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HIV-1 Tat protein binds to TLR4-MD2 and signals to induce TNF-α and IL-10.

Ben Haij N, Leghmari K, Planès R, Thieblemont N, Bahraoui E - Retrovirology (2013)

Bottom Line: HIV-1 infection results in hyper-immune activation and immunological disorders as early as the asymptomatic stage.Here, we hypothesized that during early HIV-1 infection, HIV-1 Tat protein acts on monocytes/macrophages to induce anti-inflammatory and proinflammatory cytokines and participates in immune dysregulation.Collectively, our data showed for the first time that, HIV-1 Tat interacts physically with high affinity with TLR4-MD2 to promote proinflammatory cytokines (TNF-α) and the immunosuppressive cytokine IL-10 both involved in immune dysregulation during early HIV-1 infection and AIDS progression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Université Paul Sabatier, EA 3038, 118 Route de Narbonne, 31062 Toulouse, France. bahraoui@cict.fr.

ABSTRACT

Background: HIV-1 infection results in hyper-immune activation and immunological disorders as early as the asymptomatic stage. Here, we hypothesized that during early HIV-1 infection, HIV-1 Tat protein acts on monocytes/macrophages to induce anti-inflammatory and proinflammatory cytokines and participates in immune dysregulation.

Results: In this work we showed that Tat protein: i) by its N-terminal domain induces production of both IL-10 and TNF-α in a TLR4-MD2 dependent manner, ii) interacts specifically with TLR4-MD2 and MD2 with high affinity but not with CD14, iii) induces in vivo TNF-α and IL-10 in a TLR4 dependent manner.

Conclusions: Collectively, our data showed for the first time that, HIV-1 Tat interacts physically with high affinity with TLR4-MD2 to promote proinflammatory cytokines (TNF-α) and the immunosuppressive cytokine IL-10 both involved in immune dysregulation during early HIV-1 infection and AIDS progression.

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HIV-1 Tat-protein-induced TNF-α and IL-10 is TLR4-dependent. A-B) Monocytes were pretreated or not with increasing amounts of blocking antibodies against TLR4 or TLR2 or isotype control for 1h before stimulation by GST-Tat 1–101. C) Mab anti-TLR4 (1 μg/ml) were incubated for 1 h with 106 human monocytes before stimulation with 1, 10 or 100 nM of Tat or with, D) 100 nM of GST-Tat 1–101, GST-Tat 1–45, or GST-Tat 30–72. GST was used as a control. After 24 h, TNF-α and IL-10 in the supernatant (SN) were measured by ELISA. After 24 h, TNF-α and IL-10 were measured in the SN. The values are representative of three independent experiments. E) Monocytes were treated with LPS (1 ng/ml) or GST-Tat1-101 (10 nM) in the presence or not of LPS-RS (1 μg/ml). After 24 h, TNF-α and IL-10 were quantified in the culture supernatant by ELISA. Data represent means +/− SD.
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Figure 1: HIV-1 Tat-protein-induced TNF-α and IL-10 is TLR4-dependent. A-B) Monocytes were pretreated or not with increasing amounts of blocking antibodies against TLR4 or TLR2 or isotype control for 1h before stimulation by GST-Tat 1–101. C) Mab anti-TLR4 (1 μg/ml) were incubated for 1 h with 106 human monocytes before stimulation with 1, 10 or 100 nM of Tat or with, D) 100 nM of GST-Tat 1–101, GST-Tat 1–45, or GST-Tat 30–72. GST was used as a control. After 24 h, TNF-α and IL-10 in the supernatant (SN) were measured by ELISA. After 24 h, TNF-α and IL-10 were measured in the SN. The values are representative of three independent experiments. E) Monocytes were treated with LPS (1 ng/ml) or GST-Tat1-101 (10 nM) in the presence or not of LPS-RS (1 μg/ml). After 24 h, TNF-α and IL-10 were quantified in the culture supernatant by ELISA. Data represent means +/− SD.

Mentions: To investigate the role of TLR4-MD2 as a potential receptor implicated in the production of TNF-α and IL-10 by Tat, we evaluated the inhibitory effect of the anti-TLR4 blocking monoclonal antibody (Mab), clone HTA125, on Tat-induced cytokine production. To this end, primary human monocytes were pretreated with increasing amounts of anti-TLR4 Mab (0.01–1 μg/ml) before stimulation by Tat. In these conditions anti-TLR4 antibodies inhibited Tat-induced cytokine in a dose dependent manner (Figure 1A-B). Total inhibition was obtained with anti-TLR4 Mab at 1 μg/ml. Similarly, when monocytes were pretreated with saturating amount of anti-TLR4 antibodies (1μg/ml) and then stimulated with increasing concentrations of Tat 1–101 (1–100 nM) or its deleted mutants Tat 1–45, strong inhibition of TNF-α and IL-10 were observed (Figure 1C-D). No inhibition was observed when Tat stimulation was performed in the presence of anti-TLR2 or with irrelevant IgG antibodies harbouring the same isotype as HTA125 Mab in control experiments (Figure 1A). More interestingly, we showed that LPS-RS (from Rhodobacter Sphaeroides), previously described as a potent antagonist of LPS [33], is also able to block the ability of Tat to induce TNF-α and IL-10 production (Figure 1E). Finally, as expected, we showed that HTA125 Mab also totally inhibited LPS-induced cytokine production (Additional file 1: Figure S2).


HIV-1 Tat protein binds to TLR4-MD2 and signals to induce TNF-α and IL-10.

Ben Haij N, Leghmari K, Planès R, Thieblemont N, Bahraoui E - Retrovirology (2013)

HIV-1 Tat-protein-induced TNF-α and IL-10 is TLR4-dependent. A-B) Monocytes were pretreated or not with increasing amounts of blocking antibodies against TLR4 or TLR2 or isotype control for 1h before stimulation by GST-Tat 1–101. C) Mab anti-TLR4 (1 μg/ml) were incubated for 1 h with 106 human monocytes before stimulation with 1, 10 or 100 nM of Tat or with, D) 100 nM of GST-Tat 1–101, GST-Tat 1–45, or GST-Tat 30–72. GST was used as a control. After 24 h, TNF-α and IL-10 in the supernatant (SN) were measured by ELISA. After 24 h, TNF-α and IL-10 were measured in the SN. The values are representative of three independent experiments. E) Monocytes were treated with LPS (1 ng/ml) or GST-Tat1-101 (10 nM) in the presence or not of LPS-RS (1 μg/ml). After 24 h, TNF-α and IL-10 were quantified in the culture supernatant by ELISA. Data represent means +/− SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: HIV-1 Tat-protein-induced TNF-α and IL-10 is TLR4-dependent. A-B) Monocytes were pretreated or not with increasing amounts of blocking antibodies against TLR4 or TLR2 or isotype control for 1h before stimulation by GST-Tat 1–101. C) Mab anti-TLR4 (1 μg/ml) were incubated for 1 h with 106 human monocytes before stimulation with 1, 10 or 100 nM of Tat or with, D) 100 nM of GST-Tat 1–101, GST-Tat 1–45, or GST-Tat 30–72. GST was used as a control. After 24 h, TNF-α and IL-10 in the supernatant (SN) were measured by ELISA. After 24 h, TNF-α and IL-10 were measured in the SN. The values are representative of three independent experiments. E) Monocytes were treated with LPS (1 ng/ml) or GST-Tat1-101 (10 nM) in the presence or not of LPS-RS (1 μg/ml). After 24 h, TNF-α and IL-10 were quantified in the culture supernatant by ELISA. Data represent means +/− SD.
Mentions: To investigate the role of TLR4-MD2 as a potential receptor implicated in the production of TNF-α and IL-10 by Tat, we evaluated the inhibitory effect of the anti-TLR4 blocking monoclonal antibody (Mab), clone HTA125, on Tat-induced cytokine production. To this end, primary human monocytes were pretreated with increasing amounts of anti-TLR4 Mab (0.01–1 μg/ml) before stimulation by Tat. In these conditions anti-TLR4 antibodies inhibited Tat-induced cytokine in a dose dependent manner (Figure 1A-B). Total inhibition was obtained with anti-TLR4 Mab at 1 μg/ml. Similarly, when monocytes were pretreated with saturating amount of anti-TLR4 antibodies (1μg/ml) and then stimulated with increasing concentrations of Tat 1–101 (1–100 nM) or its deleted mutants Tat 1–45, strong inhibition of TNF-α and IL-10 were observed (Figure 1C-D). No inhibition was observed when Tat stimulation was performed in the presence of anti-TLR2 or with irrelevant IgG antibodies harbouring the same isotype as HTA125 Mab in control experiments (Figure 1A). More interestingly, we showed that LPS-RS (from Rhodobacter Sphaeroides), previously described as a potent antagonist of LPS [33], is also able to block the ability of Tat to induce TNF-α and IL-10 production (Figure 1E). Finally, as expected, we showed that HTA125 Mab also totally inhibited LPS-induced cytokine production (Additional file 1: Figure S2).

Bottom Line: HIV-1 infection results in hyper-immune activation and immunological disorders as early as the asymptomatic stage.Here, we hypothesized that during early HIV-1 infection, HIV-1 Tat protein acts on monocytes/macrophages to induce anti-inflammatory and proinflammatory cytokines and participates in immune dysregulation.Collectively, our data showed for the first time that, HIV-1 Tat interacts physically with high affinity with TLR4-MD2 to promote proinflammatory cytokines (TNF-α) and the immunosuppressive cytokine IL-10 both involved in immune dysregulation during early HIV-1 infection and AIDS progression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Université Paul Sabatier, EA 3038, 118 Route de Narbonne, 31062 Toulouse, France. bahraoui@cict.fr.

ABSTRACT

Background: HIV-1 infection results in hyper-immune activation and immunological disorders as early as the asymptomatic stage. Here, we hypothesized that during early HIV-1 infection, HIV-1 Tat protein acts on monocytes/macrophages to induce anti-inflammatory and proinflammatory cytokines and participates in immune dysregulation.

Results: In this work we showed that Tat protein: i) by its N-terminal domain induces production of both IL-10 and TNF-α in a TLR4-MD2 dependent manner, ii) interacts specifically with TLR4-MD2 and MD2 with high affinity but not with CD14, iii) induces in vivo TNF-α and IL-10 in a TLR4 dependent manner.

Conclusions: Collectively, our data showed for the first time that, HIV-1 Tat interacts physically with high affinity with TLR4-MD2 to promote proinflammatory cytokines (TNF-α) and the immunosuppressive cytokine IL-10 both involved in immune dysregulation during early HIV-1 infection and AIDS progression.

Show MeSH
Related in: MedlinePlus