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HMGB1-dependent triggering of HIV-1 replication and persistence in dendritic cells as a consequence of NK-DC cross-talk.

Saïdi H, Melki MT, Gougeon ML - PLoS ONE (2008)

Bottom Line: This was associated with the defective production of IL-12 and IL-18 by infected DCs.Moreover, the crosstalk between activated NK cells and HIV-infected DCs resulted in a dramatic increase in viral replication and proviral DNA expression in DCs.HMGB1, produced both by NK cells and DCs, was found to play a pivotal role in this process, and inhibition of HMGB1 activity by glycyrrhizin, known to bind specifically to HMGB1, or blocking anti-HMGB1 antibodies, abrogated NK-dependent HIV-1 replication in DCs.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Antiviral Immunity, Biotherapy and Vaccine Unit, INSERM U668, Paris, France.

ABSTRACT

Background: HIV-1 has evolved ways to exploit DCs, thereby facilitating viral dissemination and allowing evasion of antiviral immunity. Recently, the fate of DCs has been found to be extremely dependent on the interaction with autologous NK cells, but the mechanisms by which NK-DC interaction controls viral infections remain unclear. Here, we investigate the impact of NK-DC cross-talk on maturation and functions of HIV-infected immature DCs.

Methodology/principal findings: Immature DCs were derived from primary monocytes, cultured in the presence of IL-4 and GM-CSF. In some experiments, DCs were infected with R5-HIV-1(BaL) or X4-HIV-1(NDK), and viral replication, proviral HIV-DNA and the frequency of infected DCs were measured. Autologous NK cells were sorted and either kept unstimulated in the presence of suboptimal concentration of IL-2, or activated by a combination of PHA and IL-2. The impact of 24 h NK-DC cross-talk on the fate of HIV-1-infected DCs was analyzed. We report that activated NK cells were required for the induction of maturation of DCs, whether uninfected or HIV-1-infected, and this process involved HMGB1. However, the cross-talk between HIV-1-infected DCs and activated NK cells was functionally defective, as demonstrated by the strong impairment of DCs to induce Th1 polarization of naïve CD4 T cells. This was associated with the defective production of IL-12 and IL-18 by infected DCs. Moreover, the crosstalk between activated NK cells and HIV-infected DCs resulted in a dramatic increase in viral replication and proviral DNA expression in DCs. HMGB1, produced both by NK cells and DCs, was found to play a pivotal role in this process, and inhibition of HMGB1 activity by glycyrrhizin, known to bind specifically to HMGB1, or blocking anti-HMGB1 antibodies, abrogated NK-dependent HIV-1 replication in DCs.

Conclusion: These observations provide evidence for the crucial role of NK-DC cross-talk in promoting viral dissemination, and challenge the question of the in vivo involvement of HMGB1 in the triggering of HIV-1 replication and replenishment of viral reservoirs in AIDS.

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aNK-dependent maturation of HIV-1-infected iDCs is mediated by HMGB1 and involoves RAGE.(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-1BaL (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+).
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pone-0003601-g003: aNK-dependent maturation of HIV-1-infected iDCs is mediated by HMGB1 and involoves RAGE.(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-1BaL (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+).

Mentions: To determine the possible involvement of HMGB1 in NK-dependent DC maturation, we used glycyrrhizin, known to interact specifically with soluble HMGB1 molecule [32], and anti-HMGB1 antibodies (Fig. 3a). These inhibitors, added at the initiation of the 24 h aNK-iDC coculture, reduced the proportions of mature DCs (identified as CD86brightHLA-DRbright) to the baseline level observed without aNK cells (Fig. 3a). Similar effect was obtained with infected DCs (Fig. 3a). rh-HMGB1 by itself did not induce phenotypic maturation of iDC, when treated for 24 h with 1 to 10 µg/ml rh-HMGB1, and similar data were obtained at 48 h of culture (Fig. 3b). Indeed, while spontaneous maturation of iDCs was observed after 48 h of culture in medium, as shown by the high percentage of CD86bright HLA-DRbright DCs, 10 µg/ml rh-HMGB1 only weakly increased from 65% to 71% the percentage of these cells. Interestingly, rh-HMGB1-treated DCs were not fully mature, as assessed by the lack of expression of CD80, CD83 and the weak expression of DC-lamp, all fully expressed in mDC (DC0) (Fig. 3b). However, these partially mature DCs were functionally susceptible to rh-HMGB1 as shown by the increased release of the chemokines, MCP1, MIP-1α, MIP-1β and IL-8 by hr-HMGB1-treated DCs (Fig. 3c). HMGB1 receptors include RAGE [33], [34] TLR-2 and TLR-4 [35]. RAGE was the first identified receptor for HMGB1, it is expressed by a variety of immune cells including T cells, monocytes, macrophages and DCs [36], and it is used by maturing DCs for in vivo homing to lymph nodes [37]. While TLR-2 and TLR-4 were hardly detected on iDC (not shown), RAGE was fully expressed on DCs, as shown by flow-cytometry, and its expression was even higher on mature DC0 (Fig. 3d). Following incubation of iDCs with 1 µg/ml of HMGB1, down-regulation of RAGE was observed, strongly suggesting that this receptor was used by these cells (Fig. 3d). Following DC infection with HIV-1BaL, no change in RAGE levels was detected on iDC and DC0. Incubation of infected DCs with HMGB1 induced similar down-regulation of RAGE (Fig. 3d). The possible involvement of RAGE during NK-DC cross-talk was evaluated with the same approach, comparing RAGE expression on DCs cocultured with aNK cells and DC cultured alone. After 2 h of coculture with aNK cells, DCs showed an up-regulation of RAGE expression, followed by a down-regulation at 24 h (Fig. 3e). Very similar observations were made with HIV-1-infected DCs (Fig. 3e). Thus, HMGB1 is an important factor for the maturation of both uninfected and HIV-1-infected iDCs during NK-DC cross talk, and it involves RAGE, whose expression on iDC is not altered following their productive infection.


HMGB1-dependent triggering of HIV-1 replication and persistence in dendritic cells as a consequence of NK-DC cross-talk.

Saïdi H, Melki MT, Gougeon ML - PLoS ONE (2008)

aNK-dependent maturation of HIV-1-infected iDCs is mediated by HMGB1 and involoves RAGE.(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-1BaL (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+).
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pone-0003601-g003: aNK-dependent maturation of HIV-1-infected iDCs is mediated by HMGB1 and involoves RAGE.(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-1BaL (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+).
Mentions: To determine the possible involvement of HMGB1 in NK-dependent DC maturation, we used glycyrrhizin, known to interact specifically with soluble HMGB1 molecule [32], and anti-HMGB1 antibodies (Fig. 3a). These inhibitors, added at the initiation of the 24 h aNK-iDC coculture, reduced the proportions of mature DCs (identified as CD86brightHLA-DRbright) to the baseline level observed without aNK cells (Fig. 3a). Similar effect was obtained with infected DCs (Fig. 3a). rh-HMGB1 by itself did not induce phenotypic maturation of iDC, when treated for 24 h with 1 to 10 µg/ml rh-HMGB1, and similar data were obtained at 48 h of culture (Fig. 3b). Indeed, while spontaneous maturation of iDCs was observed after 48 h of culture in medium, as shown by the high percentage of CD86bright HLA-DRbright DCs, 10 µg/ml rh-HMGB1 only weakly increased from 65% to 71% the percentage of these cells. Interestingly, rh-HMGB1-treated DCs were not fully mature, as assessed by the lack of expression of CD80, CD83 and the weak expression of DC-lamp, all fully expressed in mDC (DC0) (Fig. 3b). However, these partially mature DCs were functionally susceptible to rh-HMGB1 as shown by the increased release of the chemokines, MCP1, MIP-1α, MIP-1β and IL-8 by hr-HMGB1-treated DCs (Fig. 3c). HMGB1 receptors include RAGE [33], [34] TLR-2 and TLR-4 [35]. RAGE was the first identified receptor for HMGB1, it is expressed by a variety of immune cells including T cells, monocytes, macrophages and DCs [36], and it is used by maturing DCs for in vivo homing to lymph nodes [37]. While TLR-2 and TLR-4 were hardly detected on iDC (not shown), RAGE was fully expressed on DCs, as shown by flow-cytometry, and its expression was even higher on mature DC0 (Fig. 3d). Following incubation of iDCs with 1 µg/ml of HMGB1, down-regulation of RAGE was observed, strongly suggesting that this receptor was used by these cells (Fig. 3d). Following DC infection with HIV-1BaL, no change in RAGE levels was detected on iDC and DC0. Incubation of infected DCs with HMGB1 induced similar down-regulation of RAGE (Fig. 3d). The possible involvement of RAGE during NK-DC cross-talk was evaluated with the same approach, comparing RAGE expression on DCs cocultured with aNK cells and DC cultured alone. After 2 h of coculture with aNK cells, DCs showed an up-regulation of RAGE expression, followed by a down-regulation at 24 h (Fig. 3e). Very similar observations were made with HIV-1-infected DCs (Fig. 3e). Thus, HMGB1 is an important factor for the maturation of both uninfected and HIV-1-infected iDCs during NK-DC cross talk, and it involves RAGE, whose expression on iDC is not altered following their productive infection.

Bottom Line: This was associated with the defective production of IL-12 and IL-18 by infected DCs.Moreover, the crosstalk between activated NK cells and HIV-infected DCs resulted in a dramatic increase in viral replication and proviral DNA expression in DCs.HMGB1, produced both by NK cells and DCs, was found to play a pivotal role in this process, and inhibition of HMGB1 activity by glycyrrhizin, known to bind specifically to HMGB1, or blocking anti-HMGB1 antibodies, abrogated NK-dependent HIV-1 replication in DCs.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Antiviral Immunity, Biotherapy and Vaccine Unit, INSERM U668, Paris, France.

ABSTRACT

Background: HIV-1 has evolved ways to exploit DCs, thereby facilitating viral dissemination and allowing evasion of antiviral immunity. Recently, the fate of DCs has been found to be extremely dependent on the interaction with autologous NK cells, but the mechanisms by which NK-DC interaction controls viral infections remain unclear. Here, we investigate the impact of NK-DC cross-talk on maturation and functions of HIV-infected immature DCs.

Methodology/principal findings: Immature DCs were derived from primary monocytes, cultured in the presence of IL-4 and GM-CSF. In some experiments, DCs were infected with R5-HIV-1(BaL) or X4-HIV-1(NDK), and viral replication, proviral HIV-DNA and the frequency of infected DCs were measured. Autologous NK cells were sorted and either kept unstimulated in the presence of suboptimal concentration of IL-2, or activated by a combination of PHA and IL-2. The impact of 24 h NK-DC cross-talk on the fate of HIV-1-infected DCs was analyzed. We report that activated NK cells were required for the induction of maturation of DCs, whether uninfected or HIV-1-infected, and this process involved HMGB1. However, the cross-talk between HIV-1-infected DCs and activated NK cells was functionally defective, as demonstrated by the strong impairment of DCs to induce Th1 polarization of naïve CD4 T cells. This was associated with the defective production of IL-12 and IL-18 by infected DCs. Moreover, the crosstalk between activated NK cells and HIV-infected DCs resulted in a dramatic increase in viral replication and proviral DNA expression in DCs. HMGB1, produced both by NK cells and DCs, was found to play a pivotal role in this process, and inhibition of HMGB1 activity by glycyrrhizin, known to bind specifically to HMGB1, or blocking anti-HMGB1 antibodies, abrogated NK-dependent HIV-1 replication in DCs.

Conclusion: These observations provide evidence for the crucial role of NK-DC cross-talk in promoting viral dissemination, and challenge the question of the in vivo involvement of HMGB1 in the triggering of HIV-1 replication and replenishment of viral reservoirs in AIDS.

Show MeSH
Related in: MedlinePlus