVQSS LLIFEKFQTK AFHPSYLLSH QLIQYSSFHN LHLLFDEYTN IPVSILFNKE EADDNGD LDLQPSLIS) fragments of Tax2A protein containing NF-κB domains [28, 29] were subcloned from PET29a-Tax2-H6 [30] to pQE-30 (Amp-resistant) vector and transformed into the Esherichia coli BL21(DE3) strain (subcloning generation and protein production serviced by Promab Biotechnologies, Inc., Richmond, CA, USA). An extract was prepared in an identical manner from E. coli cells containing the empty vector for use as a mock control. Determination of protein concentrations was performed using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA). Endotoxin concentration for all protein recombinants at the concentration (100 pM) used in the in-vitro experiments were found to be endotoxin-free, as determined by the limulus amoebocyte lysate test (E-TOXATE; Sigma) [24]. Recombinant replication-deficient adenoviruses expressing Tax2B (subtype B) or green fluorescent protein (GFP), used to control the efficiency of transduction (Ad-Tax2B or Ad-GFP, respectively) [31], were propagated and titrated as described recently [25]. The recombinant adenovirus containing the dominant negative mutant of IκBα with serine to alanine substitutions at amino acids 32 and 36, and therefore resistant to phosphorylation-induced degradation (a NF-κB super-repressor designated NF-κB/SR), was obtained commercially (Vector Biolabs, Philadelphia, PA, USA).

Cellular treatment with extracellular Tax proteins or transduction with adenoviral vectors expressing Tax and luciferase reporter gene transactivation analysis

In this study the two major subtypes of HTLV-2 Tax, Tax2A (expressed as recombinant protein) and Tax2B (recombinant adenovirus) were assessed to determine whether both Tax2 subtypes were able to induce the production of CC-chemokines in peripheral blood mononuclear cells. PBMCs (1 × 106/ml) in complete RPMI medium were treated with extracellular Tax (recombinant Tax1 and Tax2A) proteins at 100 pM for 1, 2, 3, 6, 12 and 24 h to determine CC-chemokine production, and for 1 and 2 h for the determination of canonical NF-κB pathway activation. Mock-treated and untreated controls were used in all experiments. To express Tax2B via tax genes, PBMCs were transduced with the recombinant adenoviral vectors (Ad-Tax2B) at a multiplicity of infection (MOI) of 10 in Opti-MEM I reduced serum medium using lipofectamine and plus reagent (both from Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. All conditions were tested in triplicate. Cells were incubated at 37°C and 5% CO2. The efficiency of transduction was confirmed by fluorescence microscopy. A luciferase reporter assay was used as described previously [24, 32] to confirm the ability of the recombinant Tax2 proteins to regulate viral transcription.

Determination of CC-chemokines

Cell-free supernatants from Tax-treated PBMCs were harvested at various time-points and assayed for MIP-1α, MIP-1β and RANTES expression by quantitative enzyme-linked immunosorbent assay (ELISA) (R&D Systems), as described previously [24]. Absorbance values at 490 nm were used to quantify the chemokine levels in the culture supernatants from the standard concentration curve and CC-chemokine protein levels were expressed in picograms per millilitre (pg/ml).

Immunofluorescence detection of the phosphorylated p65/RelA NF-κB subunit

To determine the activation of the canonical NF-κB pathway, immunofluorescence studies were performed using Tax-treated-PBMCs for an incubation period of 1 and 2 h. Cells were harvested, washed with phosphate-buffered saline (PBS), fixed and cytocentrifuged onto clean glass slides, then permeabilized with 0·3% Triton X-100 (Sigma) and blocked with 5% normal goat serum (Sigma) for 1 h at room temperature. Phosphorylated p65/RelA was detected with phospho-p65/RelA monoclonal antibodies (mAbs) (diluted at 1:100) followed by FITC-labelled goat anti-rabbit IgG (H + L), F(ab′)2 mAbs (diluted at 1:200) and incubated at dark-room temperature for 1 h. 4′,6-Diamidino-2-phenylindole (DAPI; Sigma) was use to stain the nucleus. Slides were mounted using anti-fade fluorescent mounting media. Jurkat and MoT cells were used as negative and positive controls. Images were acquired with an Olympus BX51 epifluorescence microscope equipped with an Olympus DP-70 controller digital camera system and applicable computer capture software (Tokyo, Japan). ImageJ software was used to determine the intensity of cell fluorescence and the non-specific background was subtracted to obtain the corrected total cell fluorescence (CTCF) as integrated density – (area of selected cell × mean fluorescence of background readings) [33].

NF-κB DNA-binding ELISA-based assay

PBMCs (1 × 106/ml) were stimulated with 100 pM of recombinant Tax proteins and cells were harvested at 1 or 2 h. Nuclear extracts were obtained and tested for activation of p65/RelA and p50 subunits using the TransAM NF-κB DNA-binding ELISA kit (Active Motif, Carlsbad, CA, USA). Briefly, 10 μg of each nuclear extract were incubated in 96-well plates containing a consensus (5′-GGGACTTTCC-3′) binding site for NF-κB. The subunit binding to the oligonucleotide was detected using a primary antibody specific for the activated subunit form, visualized by incubation with anti-IgG–horseradish peroxidase (HRP) conjugate and a developing solution, and quantified at 450 nm with a reference wavelength of 655 nm. Nuclear extracts from Jurkat cells were used as negative control, and nuclear extracts from Raji cells included in the kit and from MoT cells served as positive controls in the assay.

Use of inhibitors of NF-κB pathway prior to Tax treatment

In order to address the potential cytotoxic effects of pyrrolidine dithiocarbamate (PDTC) on mononuclear cells, experiments were performed treating PBMCs with PDTC for 1 h at three different concentrations (1 μM, 10 μM, 30 μM) or left them untreated, then washed three times with RPMIc. After 3 h in culture, cell viability was measured by the trypan blue exclusion method. PDTC-treated cells were also subjected to apoptosis determination by fluorescence activated cell sorter (FACS) using the annexin-V/7-aminoactinomycin D (7AAD) kit (BD Bioscience Pharmingen). More than 95% viable cells were determined in trypan blue exclusion assay for PBMCs treated with PDTC under these concentrations. In addition, the PDTC agent did not affect the viability of the cells as assessed by annexin-V and 7AAD staining (data not shown), and therefore pretreatment of PBMCs was performed with 30 μM of PDTC. To examine the role of NF-κB in Tax-mediated CC-chemokine secretion, PBMCs were pretreated with 30 μM of PDTC, a potent inhibitor of NF-κB, for 1 h then washed three times with RPMIc, followed by treatment with Tax proteins (100 pM) for 3 h, shown to be the optimal time-point to assess levels of CC-chemokines in Tax-treated PBMCs (Fig. 1). In other experiments, PBMCs were transduced with the NF-κB super-repressor (NF-κB/SR) at an MOI of 25 using lipofectamine plus reagent (Invitrogen) for 20 h prior to Tax protein treatment (3 h). PBMCs were also co-transduced with NF-κB/SR and Ad-Tax2 or Ad-GFP. Cell-free supernatants were harvested after 24 h of incubation and assayed for MIP-1α, MIP-1β and RANTES expression, as described above.

figure

Figure 1. Macrophage inflammatory protein (MIP)-1α, MIP-1β, and regulated upon activation normal T cell expressed and secreted (RANTES) expression by peripheral blood mononuclear cells (PBMCs) after treatment with extracellular Tax proteins. PBMCs were treated once with 100 pM of Tax1, Tax2A, mock control or left untreated. Cell-free supernatants were harvested after 1, 2, 3, 6, 12 or 24 h of incubation and assayed for the detection of (a) MIP-1α, (b) MIP-1β and (c) RANTES production by enzyme-linked immunosorbent assay (ELISA). The results are shown as mean ± standard error of the mean of three separate experiments in triplicate for each data point and reported as pg/ml of each CC-chemokine determined. Statistical analyses was performed with analysis of variance (anova) and Bonferroni post-test using GraphPad Prism version 6·00 software and P-values denoted as *P < 0·05, **P < 0·01 and ***P < 0·001 versus untreated; +P < 0·05, ++P < 0·01 and +++P < 0·001 versus mock-treated control; P < 0·05, ∧∧P < 0·01 and ∧∧∧P < 0·001 Tax2 versus Tax1.

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Statistics

All statistical analyses were performed using GraphPad Prism version 6·00 for Windows (GraphPad Software http://www.graphpad.com) and the data expressed as mean ± standard error of the mean. One-way analysis of variance (anova) with Bonferroni's multiple post-test comparison were used to evaluate three or more groups. Statistical comparisons for two groups were assessed by two samples assuming equal variances Student's t-test. P-values <0·05 were considered statistically significant.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Timing of MIP-1α, MIP-1β and RANTES expression by PBMCs after treatment with recombinant Tax proteins

We have reported recently that extracellular Tax2 and Tax1 proteins induced high levels of CC-chemokines in mononuclear cells [24, 25]. The optimal dose of protein required to detect CC-chemokine secretion was determined previously by exposing PBMCs to increased concentrations of Tax proteins [24]; the concentration of 100 pM was optimal, and therefore used in all subsequent experiments. In order to determine the time of MIP-1α, MIP-1β and RANTES release, PBMCs were treated once with Tax2A (subtype A), Tax1 or mock-treated control and then cell-free supernatants were harvested after 1, 2, 3, 6, 12 or 24 h of incubation. PBMCs treated with extracellular Tax2A protein induced the release of MIP-1α, MIP-1β and RANTES by 2 h, with maximal release at 6, 12–24 and 2 h, respectively (Fig. 1a–c). Maximal levels of expression were detected at 24 h for MIP-1α and at 6 h for MIP-1β and RANTES following Tax1 treatment. Interestingly, higher levels of MIP-1α were observed at 6 and 12 h when PBMCs were treated with Tax2A compared to Tax1 (Fig. 1a), while higher levels of MIP-1β and RANTES were detected after 3 and 6 h for Tax1 treatment compared to Tax2A (Fig. 1b,c). These results indicated that HTLV-2 Tax protein induced a rapid and sustained production of MIP-1α, MIP-1β and RANTES.

HTLV-2 Tax activation of the canonical NF-κB pathway precedes CC-chemokine expression

Tax1 and Tax2A recombinant proteins were assessed for their potential to activate the p65/RelA subunit, which is a well-established indicator of the canonical NF-κB pathway [34], a rapid-acting primary transcription factor. We also employed Tax2A/1–198 and Tax2A/135–331 recombinant Tax2A fragments containing NF-κB domains [28, 29] to evaluate their potential to activate the NF-κB pathway compared to the entire Tax2A protein. Treated cells were immunolabelled for the detection of phosphorylated p65/RelA by immunofluorescence. After 1 h, both the entire Tax2A and the Tax2A/1–198 fragment induced p65/RelA activation significantly over controls (14- and 10-fold, respectively, P < 0·05) (Fig. 2a). Significantly higher levels of activation were also observed when the entire Tax2A and the Tax2A/135–331 fragment were used to treat PBMCs for 2 h (27- and ninefold, respectively, P < 0·05). The complete Tax2A protein also induced significantly higher levels of p65/RelA activation compared to Tax1 and both Tax2A fragments after 2 h of treatment (Fig. 2b). Tax1 protein induced significant levels of p65/RelA activation at 1 (12-fold) and 2 h (eightfold) (P < 0·05). The Jurkat cell line served as a negative control and the HTLV-2-infected MoT cell line, displaying constitutive activation of NF-κB [27], served as positive control in the assay (Fig. 2c). It was observed that the activation of p65/RelA (Fig. 2a,b) by Tax2A preceded the secretion of MIP-1α, MIP-1β and RANTES in all conditions tested (Fig. 1).

figure

Figure 2. Induction of nuclear factor kappa B (NF-κB) activation in peripheral blood mononuclear cells (PBMCs) treated with extracellular Tax proteins. Tax-treated-PBMCs from three human immunodeficiency virus type-1 and human T cell leukaemia virus type-1 (HIV-1/HTLV) seronegative donors were blocked and permeabilized after 1 (a) and 2 (b) h for phosphorylation of p65/RelA immunolabelling detected by immunofluorescence. Images were obtained with the Olympus BX51 epifluorescent microscope and the results are shown as mean ± standard error of the mean of the corrected total cell fluorescence (CTCF) from three independent experiments. Analysis of variance (anova) and Bonferroni post-test were used for statistical analysis. P-values < 0·05 were considered significant and denoted as *P < 0·05, **P < 0·01 and ***P < 0·001 versus untreated or as +P < 0·05, ++P < 0·01 and +++P < 0·001 versus mock control. Jurkat and MoT cells served as negative and positive controls (c).

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Next, the binding activity of p65/RelA and p50 NF-κB subunits was assessed quantitatively in nuclear extracts from PBMCs treated with Tax2A or Tax1 proteins using the TransAM assay. Tax2A significantly enhanced the activation of both p65/RelA and p50 after 1 and 2 h compared to untreated and mock-treated controls (P < 0·001). Although Tax1 also induced high levels of both p65 and p50 activation by 1 (P < 0·05) and 2 h (P < 0·001) after treatment compared to controls (Fig. 3a,c), Tax2A induced significantly higher levels of p65/RelA activation than Tax1 following 1 h of treatment (P < 0·05) (Fig. 3a). Nuclear extracts from MoT and Raji nuclear extracts, used as positive controls, induced high levels of both p65/RelA and p50 activation (Fig. 3b,d). These results provide evidence that Tax2 induces a rapid activation of the canonical NF-κB pathway, preceding the production of CC-chemokines.

figure

Figure 3. Quantification of nuclear factor kappa B (NF-κB) p65/RelA and p50 DNA-binding in nuclear extracts from Tax-treated peripheral blood mononuclear cells (PBMCs). NF-κB p65/RelA and p50 subunit were quantified independently in 10 μg nuclear extracts prepared from PBMCs of three human immunodeficiency virus type-1 and human T cell leukaemia virus type-1 (HIV-1/HTLV) seronegative donors treated during 1 (a) or 2 (c) h with Tax proteins using NF-κB DNA-binding enzyme-linked immunosorbent assay (ELISA) (TransAM, active motif). Jurkat served as negative control and MoT and Raji nuclear extracts were used as positive controls (b,d). Data are shown as mean ± standard error of the mean of arbitrary optical density (OD)450 nm triplicate values. Statistical analyses were performed with analysis of variance (anova) and Bonferroni post-test and designed as *P < 0·05, **P < 0·01 and ***P < 0·001 versus untreated; +P < 0·05, ++P < 0·01 and +++P < 0·001 versus mock-treated control; P < 0·05 Tax2 versus Tax1.

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Inhibition of the NF-κB canonical pathway abrogates Tax2-mediated CC-chemokine expression in PBMCs

The direct role of NF-κB signalling in Tax2-mediated CC-chemokine secretion in PBMCs was then examined using a potent NF-κB canonical pathway inhibitor, pyrrolidine dithiocarbamate (PDTC), which inhibits the IκB-ubiquitin ligase activity blocking the degradation of IκB; as a consequence, the IκB-p65/RelA-p50 complex remains sequestered in the cytoplasm [35, 36]. We investigated whether the inhibition of the canonical NF-κB pathway could restrain the secretion of CC-chemokines by Tax2A-treated PBMCs. Thus, cells were pretreated or not with PDTC at 30 μM for 1 h, prior to the addition of extracellular Tax1, Tax2A, Tax2A/1–198, Tax2A/135–331, PHA/PMA (5 μg/ml and 50 ng/ml, respectively) or mock control, then cell-free supernatants were taken after 3 h of incubation, a time-point shown to have significant measurable levels of CC-chemokines (Fig. 1). PBMCs pretreated with PDTC resulted in a two- to threefold reduction of MIP-1α and RANTES production (P < 0·01; Fig. 4a,c) and a four- to sevenfold inhibition of MIP-1β release (P < 0·01) using all Tax proteins tested (Fig. 4b). As a test control, PDTC pretreated PBMCs stimulated with PHA/PMA showed a statistically significant reduction of all CC-chemokines compared with the PHA/PMA-stimulated PMBCs (P < 0·05, Fig. 4a–c).

figure

Figure 4. Inhibition of Tax-mediated CC-chemokine secretion by pyrrolidine dithiocarbamate (PDTC) and by a nuclear factor kappa B (NF-κB) super-repressor (NF-κB/SR) in peripheral blood mononuclear cells (PBMCs). PBMCs were preincubated with PDTC (30μM) followed by the addition of Tax proteins or mock control for 3 h, cell-free supernatants harvested and macrophage inflammatory protein (MIP)-1α, MIP-1β and regulated upon activation normal T cell expressed and secreted (RANTES) analysed by enzyme-linked immunosorbent assay (ELISA) (a–c). PBMCs were also pretreated for 20 h with a NF-κB/SR or a mock-adenoviral control (Ad-GFP) at a multiplicity of infection (MOI) of 25 and then treated with the Tax proteins or mock control; 3 h after incubation supernatants were harvested for CC-chemokine detection (d–f). The results represent the mean ± standard error of the mean of macrophage inflammatory protein (MIP)-1α, MIP-1β and RANTES in triplicate values (pg/ml) of three independent experiments. PBMC inhibition of CC-chemokines production by PDTC or NF-κB/SR was analysed statistically (Student's t-test) by comparing each value with the correspondent Tax-treated-PBMCs to determine reduction of CC-chemokine levels. Values of P < 0·05 were considered significant. Asterisks denote different P-values as *P < 0·05, **P < 0·01 and ***P < 0·001.

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These results were confirmed using a NF-κB super-repressor (NF-κB/SR) at a MOI of 25 to pretreat PBMCs for 20 h before adding Tax proteins, and harvesting cell-free supernatants after 3 h of culture. The data showed that the NF-κB/SR pretreatment significantly reduced the expression of MIP-1α, MIP-1β and RANTES when PBMCs were treated with Tax1, the entire Tax2A protein and the Tax2A/135–331 fragment (P < 0·05, Fig. 4d–f). NF-κB/SR reduced the expression of MIP-1α significantly (P < 0·05) (Fig. 4d), but there was only a trend towards reduced levels of MIP-1β and RANTES expression in Tax2A/1–198-treated PBMCs (Fig. 4e,f).

The inhibition of CC-chemokine induction by the NF-κB/SR was also examined co-transducing PBMCs with the adenovirus expressing NF-κB/SR and Ad-Tax2B (subtype Tax2B). Tax2B expressed via the recombinant adenoviral vector retained the ability to initiate viral transcription, as determined by HTLV pLTR-Luc reporter assay in Jurkat cells (data not shown) and reported to induce high levels of all three CC-chemokines in monocyte-derived macrophages (MDMs) [25]. PBMCs transduced with Ad-Tax2B produced significant levels of MIP-1α, MIP-1β and RANTES in supernatants harvested at 24 h compared to transfected Ad-GFP-PBMCs or untreated PBMC controls (P < 0·01) (Fig. 5a). The production of MIP-1α and MIP-1β was suppressed significantly after co-transducing PBMCs with NF-κB/SR and Ad-Tax2B (P < 0·01; Fig. 5b). A slight trend towards lower RANTES production was observed when PBMCs were co-transduced with NF-κB/SR and Ad-Tax2B; however, a high background limited interpretation of these results (Fig. 5b).

figure

Figure 5. Decreased CC-chemokine expression after nuclear factor kappa B (NF-κB)/super-repressor (SR) and Ad-Tax2B co-transduction in peripheral blood mononuclear cells (PBMCs). CC-chemokines were determined by enzyme-linked immunosorbent assay (ELISA)in the supernatants collected at 24 h after PBMCs were transduced with Ad-Tax2B, Ad-GFP or left untreated (a). Cell-free cultures from PBMCs co-transfected with Ad-GFP and Ad-Tax2B or NF-κB/SR and Ad-Tax2B were assayed for CC-chemokine expression by ELISA (b). Values from three independent experiments in triplicate were expressed as the mean ± standard error of the mean and statistical analyses were performed with analysis of variance (anova) and Bonferroni post-test. Significant higher levels of CC-chemokine were determined and designed as *P < 0·05, **P < 0·01 and ***P < 0·001 versus untreated or as +P < 0·05, ++P < 0·01 and +++P < 0·001 versus Ad-GFP control (a). Significant reduction of CC-chemokine production was assessed and denoted as *P < 0·05; **P < 0·01; ***P < 0·001 versus Ad-GFP + Ad-Tax2B treatment (b).

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

In this study we aimed to assess the role of Tax2-mediated activation of the NF-κB signalling pathway on the secretion of the anti-viral CC-chemokines MIP-1α, MIP-1β and RANTES. We have reported previously that HTLV-2 Tax induces the production of high levels of MIP-1α, MIP-1β and RANTES by PBMCs and MDMs [24, 25], with the concomitant down-regulation of CCR5 expression on lymphocytes [24]. These molecules are produced by activation of macrophages, dendritic cells, T cells, natural killer cells and gamma delta (γδ) T cells, and have been shown to block the CCR5 co-receptor and prevent HIV infection in vitro [26, 37] or in vivo during simian immunodeficiency virus (SIV) infection [38]. Macaques immunized with SIV were reported to have up-regulated levels of these CC-chemokines that correlated inversely with down-modulation of CCR5 [39].

Lewis et al. [40] reported the spontaneous production of MIP-1α, MIP-1β and RANTES by individuals infected with either HTLV-2 or with HIV-1 and HTLV-2. In this study the two major subtypes of HTLV-2 Tax, Tax2A and Tax2B (expressed as recombinant protein and via recombinant adenovirus, respectively) induced the production of elevated levels of MIP-1α, MIP-1β and RANTES. Our results showed a rapid expression (starting at 2 h) of these CC-chemokines by PBMCs treated with extracellular recombinant Tax2A proteins and through transduction via the Ad-Tax2B vector. The activation of canonical NF-κB pathway was observed to precede the production of CC-chemokines. PDTC and the NF-κB super-repressor, both potent inhibitors of the canonical NF-κB pathway, lessened CC-chemokine production induced by the Tax2 protein in PMBC cultures, further implicating Tax2 in the induction of CC-chemokines through the canonical NF-κB pathway in human mononuclear cells. Furthermore, the high levels of MIP-1α, MIP-1β and RANTES secreted by PBMCs after Ad-Tax2B transduction were decreased by the specific inhibition of the canonical NF-κB pathway. These data confirm that HTLV-2 Tax alone, independent of HTLV-2 infection, induces CC-chemokine expression in PMBCs, and also provide strong evidence that Tax2 may induce the activation of the canonical NF-κB pathway in human mononuclear cells as a mechanism to regulate the production of CC-chemokines.

The data presented herein do not provide evidence to suggest that extracellular activation by Tax2 protein could be via a membrane receptor interaction activating intracellular pathways and stimulating production of CC-chemokines. We have shown that HTLV-2 Tax released in the extracellular compartment are taken up by PBMCs [24]; therefore, we think that Tax2 protein, once in the cytoplasmic compartment, may interact with proteins involved in the NF-κB canonical pathway and thus induce its activation and translocation to the nucleus to induce the transcription of CC-chemokine genes.

The use of PDTC at a concentration of 30 μM to pretreat mononuclear cells during a 1-h previous Tax protein addition did not induce significant toxic effects on cell viability. Other studies have tested the toxicity of various concentrations of PDTC in cells and found that at concentrations of between 10 and 250 μM of PDTC the number of living cells remained constant for at least 12–24 h [41, 42]. PDTC agent has been applied in numerous cell types to study NF-κB-dependent events, and the results of this study using PDTC or the NF-κB super-repressor to pretreat PBMCs before Tax addition suggested that the down-regulation in the expression of CC-chemokines relates to the inhibition of the canonical NF-κB pathway. Although the findings of this study are highly suggestive that Tax2 induces MIP-1α, MIP-1β and RANTES through activation of the canonical NF-κB, these results do not exclude the possibility that other cellular signalling pathways may also contribute to the induction of the anti-viral CC-chemokine expression. While HTLV-1 Tax has been reported to transactivate a variety of cellular genes through the NF-κB pathway, including interleukin (IL)-2, IL-2Rα, granulocyte–macrophage colony-stimulating factor (GM-CSF), TGF-β, TNF-β, c-myc, vimentin, OX40L, IL-6, IL-8, IL-15 and vascular cell adhesion protein 1 (VCAM-1) [43], Tax2 has been reported to be a less potent activator of the NF-κB pathway [19]. Tax1 also activates other several major transcription factor pathways, including the cyclic-AMP response element and activating transcription factor (ATF) binding (CREB/ATF) proteins, SRF and others [44]. CREB activators function in diverse physiological processes, including the control of cellular metabolism, growth factor-dependent cell survival, and a key event of various inflammatory and growth regulatory proteins such as IL-1β, IL-6, TNF-α and GM-CSF [45]. Tax1 activates a variety of cellular genes through its interactions with CREB/ATF proteins, such as those encoding IL-17 and cfos, but less is known regarding the ability of Tax2 to regulate phosphorylation of CREB [46, 47]. Therefore, future work will focus upon investigating if Tax2 might induce CREB activation and whether this signal pathway or another may contribute to CC-chemokine production in mononuclear cells.

In this study the relative potency of the amino- and -carboxy terminal segments of Tax2A containing NF-κB binding domains [28, 29] was compared to the entire Tax2A protein. Both Tax2A/1–198 and Tax2A/135–331 fragments induced the phosphorylation of p65/RelA and stimulated CC-chemokine secretion in PBMCs. These results are important, as the entire Tax2 protein or Tax2 fragments bearing NF-κB domains may, potentially, be employed as immunomodulators to induce the production of anti-viral CC-chemokines. These results will assist future in-vitro work testing smaller Tax2-derived peptides [28, 29] that may lead eventually to immunotherapeutic studies in animal models.

The overall results of this study have important clinical implications for HIV-1/HTLV-2 co-infected individuals and have further supported our general hypothesis that Tax2 has the potential to modify innate host-immune responses and therefore alter HIV-1 pathogenesis in this co-infected population. This study identifies Tax2 protein as an immunoregulator promoting the production of anti-viral CC-chemokines mainly through activation of the canonical NF-κB signalling pathway in PBMCs.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

This work was supported by the VA Merit Review grant (BX000488-01) and the Department of Medicine of the Medical College of Wisconsin.

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  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References
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