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Molecular mechanisms involved in HIV-1 Tat-mediated induction of IL-6 and IL-8 in astrocytes.

Nookala AR, Kumar A - J Neuroinflammation (2014)

Bottom Line: On the other hand both PI3K/Akt and p38 MAPK (β subunit) were found to be involved in activation of NF-κB that led to IL-6 and IL-8 production.Our results demonstrate HIV-1 Tat-mediated induction of both IL-6 and IL-8 in a time-dependent manner in SVG astrocytes.These results present new therapeutic targets that could be used in management of HAND.

View Article: PubMed Central - PubMed

Affiliation: Division of Pharmacology and Toxicology, UMKC-School of Pharmacy, 2464 Charlotte Street, Kansas City, MO, 64108, USA. anfh3@mail.umkc.edu.

ABSTRACT

Background: HIV-associated neurocognitive disorders (HAND) exist in approximately 50% of infected individuals even after the introduction of highly active antiretroviral therapy. HIV-1 Tat has been implicated in HIV-associated neurotoxicity mediated through production of pro-inflammatory cytokines like IL-6 and IL-8 by astrocytes among others as well as oxidative stress. However, the underlying mechanism(s) in the up-regulation of IL-6 and IL-8 are not clearly understood. The present study was designed to determine the mechanism(s) responsible for IL-6 and IL-8 up-regulation by HIV-1 Tat.

Methods: SVG astrocytes were transiently transfected with a plasmid encoding HIV-1 Tat. The HIV-1 Tat-mediated mRNA and protein expression levels of both IL-6 and IL-8 in SVG astrocytes were quantified using real time RT-PCR and multiplex cytokine assay respectively. We also employed immunocytochemistry for staining of IL-6 and IL-8. The underlying signaling mechanism(s) were identified using pharmacological inhibitors and siRNA for different intermediate steps involved in PI3K/Akt, p38 MAPK and JNK MAPK pathways. Appropriate controls were used in the experiments and the effect of pharmacological antagonists and siRNA were observed on both mRNA expression and protein levels.

Results: Both IL-6/IL-8 mRNA and protein showed peak expressions at 6 hours and 96 hours post-transfection, respectively. Elevated levels of IL-6/IL-8 were also confirmed by immunocytochemistry. Our studies indicated that both NF-kB and AP-1 transcription factors were involved in IL-6 and IL-8 expression mediated by HIV-1 Tat; however, AP-1 was differentially activated for either cytokine. In the case of IL-6, p38δ activated AP-1 whereas JNK but not p38 MAPK was involved in AP-1 activation for IL-8 production. On the other hand both PI3K/Akt and p38 MAPK (β subunit) were found to be involved in activation of NF-κB that led to IL-6 and IL-8 production.

Conclusion: Our results demonstrate HIV-1 Tat-mediated induction of both IL-6 and IL-8 in a time-dependent manner in SVG astrocytes. Furthermore, we also showed the involvement of NF-κB and AP-1 transcription factors regulated by PI3/Akt, p38 MAPK and JNK MAPK upstream signaling molecules. These results present new therapeutic targets that could be used in management of HAND.

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HIV-1 Tat induces time dependent expression of IL-6 and IL-8 in SVG astrocytes. Seven hundred thousand SVG astrocytes were transfected with 0.3 μg of HIV-1 Tat plasmid using Lipofectamine 2000. (a, d) Cells were harvested at 1, 3, 6, 12, 24, 48 and 72 hours and total RNA was isolated. The expression levels of IL-6 and IL-8 were determined by real time RT-PCR. The values represented are normalized to their mock-transfected controls. (b, e) The concentrations of IL-6 and IL-8 in cell culture supernatants were measured at 6, 12, 24, 48, 72 and 96 hours after transfection by multiplex cytokine assay. Open bars and closed bars represent protein concentrations of mock- and HIV-1 Tat- transfected cells, respectively. Each experiment was done at least in triplicate and each bar represents the ± SE of three individual experiments. (c, f) Primary astrocytes from 2 independent donors were treated with 200 ng/ml of Tat protein for 2 hours and total RNA was isolated. The expression levels of IL-6 and IL-8 were determined by real time RT-PCR. Statistical analyses was performed by Student’s t-test and ** denotes P-value of ≤ 0.01.
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Fig1: HIV-1 Tat induces time dependent expression of IL-6 and IL-8 in SVG astrocytes. Seven hundred thousand SVG astrocytes were transfected with 0.3 μg of HIV-1 Tat plasmid using Lipofectamine 2000. (a, d) Cells were harvested at 1, 3, 6, 12, 24, 48 and 72 hours and total RNA was isolated. The expression levels of IL-6 and IL-8 were determined by real time RT-PCR. The values represented are normalized to their mock-transfected controls. (b, e) The concentrations of IL-6 and IL-8 in cell culture supernatants were measured at 6, 12, 24, 48, 72 and 96 hours after transfection by multiplex cytokine assay. Open bars and closed bars represent protein concentrations of mock- and HIV-1 Tat- transfected cells, respectively. Each experiment was done at least in triplicate and each bar represents the ± SE of three individual experiments. (c, f) Primary astrocytes from 2 independent donors were treated with 200 ng/ml of Tat protein for 2 hours and total RNA was isolated. The expression levels of IL-6 and IL-8 were determined by real time RT-PCR. Statistical analyses was performed by Student’s t-test and ** denotes P-value of ≤ 0.01.

Mentions: In the present study, we first sought to confirm the earlier finding that HIV-1 Tat up-regulates IL-6 and IL-8 expression in astrocytes. The SVG astrocytes were transfected with a plasmid encoding HIV-1 Tat and the transfection efficiency was monitored by setting a parallel transfection with a plasmid encoding green florescent protein [27]. The efficiency as measured and analyzed by flow cytometry ranged between 50 to 65% (data not shown). We first performed a time kinetics experiment to determine the peak expression levels of IL-6 and IL-8 at mRNA and protein levels. Compared to mock-transfected cells, the levels of IL-6 and IL-8 mRNA increased as early as 1 hour, peaked at 6 hours and declined steadily until 72 hours (Figure 1a, d). The peak expression levels of IL-6 and IL-8 after 6 hours of transfection was found to be 29.2 ± 2.5 and 26.2 ± 1.7 fold, respectively.Figure 1


Molecular mechanisms involved in HIV-1 Tat-mediated induction of IL-6 and IL-8 in astrocytes.

Nookala AR, Kumar A - J Neuroinflammation (2014)

HIV-1 Tat induces time dependent expression of IL-6 and IL-8 in SVG astrocytes. Seven hundred thousand SVG astrocytes were transfected with 0.3 μg of HIV-1 Tat plasmid using Lipofectamine 2000. (a, d) Cells were harvested at 1, 3, 6, 12, 24, 48 and 72 hours and total RNA was isolated. The expression levels of IL-6 and IL-8 were determined by real time RT-PCR. The values represented are normalized to their mock-transfected controls. (b, e) The concentrations of IL-6 and IL-8 in cell culture supernatants were measured at 6, 12, 24, 48, 72 and 96 hours after transfection by multiplex cytokine assay. Open bars and closed bars represent protein concentrations of mock- and HIV-1 Tat- transfected cells, respectively. Each experiment was done at least in triplicate and each bar represents the ± SE of three individual experiments. (c, f) Primary astrocytes from 2 independent donors were treated with 200 ng/ml of Tat protein for 2 hours and total RNA was isolated. The expression levels of IL-6 and IL-8 were determined by real time RT-PCR. Statistical analyses was performed by Student’s t-test and ** denotes P-value of ≤ 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4302610&req=5

Fig1: HIV-1 Tat induces time dependent expression of IL-6 and IL-8 in SVG astrocytes. Seven hundred thousand SVG astrocytes were transfected with 0.3 μg of HIV-1 Tat plasmid using Lipofectamine 2000. (a, d) Cells were harvested at 1, 3, 6, 12, 24, 48 and 72 hours and total RNA was isolated. The expression levels of IL-6 and IL-8 were determined by real time RT-PCR. The values represented are normalized to their mock-transfected controls. (b, e) The concentrations of IL-6 and IL-8 in cell culture supernatants were measured at 6, 12, 24, 48, 72 and 96 hours after transfection by multiplex cytokine assay. Open bars and closed bars represent protein concentrations of mock- and HIV-1 Tat- transfected cells, respectively. Each experiment was done at least in triplicate and each bar represents the ± SE of three individual experiments. (c, f) Primary astrocytes from 2 independent donors were treated with 200 ng/ml of Tat protein for 2 hours and total RNA was isolated. The expression levels of IL-6 and IL-8 were determined by real time RT-PCR. Statistical analyses was performed by Student’s t-test and ** denotes P-value of ≤ 0.01.
Mentions: In the present study, we first sought to confirm the earlier finding that HIV-1 Tat up-regulates IL-6 and IL-8 expression in astrocytes. The SVG astrocytes were transfected with a plasmid encoding HIV-1 Tat and the transfection efficiency was monitored by setting a parallel transfection with a plasmid encoding green florescent protein [27]. The efficiency as measured and analyzed by flow cytometry ranged between 50 to 65% (data not shown). We first performed a time kinetics experiment to determine the peak expression levels of IL-6 and IL-8 at mRNA and protein levels. Compared to mock-transfected cells, the levels of IL-6 and IL-8 mRNA increased as early as 1 hour, peaked at 6 hours and declined steadily until 72 hours (Figure 1a, d). The peak expression levels of IL-6 and IL-8 after 6 hours of transfection was found to be 29.2 ± 2.5 and 26.2 ± 1.7 fold, respectively.Figure 1

Bottom Line: On the other hand both PI3K/Akt and p38 MAPK (β subunit) were found to be involved in activation of NF-κB that led to IL-6 and IL-8 production.Our results demonstrate HIV-1 Tat-mediated induction of both IL-6 and IL-8 in a time-dependent manner in SVG astrocytes.These results present new therapeutic targets that could be used in management of HAND.

View Article: PubMed Central - PubMed

Affiliation: Division of Pharmacology and Toxicology, UMKC-School of Pharmacy, 2464 Charlotte Street, Kansas City, MO, 64108, USA. anfh3@mail.umkc.edu.

ABSTRACT

Background: HIV-associated neurocognitive disorders (HAND) exist in approximately 50% of infected individuals even after the introduction of highly active antiretroviral therapy. HIV-1 Tat has been implicated in HIV-associated neurotoxicity mediated through production of pro-inflammatory cytokines like IL-6 and IL-8 by astrocytes among others as well as oxidative stress. However, the underlying mechanism(s) in the up-regulation of IL-6 and IL-8 are not clearly understood. The present study was designed to determine the mechanism(s) responsible for IL-6 and IL-8 up-regulation by HIV-1 Tat.

Methods: SVG astrocytes were transiently transfected with a plasmid encoding HIV-1 Tat. The HIV-1 Tat-mediated mRNA and protein expression levels of both IL-6 and IL-8 in SVG astrocytes were quantified using real time RT-PCR and multiplex cytokine assay respectively. We also employed immunocytochemistry for staining of IL-6 and IL-8. The underlying signaling mechanism(s) were identified using pharmacological inhibitors and siRNA for different intermediate steps involved in PI3K/Akt, p38 MAPK and JNK MAPK pathways. Appropriate controls were used in the experiments and the effect of pharmacological antagonists and siRNA were observed on both mRNA expression and protein levels.

Results: Both IL-6/IL-8 mRNA and protein showed peak expressions at 6 hours and 96 hours post-transfection, respectively. Elevated levels of IL-6/IL-8 were also confirmed by immunocytochemistry. Our studies indicated that both NF-kB and AP-1 transcription factors were involved in IL-6 and IL-8 expression mediated by HIV-1 Tat; however, AP-1 was differentially activated for either cytokine. In the case of IL-6, p38δ activated AP-1 whereas JNK but not p38 MAPK was involved in AP-1 activation for IL-8 production. On the other hand both PI3K/Akt and p38 MAPK (β subunit) were found to be involved in activation of NF-κB that led to IL-6 and IL-8 production.

Conclusion: Our results demonstrate HIV-1 Tat-mediated induction of both IL-6 and IL-8 in a time-dependent manner in SVG astrocytes. Furthermore, we also showed the involvement of NF-κB and AP-1 transcription factors regulated by PI3/Akt, p38 MAPK and JNK MAPK upstream signaling molecules. These results present new therapeutic targets that could be used in management of HAND.

Show MeSH
Related in: MedlinePlus