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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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Tat protein fails to stimulate TNF-α and IL-10 in macrophages from TLR4−/− MD2−/−, and CD14−/− mice. A-B) Tat induces mTNFα and mIL-10 production in wt C57Bl/6 murine macrophages. Peritoneal macrophages (5.105/well) from wild type C57BL/6 mice were stimulated for 24 h with increasing concentration of GST-Tat 1–101, GST-Tat 1–45, GST-Tat 30–72 or GST as control. LPS was used as a positive control. C-F) TLR4, CD14 and MD2 cell surface membrane proteins are essential for TNF-α and IL-10 production by Tat. Macrophages were isolated from wt mice or mice KO for TLR4, TLR2 (C-D) or CD14 or MD2 (E-F). The cells were stimulated with increasing concentrations of GST-Tat 1–101, GST-Tat 1–45 or GST as control. Positive control experiments were performed by using the following TLR ligands: LPS (TLR4-CD14-MD2) and Pam3CsK4 (TLR2-CD14). G-H) Human monocytes were pretreated with blocking antibodies, anti-CD14 or anti-MD2 (0.1 - 1 μg/ml) for 60 min. Cells were then stimulated with GST or GST-Tat 1–101 (100 nM). I-J) TNF-α and IL-10 production in peritoneal washes from wt (I) or TLR4 KO mice (J) injected with Tat protein. The data represent means +/− SD of three independent experiments.
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Figure 4: Tat protein fails to stimulate TNF-α and IL-10 in macrophages from TLR4−/− MD2−/−, and CD14−/− mice. A-B) Tat induces mTNFα and mIL-10 production in wt C57Bl/6 murine macrophages. Peritoneal macrophages (5.105/well) from wild type C57BL/6 mice were stimulated for 24 h with increasing concentration of GST-Tat 1–101, GST-Tat 1–45, GST-Tat 30–72 or GST as control. LPS was used as a positive control. C-F) TLR4, CD14 and MD2 cell surface membrane proteins are essential for TNF-α and IL-10 production by Tat. Macrophages were isolated from wt mice or mice KO for TLR4, TLR2 (C-D) or CD14 or MD2 (E-F). The cells were stimulated with increasing concentrations of GST-Tat 1–101, GST-Tat 1–45 or GST as control. Positive control experiments were performed by using the following TLR ligands: LPS (TLR4-CD14-MD2) and Pam3CsK4 (TLR2-CD14). G-H) Human monocytes were pretreated with blocking antibodies, anti-CD14 or anti-MD2 (0.1 - 1 μg/ml) for 60 min. Cells were then stimulated with GST or GST-Tat 1–101 (100 nM). I-J) TNF-α and IL-10 production in peritoneal washes from wt (I) or TLR4 KO mice (J) injected with Tat protein. The data represent means +/− SD of three independent experiments.

Mentions: To confirm the involvement of Tat-TLR4 interaction in the signalling pathways leading to cytokine production, we used genetically engineered mice deficient in various TLR or their cofactors, including MD2 and CD14. Firstly, we validated the ability of Tat protein to stimulate the production of TNF-α and IL-10 in peritoneal macrophages. Our results showed that Tat protein and its N-terminal Tat 1–45, but not Tat 30–72, stimulated specifically and in a dose dependent manner TNF-α and IL-10 production in murine wt macrophages (Figure 4A-B). In agreement with the implication of TLR4-MD2, we showed that, when murine macrophages from TLR4−/− mice were stimulated in the same conditions, no production of TNF-α and IL-10 was observed (Figure 4C-D). Similar results were obtained with macrophages from C3H/HeJ mice, which have a missense mutation in the third exon of TLR4 (Additional file 4: Figure S4). In accordance with the selective involvement of TLR4, our results showed that Tat protein and Tat 1–45, continued to stimulate TNF-α and IL-10 production in macrophages from mice deficient for TLR2−/− TLR3−/−, TLR7−/− or TLR9−/− (data not shown). As a positive control we showed that TLR2 pathway was not altered in macrophages obtained from TLR4 KO mice as shown by cytokines production following stimulation with Pam3CsK4 ligand (Figure 4C-D). Most interestingly, in vivo data, showed that intraperitoneal administration of Tat protein leads to the production of TNF-α and IL-10 in the peritoneal washes of wt mice whereas these cytokines were greatly reduced by 75% for TNF-α and remained undetectable for IL-10, in TLR4 KO mice ( Figure 4I-J).


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)

Tat protein fails to stimulate TNF-α and IL-10 in macrophages from TLR4−/− MD2−/−, and CD14−/− mice. A-B) Tat induces mTNFα and mIL-10 production in wt C57Bl/6 murine macrophages. Peritoneal macrophages (5.105/well) from wild type C57BL/6 mice were stimulated for 24 h with increasing concentration of GST-Tat 1–101, GST-Tat 1–45, GST-Tat 30–72 or GST as control. LPS was used as a positive control. C-F) TLR4, CD14 and MD2 cell surface membrane proteins are essential for TNF-α and IL-10 production by Tat. Macrophages were isolated from wt mice or mice KO for TLR4, TLR2 (C-D) or CD14 or MD2 (E-F). The cells were stimulated with increasing concentrations of GST-Tat 1–101, GST-Tat 1–45 or GST as control. Positive control experiments were performed by using the following TLR ligands: LPS (TLR4-CD14-MD2) and Pam3CsK4 (TLR2-CD14). G-H) Human monocytes were pretreated with blocking antibodies, anti-CD14 or anti-MD2 (0.1 - 1 μg/ml) for 60 min. Cells were then stimulated with GST or GST-Tat 1–101 (100 nM). I-J) TNF-α and IL-10 production in peritoneal washes from wt (I) or TLR4 KO mice (J) injected with Tat protein. The data represent means +/− SD of three independent experiments.
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Figure 4: Tat protein fails to stimulate TNF-α and IL-10 in macrophages from TLR4−/− MD2−/−, and CD14−/− mice. A-B) Tat induces mTNFα and mIL-10 production in wt C57Bl/6 murine macrophages. Peritoneal macrophages (5.105/well) from wild type C57BL/6 mice were stimulated for 24 h with increasing concentration of GST-Tat 1–101, GST-Tat 1–45, GST-Tat 30–72 or GST as control. LPS was used as a positive control. C-F) TLR4, CD14 and MD2 cell surface membrane proteins are essential for TNF-α and IL-10 production by Tat. Macrophages were isolated from wt mice or mice KO for TLR4, TLR2 (C-D) or CD14 or MD2 (E-F). The cells were stimulated with increasing concentrations of GST-Tat 1–101, GST-Tat 1–45 or GST as control. Positive control experiments were performed by using the following TLR ligands: LPS (TLR4-CD14-MD2) and Pam3CsK4 (TLR2-CD14). G-H) Human monocytes were pretreated with blocking antibodies, anti-CD14 or anti-MD2 (0.1 - 1 μg/ml) for 60 min. Cells were then stimulated with GST or GST-Tat 1–101 (100 nM). I-J) TNF-α and IL-10 production in peritoneal washes from wt (I) or TLR4 KO mice (J) injected with Tat protein. The data represent means +/− SD of three independent experiments.
Mentions: To confirm the involvement of Tat-TLR4 interaction in the signalling pathways leading to cytokine production, we used genetically engineered mice deficient in various TLR or their cofactors, including MD2 and CD14. Firstly, we validated the ability of Tat protein to stimulate the production of TNF-α and IL-10 in peritoneal macrophages. Our results showed that Tat protein and its N-terminal Tat 1–45, but not Tat 30–72, stimulated specifically and in a dose dependent manner TNF-α and IL-10 production in murine wt macrophages (Figure 4A-B). In agreement with the implication of TLR4-MD2, we showed that, when murine macrophages from TLR4−/− mice were stimulated in the same conditions, no production of TNF-α and IL-10 was observed (Figure 4C-D). Similar results were obtained with macrophages from C3H/HeJ mice, which have a missense mutation in the third exon of TLR4 (Additional file 4: Figure S4). In accordance with the selective involvement of TLR4, our results showed that Tat protein and Tat 1–45, continued to stimulate TNF-α and IL-10 production in macrophages from mice deficient for TLR2−/− TLR3−/−, TLR7−/− or TLR9−/− (data not shown). As a positive control we showed that TLR2 pathway was not altered in macrophages obtained from TLR4 KO mice as shown by cytokines production following stimulation with Pam3CsK4 ligand (Figure 4C-D). Most interestingly, in vivo data, showed that intraperitoneal administration of Tat protein leads to the production of TNF-α and IL-10 in the peritoneal washes of wt mice whereas these cytokines were greatly reduced by 75% for TNF-α and remained undetectable for IL-10, in TLR4 KO mice ( Figure 4I-J).

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