Etude du rôle du TLR4 comme récepteur de la protéine Tat du VIH-1 dans l'induction de l'IL-10 et du TNF-alpha par les monocytes humains

Chez les personnes infectées par le VIH-1, on observe dès le stade asymptomatique une dérégulation du réseau de cytokines avec une forte production de cytokines proinflammatoires et immunosuppressives comme le TNF-alpha et l'IL-10. Le TNF-alpha est associé à l'hyperactivation du système immunitaire, la destruction de la barrière gastro-intestinale et la pathogénèse associée à l'infection. L'IL-10, elle, est une cytokine immunosuppressive fortement associée à la progression de la maladie vers le stade SIDA. Nous avons montré que la protéine Tat du VIH-1, par son domaine N-terminal 1-45, agit à la membrane des monocytes pour produire le TNF-a et l'IL-10 mais aussi l'IL-6, l'IL-8 et l'IFN-gamma. Au cours de ma thèse nous avons mis en évidence l'implication du " Toll Like Receptor 4 " (TLR4) dans la sécrétion de ces cytokines induite par Tat. Cette sécrétion se fait par une interaction directe, spécifique et forte du domaine 1-45 de Tat avec l'ectodomaine du TLR4-MD2 et du MD2 (K0.5 entre 4.10-9 à 10-9 M). Cette interaction a été démontrée dans des tests de fixation direct, d'immunoprecipitation, de GST-pull down et de colocalisation par microscopie confocale. Tat induit aussi l'internalisation dynamine dépendante du complexe TLR4-MD2-CD14. Cette dernière est essentielle à la signalisation et à la synthèse de cytokines induites par Tat. En effet, nous avons montré l'implication du TLR4-MD2-CD14 dans l'activation des MAP kinases et du facteur de transcription NF-kB induit par Tat pour la synthèse de cytokines. Ce travail montre aussi que Tat active les voies dépendantes de Myd88 et de TRIF du TLR4 afin d'activer les MAP kinases et NF-kB, responsables de la production de cytokines. De façon intéressante, la protéine Tat 1-45, détourne l'activation du TLR4 afin d'induire l'expression de SOCS1 et SOCS3, deux régulateurs des voies du TLR4 et des voies de l'IFN antiviral. Nos travaux ont démontré pour la première fois, le détournement d'un récepteur de l'immunité innée, le TLR4, par la protéine Tat. Ce mécanisme permettrait au VIH de déréguler le réseau de cytokines très précocement au cours de l'infection et ainsi participer à l'évolution de la maladie vers la phase SIDA. Cette étude pourrait contribuer à la compréhension des mécanismes mis en place par le VIH pour induire l'hyperactivation et l'immunosuppression du système immunitaire et permettrait aussi d'identifier de nouvelles cibles thérapeutiques.In HIV-1 infected persons, we observed since the asymptomatic phase a deregulation of the cytokine network with a high production of TNF-alpha and IL-10. The proinflammatory cytokine, TNF-alpha, is linked to the hyperactivation of the immune system, the destruction of the gastrointestinal barrier and the pathogenesis associated with HIV-1 infection. The immunosuppressive cytokine, IL-10, is strongly associated with the weakening of the immune system and the progression of the infection toward AIDS. Our group have shown that HIV-1 Tat protein, by the N-terminal domain 1-45, stimulates the production of TNF-a, IL-10, IL-6, IL-8, and IFN-alpha by monocytes. In my thesis, we demonstrated the involvement of the Toll Like Receptor 4 (TLR4), in the secretion of TNF-a, IL-6, IL-8, IFN- gamma and IL-10 by HIV-1 Tat. These cytokines are produced after a direct, specific and strong interaction of Tat 1-45 with TLR4-MD2 and MD2 (K0.5 between 4.10-9 to 10-9 M). This interaction was demonstrated by complementary approaches including direct binding assay, immunoprecipitation, GST-pull-down and colocalization by confocal microscopy. We showed that Tat 1-45 activates TLR4-MD2 dynamin endocytosis which is essential for the signalisation and cytokine synthesis induced by Tat in monocytes. Indeed, we have demonstrated that TLR4-MD2-CD14 is essential for the activation of MAPkinases and NF-kB induced by Tat in primary monocytes/macrophages. In addition, our work shows that Tat, by its 1-45 fragment, uses MyD88 and/or TRIF dependent pathways of the TLR4 to activate MAPkinases and NF-kB which are essential for cytokines production. Interestingly, Tat protein by its interaction with TLR4-MD2 induces also the expression of SOCS1 and SOCS3, two negative regulators of the TLR4 and antiviral IFN pathways. Thus, in our work we have demonstrated for the first time, the hijacking of an innate receptor: TLR4 by the viral Tat protein. Very early during infection, this interaction causes the cytokine network deregulation and contributes to the disease progression toward AIDS. This study may contribute to understand the mechanisms put in place by HIV to induce immunosuppression and also to identify new therapeutic targets for future treatments

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

International audienceBACKGROUND: 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-alpha 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-alpha 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-alpha) and the immunosuppressive cytokine IL-10 both involved in immune dysregulation during early HIV-1 infection and AIDS progression

HIV-1 Tat Protein Activates both the MyD88 and TRIF Pathways To Induce Tumor Necrosis Factor Alpha and Interleukin-10 in Human Monocytes

In this study, we show that the HIV-1 Tat protein interacts with rapid kinetics to engage the Toll-like receptor 4 (TLR4) pathway, leading to the production of proinflammatory and anti-inflammatory cytokines. The pretreatment of human monocytes with Tat protein for 10 to 30 min suffices to irreversibly engage the activation of the TLR4 pathway, leading to the production of tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10), two cytokines strongly implicated in the chronic activation and dysregulation of the immune system during HIV-1 infection. Therefore, this study analyzed whether the HIV-1 Tat protein is able to activate these two pathways separately or simultaneously. Using three complementary approaches, including mice deficient in the MyD88, TIRAP/MAL, or TRIF adaptor, biochemical analysis, and the use of specific small interfering RNAs (siRNAs), we demonstrated (i) that Tat was able to activate both the MyD88 and TRIF pathways, (ii) the capacity of Tat to induce TIRAP/MAL degradation, (iii) the crucial role of the MyD88 pathway in the production of Tat-induced TNF-α and IL-10, (iv) a reduction but not abrogation of IL-10 and TNF-α by Tat-stimulated macrophages from mice deficient in TIRAP/MAL, and (v) the crucial role of the TRIF pathway in Tat-induced IL-10 production. Further, we showed that downstream of the MyD88 and TRIF pathways, the Tat protein activated the protein kinase C (PKC) βII isoform, the mitogen-activated protein (MAP) kinases p38 and extracellular signal-regulated kinase 1/2 (ERK1/2), and NF-κB in a TLR4-dependent manner. Collectively, our data show that by recruiting the TLR4 pathway with rapid kinetics, the HIV-1 Tat protein leads to the engagement of both the MyD88 and TRIF pathways and to the activation of PKC, MAP kinase, and NF-κB signaling to induce the production of TNF-α and IL-10. IMPORTANCE In this study, we demonstrate that by recruiting the TLR4 pathway with rapid kinetics, the HIV-1 Tat protein leads to the engagement of both the MyD88 and TRIF pathways and to the activation of PKC-βII, MAP kinase, and NF-κB signaling to induce the production of TNF-α and IL-10, two cytokines strongly implicated in the chronic activation and dysregulation of the immune system during HIV-1 infection. Thus, it may be interesting to target Tat as a pathogenic factor early after HIV-1 infection. This could be achieved either by vaccination approaches including Tat as an immunogen in potential candidate vaccines or by developing molecules capable of neutralizing the effect of the Tat protein

HIV-1 Tat induces IL-6 and IL-8 production in monocytes/macrophages.

<p>(<b>A-B</b>) and <b>(C-D)</b> Human monocytes (0.5x10⁶) or <b>(E-F)</b> Wt peritoneal mice macrophages (0.5x10⁶) were incubated with increasing amounts of recombinant GST-Tat 1–101 protein (Tat), a deleted mutant GST-Tat 1–45 protein carrying the first 45 amino acid (Tat 1–45) or an equal amount of GST protein alone (GST). Untreated cells were used as negative controls. Tat heat-inactivated for 20 min at 95°C or Tat previously incubated with mAb anti-Tat for 60 min at 37°C, or GST were used for the control of the specificity. <b>(C-D)</b> Monocytes were isolated from PBMC using either adherence protocol as described in Material and Methods, or positive selection using CD14 MicroBead according to the manufacturer instruction (Miltenyi Biotec), CD14 negative fraction of PBMC was used as control. Cells were either kept untreated or treated with Synthetic Tat. After 24 h of treatment, human or mouse IL-6 and human IL-8 and mouse CXCL1/KC cytokines were measured in the culture supernatants by ELISA. Cytokine production is expressed in ng/ml. The data represent means and standard deviation (SD) of three independent experiments. Asterisks represent <i>P</i> values comparing "untreated" group versus "Treated (as indicated)" group * for p < 0.05, ** p < 0.01, *** p < 0.001, ns non significant. Statistical significance comparing different group linked with a black line above the compared bar and are denoted with # for p < 0.05, ## p < 0.01, ### p < 0.001, ns non significant.</p

HIV-1 Tat activates NF-κB pathway ina TLR4 dependent manner.

<p>(<b>A</b>) HEK null, HEK-TLR4, HEK-TLR4-CD14-MD2, (<b>B</b>) primary human monocytes, pretreated or not with anti-TLR4 (1μg/ml) or, (<b>C</b>) peritoneal macrophages from wt or TLR4 KO mice cells were stimulated during 30 or 60 minutes with total Tat protein (10 nM). Similar stimulations were also conducted with GST, or Pam3CSK or LPS TLR ligands. p65 subunit of NF-κB was detected by Western-Blot in the nucleic fraction of the cells. Quantification of the band obtained from 3 independent experiment was performed using Image J Software. (<b>D-E</b>) HEK TLR4 or HEK expressing TLR4-CD14-MD2 cell lines were co-transfected with same amounts of the NF-κB reporter plasmid together with pORF-LacZ and then stimulated with increasing amounts of GST-Tat 1–101, GST-Tat 1–45, GST-Tat 30–72 or GST alone as negative control (<b>D</b>). In (<b>E</b>), cells were also co-transfected with the indicated amounts of the plasmid encoding siRNA or Dominant Negative (control, TLR4 or TLR2). After 24h, cells were stimulated with total Tat or its deleted mutants. After 24h of stimulation, NF-κB driven SEAP-reporter gene expression was measured in the culture supernatants. For normalization, cells were lysed and expression of β-galactosidase gene was analyzed. (<b>F-H</b>) IL-6 and IL-8 cytokine production was analyzed in the culture supernatants of HEK-TLR4-MD2-CD14 cells (<b>F</b>) and human monocytes (<b>G-H</b>) previously treated with non-toxic concentrations of Bay11-7082 (0.5–10μM). Asterisks represent <i>P</i> values: *, <i>P ˂</i> 0.05; **, <i>P ˂</i> 0.01; ***, <i>P ˂</i> 0.001, ns non significant.</p

HIV-1 Tat induces IL-6 and IL-8 production in a TLR4-CD14-MD2 dependent manner.

<p>(<b>A-B</b>) Human monocytes were pretreated or not 1h, with increasing amounts of blocking antibodies against TLR4 (0.01–1μg/ml), or TLR2 (1μg/ml) before stimulation with GST-Tat 101 protein (10nM, 100nM). Culture supernatants were recovered after 24 h and IL-6 and IL-8 production was measured by ELISA. (<b>C-D</b>) Monocytes derived Dendritic Cells (MoDC) were treated with increasing amounts of GST-Tat 1–101 protein pre-incubated (light grey histograms) or not (dark grey histograms) with recombinant human TLR4/MD2 (10μg/ml) for 1h at 37°C. After 24 hrs., culture supernatants were recovered and IL-6 and IL-8 production was measured by ELISA. (<b>E-F</b>) Peritoneal macrophages from wild type (dark grey histograms) or TLR4 KO mice (light grey histograms) were stimulated for 24 h with increasing concentrations of GST-Tat 1–101 protein. Control experiments were performed by using the following TLR ligands: LPS (TLR4-CD14-MD2) and Pam3CSK4 (TLR2-CD14). Mouse IL-6 and CXCL1/KC production was determined by ELISA. The data represent means +/- SD of three independent experiments. Statistical significance comparing "untreated" group versus "Treated (as indicated)" group are denoted with * for p < 0.05, ** p < 0.01, *** p < 0.001, ns non significant. Statistical significance comparing different groups linked with a black line above the compared bar and are denoted with # for p < 0.05, ## p < 0.01, ### p < 0.001, ns non significant.</p

HIV-1 Tat induces SOCS1 expression.

<p>(<b>A</b>) Human monocytes, dendritic cells or HEK-TLR4-CD14-MD2 cells (10⁶) were stimulated or not for 24 h with Tat (100 nM) and tested for SOCS1 expression. LPS (10 ng/mL) or LPS + IFN-γ (10 ng/mL) were used as positive control. Actin was used as a loading control. (<b>B</b>) HEK-Null used as control or HEK cell line expressing TLR4-CD14-MD2 were stimulated with GST-Tat 1–45 (100 nM), GST-Tat 30–72 (100 nM) or LPS (10 ng/mL) +/- IFN-γ (10 ng/ml). The specificity of the Tat effect was tested in the presence of anti-Tat antibodies at 1 μg/ml. Stimulation with GST or with anti-Tat alone was used as controls. Quantification of the band obtained from 3 independent experiments was performed using Image J Software. Asterisks represent <i>P</i> values: *, <i>P ˂</i> 0.05; **, <i>P ˂</i> 0.01; ***, <i>P ˂</i> 0.001, ns non significant.</p

HIV-1 Tat induces production of IL-8 production in a TLR4-CD14-MD2 dependent manner.

<p>(<b>A</b>) HEK cell lines expressing TLR4 (grey histograms), TLR4-CD14-MD2 (black histograms), or HEK null, carrying an empty plasmid (white histograms) were pretreated or not with 10 and 100 nM of GST-Tat 1–101 or its deleted mutants 1–45 or 30–72. After 24 hrs., culture supernatants were recovered and IL-8 production was measured by ELISA. The data represent means +/- SD of three independent experiments. (<b>B</b>) HEK Null or HEK cell expressing TLR2-CD14 were pretreated or not with indicated amounts of GST-Tat 1–101 or LPS or Pam3CSK4. After 24h, IL-8 production in culture supernatant was analyzed by ELISA. The data represent means +/- SD of three independent experiments. (<b>C</b>) HEK-TLR4-CD14-MD2 cells were stimulated with Tat protein (Black histograms) or Tat protein previously incubated with 5 μg/mL of anti-Tat blocking antibodies (hatched histograms). After 24h of stimulation, IL-8 production induced by Tat was quantified in the culture supernatant by ELISA. The data represent means +/- SD of three independent experiments. (<b>D</b>) HEK-TLR4-CD14-MD2 cells were previously treated with increasing amounts of mAb anti-TLR4 or anti-TLR2 or isotypes control for 60 min before stimulation with Tat. After 24h, IL-8 production was measured in the culture supernatants by ELISA. The results are expressed in pg/ml. (E) HEK cell lines stably expressing TLR4/MD2-CD14 (HEK TLR4/MD2-CD14) or an empty plasmid (HEK Null) from InvivoGen were either kept untreated or treated with escalating doses of Synthetic Tat, or LPS as a positive control. After 24h of incubation, cell supernatant was collected and used for cytokine quantification by ELISA as described in Materials and Methods. The values are representative of at least three independent experiments. Statistical significance comparing "untreated" group versus "Treated (as indicated) " group are denoted with * for p < 0.05, ** p < 0.01, *** p < 0.001, ns non significant. Statistical significance comparing different group linked with a black line above the compared bar and are denoted with # for p < 0.05, ## p < 0.01, ### p < 0.001, ns non significant.</p