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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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HMGB1-dependent triggering of HIV replication in DC as a consequence of NK-DC cross talk.(a) Flow cytometry analysis of p24 intracellular expression in iDCs (CD40+), either uninfected (upper panel) or infected with HIV-1BaL (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-1BaL 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-1NDK 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.
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pone-0003601-g005: HMGB1-dependent triggering of HIV replication in DC as a consequence of NK-DC cross talk.(a) Flow cytometry analysis of p24 intracellular expression in iDCs (CD40+), either uninfected (upper panel) or infected with HIV-1BaL (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-1BaL 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-1NDK 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.

Mentions: Since we showed that the impairment of Th1 polarization by NK-sensitized HIV-1-infected DCs was dependent on HIV-1 replication (Fig. 4d), we tested whether aNK-iDC interaction could trigger HIV-1 replication in DCs. iDCs were infected for 3 h with R5- or X4-HIV-1 (1 ng/ml of p24) and further cultured either alone or in the presence of rNK or aNK for 18 h, and the frequency of DCs with intracellular expression of p24 was determined by flow cytometry. While the percentage of p24+ DCs was quite low when HIV-infected iDCs were cultured alone, it significantly increased following their interaction with aNK cells, the p24+ DCs representing almost one third of all DCs as compared to only 4% in the absence of NK cells (Fig. 5a). Under the same conditions, rNK cells had no effect on HIV replication in iDCs (Fig. 5a). aNK-dependent increased HIV replication in infected DCs was confirmed by p24 antigen detection in culture supernatants, and a statistically significant increase of p24 production was detected in cocultures of aNK with HIV-1-iDC as compared to infected iDCs cultured alone or with rNK cells (Fig. 5b). The dramatic effect of NK-DC interaction on the frequency of p24-expressing DCs was confirmed by confocal microscopy with p24-specific antibodies. While very rare DCs were stained for intracellular p24 on the day following their infection, a high number of p24+ DC were observed after their culture with aNK cells (Fig. 5c). Interestingly, the positive influence of aNK cells on HIV replication in iDCs was similarly observed on mature DCs. An increased frequency of p24+ DCs, detected by FACS, was found in HIV-1-infected DC0 cocultured during 24 h with aNK cells as compared to DC0 cultured alone (Fig. 5d), and p24 detection in culture supernatants from HIV-1-infected mature DC0, DC1 and DC2 cocultured with aNK cells, confirmed the significant stimulating effect of aNK cells on HIV-1 replication in mature DCs. This was observed whether DCs were infected with X4- or R5-HIV-1) (Fig. 5f and 5g). Of note, rNK cells had a weak impact on HIV-1 replication in mature infected-DCs (Fig. 5f and 5g). We then tested whether aNK cells had an influence on the expression of proviral DNA in iDCs. Data in Figure 5e show that a very high increase in the number of HIV-1 proviral DNA copies was detected in cultures of infected iDCs with aNK cells, as compared with that of infected iDCs with rNK cells or infected iDCs alone.


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)

HMGB1-dependent triggering of HIV replication in DC as a consequence of NK-DC cross talk.(a) Flow cytometry analysis of p24 intracellular expression in iDCs (CD40+), either uninfected (upper panel) or infected with HIV-1BaL (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-1BaL 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-1NDK 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.
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pone-0003601-g005: HMGB1-dependent triggering of HIV replication in DC as a consequence of NK-DC cross talk.(a) Flow cytometry analysis of p24 intracellular expression in iDCs (CD40+), either uninfected (upper panel) or infected with HIV-1BaL (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-1BaL 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-1NDK 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.
Mentions: Since we showed that the impairment of Th1 polarization by NK-sensitized HIV-1-infected DCs was dependent on HIV-1 replication (Fig. 4d), we tested whether aNK-iDC interaction could trigger HIV-1 replication in DCs. iDCs were infected for 3 h with R5- or X4-HIV-1 (1 ng/ml of p24) and further cultured either alone or in the presence of rNK or aNK for 18 h, and the frequency of DCs with intracellular expression of p24 was determined by flow cytometry. While the percentage of p24+ DCs was quite low when HIV-infected iDCs were cultured alone, it significantly increased following their interaction with aNK cells, the p24+ DCs representing almost one third of all DCs as compared to only 4% in the absence of NK cells (Fig. 5a). Under the same conditions, rNK cells had no effect on HIV replication in iDCs (Fig. 5a). aNK-dependent increased HIV replication in infected DCs was confirmed by p24 antigen detection in culture supernatants, and a statistically significant increase of p24 production was detected in cocultures of aNK with HIV-1-iDC as compared to infected iDCs cultured alone or with rNK cells (Fig. 5b). The dramatic effect of NK-DC interaction on the frequency of p24-expressing DCs was confirmed by confocal microscopy with p24-specific antibodies. While very rare DCs were stained for intracellular p24 on the day following their infection, a high number of p24+ DC were observed after their culture with aNK cells (Fig. 5c). Interestingly, the positive influence of aNK cells on HIV replication in iDCs was similarly observed on mature DCs. An increased frequency of p24+ DCs, detected by FACS, was found in HIV-1-infected DC0 cocultured during 24 h with aNK cells as compared to DC0 cultured alone (Fig. 5d), and p24 detection in culture supernatants from HIV-1-infected mature DC0, DC1 and DC2 cocultured with aNK cells, confirmed the significant stimulating effect of aNK cells on HIV-1 replication in mature DCs. This was observed whether DCs were infected with X4- or R5-HIV-1) (Fig. 5f and 5g). Of note, rNK cells had a weak impact on HIV-1 replication in mature infected-DCs (Fig. 5f and 5g). We then tested whether aNK cells had an influence on the expression of proviral DNA in iDCs. Data in Figure 5e show that a very high increase in the number of HIV-1 proviral DNA copies was detected in cultures of infected iDCs with aNK cells, as compared with that of infected iDCs with rNK cells or infected iDCs alone.

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