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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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Related in: MedlinePlus

aNK cells induce the maturation of primary immature HIV-1-infected DCs.(a) iDCs, generated from purified CD14+ monocytes in the presence of IL-4 and GM-CSF, were cocultured during 24 h with aNK cells at different ratios. DC survival was determined by flow cytometry with the 7-AAD assay. Surviving DCs were identified as 7AAD− CD56− cells. Data represent three independent experiments and values are means±sd. (b) aNK cells induce the maturation of iDCs. Flow cytometry analysis of iDCs, which were either infected with HIV-1BaL (1 ng/ml of p24) for 3 h or uninfected, were incubated with rNK cells or aNK cells at a ratio of 1∶5. Co-staining with HLA-DR and CD86 specific antibodies allowed the identification of mature DCs (CD86brightHLA-DRbright). Data from a representative experiment out of three independent experiments are shown. (c) The conditions of infection used in this study were those of a productive infection of iDCs, as shown at day 3 by a significant p24 detection in culture supernatant of infected iDCs and intracellular detection by flow cytometry of p24 in DC targeted by CD40 expression. Experiments were performed on DCs from three independent donors, and values are means±sd. (d) HIV-1 infection does not induce by itself the maturation of iDC, as shown by CD86/HLA-DR dual staining of iDCs infected with 0.001 to 10 ng/ml p24 HIV-1BaL. The proportion of mDCs induced by LPS (DC0) (78.1% CD86brightHLA-DRbright) is shown as a positive control. (e) The proportion of mature CD86brightHLA-DRbright DCs induced in the indicated cocultures of infected or uninfected iDCs with either rNK or aNK cells are shown. These experiments have been performed on primary cells from a number of donors, and representative data from three of them are shown. When indicated, statistical analyses were made with the non-parametric Mann-Whitney test. * p<0.05, ** p = 0.02.
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pone-0003601-g001: aNK cells induce the maturation of primary immature HIV-1-infected DCs.(a) iDCs, generated from purified CD14+ monocytes in the presence of IL-4 and GM-CSF, were cocultured during 24 h with aNK cells at different ratios. DC survival was determined by flow cytometry with the 7-AAD assay. Surviving DCs were identified as 7AAD− CD56− cells. Data represent three independent experiments and values are means±sd. (b) aNK cells induce the maturation of iDCs. Flow cytometry analysis of iDCs, which were either infected with HIV-1BaL (1 ng/ml of p24) for 3 h or uninfected, were incubated with rNK cells or aNK cells at a ratio of 1∶5. Co-staining with HLA-DR and CD86 specific antibodies allowed the identification of mature DCs (CD86brightHLA-DRbright). Data from a representative experiment out of three independent experiments are shown. (c) The conditions of infection used in this study were those of a productive infection of iDCs, as shown at day 3 by a significant p24 detection in culture supernatant of infected iDCs and intracellular detection by flow cytometry of p24 in DC targeted by CD40 expression. Experiments were performed on DCs from three independent donors, and values are means±sd. (d) HIV-1 infection does not induce by itself the maturation of iDC, as shown by CD86/HLA-DR dual staining of iDCs infected with 0.001 to 10 ng/ml p24 HIV-1BaL. The proportion of mDCs induced by LPS (DC0) (78.1% CD86brightHLA-DRbright) is shown as a positive control. (e) The proportion of mature CD86brightHLA-DRbright DCs induced in the indicated cocultures of infected or uninfected iDCs with either rNK or aNK cells are shown. These experiments have been performed on primary cells from a number of donors, and representative data from three of them are shown. When indicated, statistical analyses were made with the non-parametric Mann-Whitney test. * p<0.05, ** p = 0.02.

Mentions: To investigate the role of NK cells on DC maturation, we generated monocyte-derived DCs from isolated monocytes and cocultured them with NK cells purified from the same donor. NK cells were either resting (rNK) or activated by a combination of PHA and IL-2 (aNK). 24 h of coculture of aNK cells with autologous immature DC (iDC) induced either the survival or apoptosis of iDCs, dependent on NK-DC ratio, consistent with previous reports [14]. Indeed, aNK-DC ratio of 5∶1 induced DC apoptosis, while 1∶5 ratio induced DC survival. (Fig. 1a). iDCs survival at a NK-DC ratio of 1∶5 was associated with their maturation, as shown by the increased coexpression of the maturation markers CD86 and HLA-DR (72.1% of CD86brightHLA-DRbright DCs were induced by aNK cells compared to 15.3% at baseline) (Fig. 1b), a feature of mature DCs. Under the same experimental conditions, rNK cells had a weaker effect on DC maturation, as judged by the proportion of CD86brightHLA-DRbright DCs (Fig. 1b,e). Following infection of iDC with HIV-1BaL, NK-dependent maturation of iDCs was not altered (Fig. 1b), under conditions of productive infection of iDCs, measured at day 3 by p24 release in culture supernatant and intracellular staining of iDC for p24 (Fig. 1c). Investigating the direct effect of HIV-1 on DC maturation, we found that, at concentrations ranging from 0.001 to 10 ng/ml, HIV-1BaL was unable to increase the expression of the maturation markers CD86 and HLA-DR, in contrast to LPS, used as a positive control as a strong inducer of DC maturation (Fig. 1b,d). Data from three representative donors, shown in Fig. 1e, confirm the high impact of aNK cells on maturation of iDC after 24 h of coculture, whatever the infected or uninfected status of iDC. These experiments have all been reproduced with X4-HIV-1 and very similar data were obtained (data not shown). From these data, we conclude that productively HIV-1-infected iDCs have maintained a normal susceptibility to maturation induced by NK cells during the NK-DC cross-talk.


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 cells induce the maturation of primary immature HIV-1-infected DCs.(a) iDCs, generated from purified CD14+ monocytes in the presence of IL-4 and GM-CSF, were cocultured during 24 h with aNK cells at different ratios. DC survival was determined by flow cytometry with the 7-AAD assay. Surviving DCs were identified as 7AAD− CD56− cells. Data represent three independent experiments and values are means±sd. (b) aNK cells induce the maturation of iDCs. Flow cytometry analysis of iDCs, which were either infected with HIV-1BaL (1 ng/ml of p24) for 3 h or uninfected, were incubated with rNK cells or aNK cells at a ratio of 1∶5. Co-staining with HLA-DR and CD86 specific antibodies allowed the identification of mature DCs (CD86brightHLA-DRbright). Data from a representative experiment out of three independent experiments are shown. (c) The conditions of infection used in this study were those of a productive infection of iDCs, as shown at day 3 by a significant p24 detection in culture supernatant of infected iDCs and intracellular detection by flow cytometry of p24 in DC targeted by CD40 expression. Experiments were performed on DCs from three independent donors, and values are means±sd. (d) HIV-1 infection does not induce by itself the maturation of iDC, as shown by CD86/HLA-DR dual staining of iDCs infected with 0.001 to 10 ng/ml p24 HIV-1BaL. The proportion of mDCs induced by LPS (DC0) (78.1% CD86brightHLA-DRbright) is shown as a positive control. (e) The proportion of mature CD86brightHLA-DRbright DCs induced in the indicated cocultures of infected or uninfected iDCs with either rNK or aNK cells are shown. These experiments have been performed on primary cells from a number of donors, and representative data from three of them are shown. When indicated, statistical analyses were made with the non-parametric Mann-Whitney test. * p<0.05, ** p = 0.02.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2571988&req=5

pone-0003601-g001: aNK cells induce the maturation of primary immature HIV-1-infected DCs.(a) iDCs, generated from purified CD14+ monocytes in the presence of IL-4 and GM-CSF, were cocultured during 24 h with aNK cells at different ratios. DC survival was determined by flow cytometry with the 7-AAD assay. Surviving DCs were identified as 7AAD− CD56− cells. Data represent three independent experiments and values are means±sd. (b) aNK cells induce the maturation of iDCs. Flow cytometry analysis of iDCs, which were either infected with HIV-1BaL (1 ng/ml of p24) for 3 h or uninfected, were incubated with rNK cells or aNK cells at a ratio of 1∶5. Co-staining with HLA-DR and CD86 specific antibodies allowed the identification of mature DCs (CD86brightHLA-DRbright). Data from a representative experiment out of three independent experiments are shown. (c) The conditions of infection used in this study were those of a productive infection of iDCs, as shown at day 3 by a significant p24 detection in culture supernatant of infected iDCs and intracellular detection by flow cytometry of p24 in DC targeted by CD40 expression. Experiments were performed on DCs from three independent donors, and values are means±sd. (d) HIV-1 infection does not induce by itself the maturation of iDC, as shown by CD86/HLA-DR dual staining of iDCs infected with 0.001 to 10 ng/ml p24 HIV-1BaL. The proportion of mDCs induced by LPS (DC0) (78.1% CD86brightHLA-DRbright) is shown as a positive control. (e) The proportion of mature CD86brightHLA-DRbright DCs induced in the indicated cocultures of infected or uninfected iDCs with either rNK or aNK cells are shown. These experiments have been performed on primary cells from a number of donors, and representative data from three of them are shown. When indicated, statistical analyses were made with the non-parametric Mann-Whitney test. * p<0.05, ** p = 0.02.
Mentions: To investigate the role of NK cells on DC maturation, we generated monocyte-derived DCs from isolated monocytes and cocultured them with NK cells purified from the same donor. NK cells were either resting (rNK) or activated by a combination of PHA and IL-2 (aNK). 24 h of coculture of aNK cells with autologous immature DC (iDC) induced either the survival or apoptosis of iDCs, dependent on NK-DC ratio, consistent with previous reports [14]. Indeed, aNK-DC ratio of 5∶1 induced DC apoptosis, while 1∶5 ratio induced DC survival. (Fig. 1a). iDCs survival at a NK-DC ratio of 1∶5 was associated with their maturation, as shown by the increased coexpression of the maturation markers CD86 and HLA-DR (72.1% of CD86brightHLA-DRbright DCs were induced by aNK cells compared to 15.3% at baseline) (Fig. 1b), a feature of mature DCs. Under the same experimental conditions, rNK cells had a weaker effect on DC maturation, as judged by the proportion of CD86brightHLA-DRbright DCs (Fig. 1b,e). Following infection of iDC with HIV-1BaL, NK-dependent maturation of iDCs was not altered (Fig. 1b), under conditions of productive infection of iDCs, measured at day 3 by p24 release in culture supernatant and intracellular staining of iDC for p24 (Fig. 1c). Investigating the direct effect of HIV-1 on DC maturation, we found that, at concentrations ranging from 0.001 to 10 ng/ml, HIV-1BaL was unable to increase the expression of the maturation markers CD86 and HLA-DR, in contrast to LPS, used as a positive control as a strong inducer of DC maturation (Fig. 1b,d). Data from three representative donors, shown in Fig. 1e, confirm the high impact of aNK cells on maturation of iDC after 24 h of coculture, whatever the infected or uninfected status of iDC. These experiments have all been reproduced with X4-HIV-1 and very similar data were obtained (data not shown). From these data, we conclude that productively HIV-1-infected iDCs have maintained a normal susceptibility to maturation induced by NK cells during the NK-DC cross-talk.

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