Exogenous rh-HMGB1 triggers HIV-1 replication in iDC.

<p>(a) iDC -infected with HIV-1_BaL were cultured either alone or in the presence of aNK cells for 3 days. rh-HMGB1 (1 µg/ml) was added in some cultures. HIV replication was measured by p24 quantification in culture supernatant. (b) HIV-1-infected iDC were cultured either alone or in the presence of aNK cells for 3 days. Blocking anti-HMGB1 antibodies (10 µg/ml) or glycyrrhizin (10 µg/ml) were added at culture initiation. HIV replication was measured by p24 quantification in culture supernatant. The mean±sd of three independent experiments is shown. Statistical comparisons were made with the non-parametric Mann-Whitney test. * p<0.05.</p

Impairment of NK-triggered Th1 polarization by DCs following HIV-1 infection is associated to altered IL-12 and IL-18 production.

<p>(a) Th1 polarization by DCs triggered by NK cells was tested by incubating iDC (10⁶/ml) for 30 mn in the presence of rNK or aNK cells (2×10⁵/ml). Naïve CD4 T cells (10⁶/ml) were added to the cocultures and the frequency of T cells producing IFN-γ or IL-4 was determined by flow cytometry 8 days later. The experiment was performed with either uninfected iDCs (b) iDCs infected with HIV-1_BaL (c), or iDC infected with HIV-1_BaL in the presence of AZT (1 mM) (d). Culture supernatants of indicated cultures were tested for IL-12 (e), IL-18 (f), and IFN-g (g) content. Data represent the mean±sd of five independent experiments. Statistical comparisons were made with the non-parametric Mann-Whitney test. * p<0.05, **p = 0.03.</p

aNK-dependent maturation of HIV-1-infected iDCs is mediated by HMGB1 and involoves RAGE.

<p>(a) Left panel: iDCs were cultured for 24 h either alone or with aNK cells, in the presence of blocking anti-HMGB1 antibodies (10 µg/ml) or glycyrrhizin (10 µg/ml). The maturation status of DCs was determined by flow cytometry with CD86 and HLA-DR –specific antibodies. Right panel: same experiment, but performed with HIV-1 infected iDCs. Data represent mean±sd of at least three independent experiments, and statistical comparisons were made with the non-parametric Mann-Whitney test. * p<0.05. (b) iDC (10⁶ cells/ml) were cultured for 48 h with increasing concentrations (1–10 µg/ml) of rh-HMGB1. Cells were then stained with anti-CD86, -HLA-DR, -CD80, -CD83, DC-LAMP and -CD40 antibodies and analysed by flow cytometry. (c) Influence of rh-HMGB1 on cytokine and chemokine production (determined by MAP) by DCs. iDCs (10⁶ cells/ml) were incubated for 48 h in medium or in presence of rh-HMGB1 (1 or 10 µg/ml). As a positive control, iDCs were stimulated with LPS (DC0). (d) Flow cytometry detection of surface expression of RAGE by iDCs, DC0, or iDCs incubated with rh-HMGB1 (1 µg/ml). iDCs were either non infected or infected with HIV-1_BaL (1 ng/ml p24 for 3 h). (e) iDC, DC0, uninfected or HIV-1-infected iDC cocultured for 24 h with aNK cells, were incubated with rh-HMGB1 (1 µg/ml) and subsequently stained with anti-RAGE antibodies and analyzed by flow cytometry. NK cells were excluded from the analysis through the co-staining with CD3- and CD56-specific antibodies (CD3⁻CD56⁺).</p

aNK-DC cross-talk triggers HMGB1 expression in both aNK cells and DCs.

<p>(a) 24 h cell-free culture supernatants of iDCs, rNK cells, aNK cells (10⁶/ml), or cocultures of aNK cells and iDCs (ratio 1∶5) were tested for cytokine content. MAP technology was used to quantify IL-1β, IL-6, IL-10, TNF-α, IL-12 and IFN-γ, whereas HMGB1 was quantified by ELISA. * p<0.05 (non-parametric Mann-Whitney test). (b) HMGB1 expression was detected by immunofluorescence (in red) in freshly sorted blood NK cells. Counterstaining with DAPI (in blue) showed the nuclear localisation of HMGB1. (c) Incubation of aNK cells with HIV-1 inhibits HMGB1 secretion. Left panel: aNK cells (10⁶ cells/ml) were incubated in medium or with HIV-1_BaL (1 ng/ml of p24) for 3 h and tested for HMGB1 production 21 h later. Data represent three independent experiments and values are means±sd. Right panel: immunofluorescence analysis of HMGB1 expression in the same preparations of aNK cells. (d) HMGB1 production during aNK-iDC cross-talk is not inhibited by HIV-1 infection of iDCs. iDCs were incubated for 3 h in medium or with HIV-1_BaL (1 ng/ml of p24) and further cocultured for 21 h with aNK cells (aNK∶iDC ratio 1∶5). HMGB1 concentration was then measured in culture supernatants. Data represent the mean±sd of three independent experiments. (e) Immunofluorescence confocal analysis of HMGB1expression in uninfected or HIV-1-infected iDCs. Upper panel: non infected iDCs; middle panel: HIV-1-infected and replicating iDCs, as shown by intracellular p24 staining; lower panel: iDCs incubated with HIV-1 but negative for intracellular p24 expression. (f) Mature DCs were generated by 48 h stimulation of iDCs with LPS (DC0), soluble CD40L (DC1) or LPS+PGE2 (DC2). DC0, DC1 and DC2 were incubated for 3 h in medium or infected with HIV-1_BaL (1 ng/ml of p24) and further incubated in medium for 21 h. HMGB1 quantification in culture supernatants was performed. The mean±sd of three independent experiments is shown. (g) Immunofluorescence analysis of HMGB1 expression in conjugates of aNK cells and uninfected (upper panel) or HIV-1-infected DCs (lower panel) in a 24 h coculture. DCs are DC-SIGN⁺ and both aNK cells and DCs express HMGB1 in these conjugates. Pictures from one representative experiment out of three conducted with different primary cell preparations are shown.</p

HMGB1-dependent triggering of HIV replication in DC as a consequence of NK-DC cross talk.

<p>(a) Flow cytometry analysis of p24 intracellular expression in iDCs (CD40⁺), either uninfected (upper panel) or infected with HIV-1_BaL (lower panel) following 3 day-incubation at 10⁶/ml, either alone, or in the presence of rNK or aNK cells (2×10⁵/ml). (b) p24 concentration in culture supernatants of same cultures. Mean±sd of three independent experiments. *p<0.05, non-parametric Mann-Whitney test. (c) Immunofluorescence analysis of intracellular p24 expression in HIV-1-infected iDCs cultured for 3 days either alone or in the presence of aNK cells. Nuclei are stained with DAPI. (d) Flow cytometry intracellular p24 expression in HIV-1-infected DC0 (10⁶/ml) cultured either alone or in the presence of aNK cells for 6 days. (e) HIV-1 proviral DNA levels, determined by light cycler analysis on cells from indicated cultures. One representative experiment out of three conducted with different primary cells preparations is shown. (f) p24 concentration in culture supernatants of mature DCs infected with HIV-1_BaL and cultured for 6 days either alone or in the presence of rNK or aNK cells Mean±sd of three independent experiments. Statistical comparisons were made with the non-parametric Mann-Whitney test. * p<0.05, **p = 0.03. (g) p24 concentration in culture supernatants of either iDCs or mature DCs infected with HIV-1_NDK and cultured under the same conditions as in (f). Mean±sd of three independent experiments. Statistical comparisons were made with the non-parametric Mann-Whitney test. * p<0.05.</p

HIV-1 triggers the translocation of TRAIL from the cytoplasm to the membrane of pDCs.

<p>(A): pDCs were exposed to CpG (3 μg/ml) or increasing concentrations of HIV-1 (1 to 20 ng p24/ml) for 24 h. The expression of mTRAIL at the surface of pDCs was monitored by flow cytometry. The mean values ± SD of five independent experiments are shown (p-value ≤ 0.05 was considered significant; non-parametric Mann-Whitney test). (B): To characterize TRAIL and HMGB1 localization on pDCs exposed to HIV-1 (1ng/ml p24) or CpG (3 μg/ml), images were analysed using 3D interactive surface plot analysis. Deconvolution overlays of representative 2D red cross axis XY focal plan with XZ/YZ view of focal plane and projection overlay of cell stainings (analyzed by Metamorph software) are shown. (C): Flow cytometry analysis of mTRAIL expression and HMGB1 intracellular staining of pDCs cultured in medium, exposed to HIV-1 (1 ng p24/ml) or stimulated with CpG (3 μg/ml). Dot plots from one representative experiment out of three independent experiments are shown.</p

Triggering by HIV-1 of HMGB1/RAGE autocrine loop during NK-pDC interaction.

<p>(A): Cytokines and chemokines content were quantified by MAP technology in 24 h cell-free culture supernatants of NK-pDCs co-cultures after exposure of pDCs to HIV-1 (20 ng p24/ml). The IFN-α inhibitor CB (1μM) was added at the initiation of the coculture. The mean values ± SD from three independent experiments are shown. The asterisks indicate statistically significant values (p <0.05) (non-parametric Wilcoxon test). (B): IL-12p70, IL-12p40, IFN-α and IFN-γ were quantified by MAP technology in 24 h cell-free culture supernatants of pDCs stimulated with control DNA, CpG (A) or CpG (B) (3μg/ml) in the absence or presence of two concentrations of recombinant HMGB1 (1 and 2.5 μg/ml). (C): pDCs exposed or not to HIV-1 (20 ng p24/ml) were cultured for 24 h with aNK cells, and a number of inhibitors were added at the initiation of the coculture: glycyrrhizin at 10μg/ml, N-ethyl pyruvate at 10μM, anti-RAGE antibodies at 10 μg/ml, the IFN-α inhibitor CB at 1μM, and anti-IFN-α antibodies IFN-α at 10 μg/ml. IFN-α was quantified by specific ELISA in 24 h cell-free culture supernatants. Histograms show the mean values ± SD (triplicates) from one representative experiment, out of three independent experiments. (D): A schematic representation illustrating the requirement for HMGB1/RAGE autocrine loop for triggering NK-mediated activation of pDCs and IFN-α production in the context of HIV-1 infection.</p

HIV-1 mediates phenotypic maturation of pDCs in a dose-dependent manner.

<p>(A): Phenotypic characterization of freshly sorted pDCs. The expression of HIV-1- receptor CD4, co-receptors CCR5 and CXCR4, maturation markers CD40, CD80, CD86, CD83, HLA-DR and chemokine-receptor CCR7 were analyzed by flow cytometry on gated CD123⁺CD303⁺ pDCs. (B): Sorted pDCs were either incubated with medium (NS) or stimulated with CpG (3μg/ml) for 24 hours and the expression of the indicated markers was analyzed. (C): pDCs were exposed to indicated concentrations of HIV-1 and the expression of the indicated markers was analyzed. The percentage of cells expressing the indicated marker and mean fluorescence intensity (in brackets) are indicated. (D): HIV-1-induced phenotypic maturation of pDCs was demonstrated following the identification of mature cells, i.e. CD123^brightHLA-DR^brightFSC^high cells. These results are representative of at least three different experiments conducted with primary cells from distinct donors.</p

HIV-1 triggers mTRAIL expression on both pDCs and NK cells during NK-pDC crosstalk.

<p>(A): pDCs were exposed or not to HIV-1 (1 ng/ml or 20 ng/ml p24) and further cultured for 24 h in the presence or not of aNK cells. Stimulation with CpG (3 μg/ml) was used as positive control. mTRAIL expression at the surface of pDCs was monitored by flow cytometry. (B): Resting NK cells (rNK cells) were cultured alone or with pDCs after their exposure or not to HIV-1 (1 ng/ml or 20 ng/ml). mTRAIL expression at the surface of NK cells was assessed by flow cytometry. The mean values ± SD of seven independent experiments are shown (non-parametric Mann-Whitney test). (C) Resting NK cells (rNK cells) were cultured alone or with pDCs after their exposure or not to HIV-1 (1 ng/ml or 20 ng/ml). The expression of the cytotoxic marker CD107 or the activation marker CD69 was monitored by flow cytometry. The mean values ± SD of seven independent experiments are shown (non-parametric Mann-Whitney test).</p

Impact of aNK cells on the proinflammatory array of cytokines and chemokines released by pDCs in the context of HIV-1 infection.

<p>Cytokines (A, B) and chemokines (C, D) content were quantified by MAP technology in 24 hour cell-free culture supernatants of pDCs exposed to low (1 ng p24/ml) or high (20 ng p24/ml) concentration of HIV-1, and further cocultured or not with aNK cells. Heat-maps (A, C) from one representative experiment out of four conducted with different primary cell preparations are shown. Mean values ± SD for indicated cytokines and chemokines from six independent experiments are shown (B, D) (non-parametric Wilcoxon test).</p