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 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 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

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 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

Demographic and clinical data for the patients according to liver fibrosis.

<p><i>Abbreviations</i>: ART: antiretroviral therapy; CDC: Centers for Disease Control; HOMA: homeostasis model assessment of insulin resistance score; PI: protease inhibitors; NNRTI: non-nucleoside reverse-transcriptase inhibitors; NRTI: nucleoside reverse-transcriptase inhibitors; IQR: inter-quartile range; IVDU: intravenous drug user.</p

Factors associated with severe fibrosis (F3–F4).

<p><i>Abbreviations</i>: ART: antiretroviral therapy; CDC: Centers for Disease Control; HOMA: homeostasis model assessment of insulin resistance score; PI: protease inhibitors; NNRTI: non-nucleoside reverse-transcriptase inhibitors; NRTI: nucleoside reverse-transcriptase inhibitors; IQR: inter-quartile range; IVDU: intravenous drug user.</p

HCV RNA concentrations according to HIV-1 tropism.

<p>HCV RNA concentrations according to HIV-1 tropism.</p

Changes in liver stiffness according to HIV-1 tropism of the 34 patients in the longitudinal study (medians are indicated by bars).

<p>Changes in liver stiffness according to HIV-1 tropism of the 34 patients in the longitudinal study (medians are indicated by bars).</p

Factors associated with the presence of CXCR4-using viruses.

<p><i>Abbreviations</i>: ART: antiretroviral therapy; CDC: Centers for Disease Control; PI: protease inhibitors; NNRTI: non-nucleoside reverse-transcriptase inhibitors; NRTI: nucleoside reverse-transcriptase inhibitors; IQR: inter-quartile range; IVDU: intravenous drug user.</p