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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-DC cross-talk triggers HMGB1 expression in both aNK cells and DCs.(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-1BaL (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-1BaL (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-1BaL (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.
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pone-0003601-g002: aNK-DC cross-talk triggers HMGB1 expression in both aNK cells and DCs.(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-1BaL (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-1BaL (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-1BaL (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.

Mentions: In order to identify the molecules involved in aNK-dependent maturation of iDC, we used multianalyte profiling (MAP) to map the key cytokines produced in 24 h culture of iDC, NK cells and aNK∶iDC. iDC released low amounts of IL-1ß, IL-6 and IL-12, and they did not produce IL-10 or TNF-α. Following their coculture with aNK cells, a proinflammatory cytokine profile was induced, with a high increase in IL-12 secretion, significant levels of TNF-α and IFN-γ, both derived from NK cells, and no production of IL-10 (Fig. 2a). Interestingly, high levels of HMGB1 were detected in those culture supernatants, originating both from iDC and NK cells, and aNK∶iDC cocultures resulted in a strong enhancement of HMGB1 concentration in culture supernatants (Fig. 2a). We confirmed at the single cell level, by confocal microscopy, that NK cells were able to produce HMGB1, detected in the nucleus of freshly isolated NK cells (Fig. 2b), and further translocated to the cytoplasm in aNK cells (Fig. 2c). Following 3 h incubation with HIV-1BaL, aNK cells showed a strong decrease in HMGB1 expression, detected both in culture supernatants and by confocal microscopy (Fig. 2c), possibly due to a direct inhibitory effect of HIV virions on NK cell functions through the binding of gp120 [31]. HMGB1 level reached then a level comparable to that of rNK cells (Fig. 2a). We checked that NK cells were not able to replicate HIV-1, as shown by the lack of p24 detection in culture supernatant and the lack of intracellular p24 staining (detected by FACS) in NK cells (data not shown). HMGB1 was also secreted by iDCs and, once infected, they still produced comparable amount of the cytokine in culture supernatants (Fig. 2d). HMGB1 was mostly detected in the cytoplasm of iDCs, whether infected by HIV-1 or not (Fig. 2e), and p24 expression in infected DCs did not alter HMGB1 expression, as shown by dual intracellular staining for p24 and HMGB1 (Fig. 2e). When iDCs were cocultured with aNK cells, a strong induction of HMGB1 secretion in culture supernatants was observed (Fig. 2d), reaching levels comparable to those produced by mature DCs, i.e. DC0, DC1 and DC2 (Fig. 2f). Strikingly, HIV-1 infection of iDC did not affect the amount of HMGB1 produced in NK-DC cocultures (Fig. 2d) and in cultures of mature DCs (Fig. 2f). Confocal microscopy analysis showed the formation of conjugates between aNK cells and iDCs, which were also observed when aNK cells were cocultured with HIV-1-infected DCs, and both cells expressed HMGB1, whatever the infected status of DCs (Fig. 2g). These results demonstrate that HMGB1 is expressed both by NK cells and iDCs during NK-DC cross-talk, and this process is not altered by HIV-1 infection of iDCs.


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-DC cross-talk triggers HMGB1 expression in both aNK cells and DCs.(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-1BaL (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-1BaL (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-1BaL (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.
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pone-0003601-g002: aNK-DC cross-talk triggers HMGB1 expression in both aNK cells and DCs.(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-1BaL (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-1BaL (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-1BaL (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.
Mentions: In order to identify the molecules involved in aNK-dependent maturation of iDC, we used multianalyte profiling (MAP) to map the key cytokines produced in 24 h culture of iDC, NK cells and aNK∶iDC. iDC released low amounts of IL-1ß, IL-6 and IL-12, and they did not produce IL-10 or TNF-α. Following their coculture with aNK cells, a proinflammatory cytokine profile was induced, with a high increase in IL-12 secretion, significant levels of TNF-α and IFN-γ, both derived from NK cells, and no production of IL-10 (Fig. 2a). Interestingly, high levels of HMGB1 were detected in those culture supernatants, originating both from iDC and NK cells, and aNK∶iDC cocultures resulted in a strong enhancement of HMGB1 concentration in culture supernatants (Fig. 2a). We confirmed at the single cell level, by confocal microscopy, that NK cells were able to produce HMGB1, detected in the nucleus of freshly isolated NK cells (Fig. 2b), and further translocated to the cytoplasm in aNK cells (Fig. 2c). Following 3 h incubation with HIV-1BaL, aNK cells showed a strong decrease in HMGB1 expression, detected both in culture supernatants and by confocal microscopy (Fig. 2c), possibly due to a direct inhibitory effect of HIV virions on NK cell functions through the binding of gp120 [31]. HMGB1 level reached then a level comparable to that of rNK cells (Fig. 2a). We checked that NK cells were not able to replicate HIV-1, as shown by the lack of p24 detection in culture supernatant and the lack of intracellular p24 staining (detected by FACS) in NK cells (data not shown). HMGB1 was also secreted by iDCs and, once infected, they still produced comparable amount of the cytokine in culture supernatants (Fig. 2d). HMGB1 was mostly detected in the cytoplasm of iDCs, whether infected by HIV-1 or not (Fig. 2e), and p24 expression in infected DCs did not alter HMGB1 expression, as shown by dual intracellular staining for p24 and HMGB1 (Fig. 2e). When iDCs were cocultured with aNK cells, a strong induction of HMGB1 secretion in culture supernatants was observed (Fig. 2d), reaching levels comparable to those produced by mature DCs, i.e. DC0, DC1 and DC2 (Fig. 2f). Strikingly, HIV-1 infection of iDC did not affect the amount of HMGB1 produced in NK-DC cocultures (Fig. 2d) and in cultures of mature DCs (Fig. 2f). Confocal microscopy analysis showed the formation of conjugates between aNK cells and iDCs, which were also observed when aNK cells were cocultured with HIV-1-infected DCs, and both cells expressed HMGB1, whatever the infected status of DCs (Fig. 2g). These results demonstrate that HMGB1 is expressed both by NK cells and iDCs during NK-DC cross-talk, and this process is not altered by HIV-1 infection of iDCs.

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