Although direct infection of DCs is less efficient than infection of CD4+
T cells 
an increasing amount of evidence indicates that long-term HIV transmission that is mediated by DCs depends on viral production by the DCs 
, and HIV-infected DCs in vivo
might function as viral reservoirs during migration to the lymphoid tissues, thereby helping to spread viral infection. Here we have shown for the first time that activated NK cells stimulate HIV-1 replication in DCs and thus might contribute to the establishment of viral reservoirs in these cells. We demonstrated the NK-cell activating capacity of HIV-1-infected iDCs and the crucial involvement of HMGB1, produced during aNK-iDC cross-talk, in the stimulation of HIV-1 replication and proviral DNA expression in DCs. We also showed a strong impairment of mature infected DCs to induce Th1 polarization following their cross-talk with NK cells. Our observations reveal novel HIV strategies to promote efficiently viral dissemination and escape the immune system.
Interaction of NK cells with autologous iDCs results in reciprocal activation, and this interaction seems crucial in the initiation/amplification of the early phases of an immune response, before T cells are generated 
. NK cells trigger iDCs to mature, and this occurs through an HMGB1-dependent mechanism 
. NK-dependent maturation of iDCs was reported to involve a functional polarization of DCs, with increase in intracellular free Ca2+
concentration, cytoskeleton rearrangement, accumulation of secretory lysosomes at the NK/DC synapse, and regulated expression of IL-18 toward the interacting NK cells. In turn, NK cells secrete large amounts of HMGB1, which induces maturation of DCs 
. Here, we confirm the involvement of HMGB1 in NK-dependent DC maturation during NK-DC contact, as shown by the inhibitory effect of anti-HMGB1 antibodies or glycyrrhizin, known to interact specifically with HMGB1 
. Confocal microscopy analyses and HMGB1 detection in cell-free culture supernatants demonstrated that HMGB1 was not only expressed and secreted by primary NK cells, as reported 
, but it was also produced by isolated DCs, the level of HMGB1 release being linked to their maturation stage. An extremely high level of HMGB1 was detected when iDCs where put in contact with aNK cells, similar to the one released by mature DCs. Interestingly, confocal microscopy analysis of NK-DC conjugates showed that both cells expressed the cytokine. HMGB1 receptor, RAGE, was found rapidly induced following DC interaction with aNK cells, and was further down-regulated, compatible with the implication of HMGB1 in NK-dependent DC maturation. In addition to contributing to DC maturation, HMGB1 has been shown to act as a chemoattractant on iDCs 
, and to be also required for migration of mature DCs in response to CCR7 and CCR4 ligands 
, both activities being mediated by RAGE 
. Thus HMGB1 acts as an alarmin, having activating and chemotactic effects on DCs, and stimulating then the migration of DCs from inflamed tissues to the draining lymph nodes 
. These properties of HMGB1 have to be taken into consideration in the context of an uncontrolled viral infection, such as that induced by HIV.
Productive infection of iDCs with R5 or X4 strains of HIV-1 preserved NK-dependent phenotypic DC maturation, as shown by the frequency of CD86bright
DCs, while HIV itself didn't induce DC maturation in the range of p24 concentrations used (0.001 to 10 ng/ml). However, the consequence of aNK-DC interaction was a significant enhancement of HIV-1 infection in iDCs. This was shown by several means, indicating an increased frequency of p24+
DC, associated with a significant enhancement of p24 release in NK-DC culture supernatant, and this was confirmed by immunofluorescence at the single cell level. Moreover, NK-DC cross-talk resulted in a dramatic increase in HIV-1 DNA expression in DCs. Considering the crucial role of HMGB1 during the reciprocal activation of DCs and NK cells, we evaluated its contribution to the triggering of HIV-1 replication in iDCs with blocking anti-HMGB1 antibodies or glycyrrhizin. The strong blocking effect of these inhibitors on p24 release indicates the involvement of HMGB1 in the process. It is noteworthy that both inhibitors also decreased significantly HIV-1 replication in 24 h cultures of infected iDCs, in the absence of NK cells. This is likely due to the spontaneous release of HMGB1 by iDC, shown here and previously reported 
, which was preserved following their infection with HIV-1. These observations reveal a pivotal role for HMGB1 in controlling HIV-1 replication in DCs. As a corollary, we demonstrated that rh-HMGB1 significantly increased p24 release in culture supernatants of infected DCs and of aNK-infected DC cocultures. These data may have important implications in the understanding of HIV pathogenesis, since plasma HMGB1 levels were found elevated in chronically HIV-1-infected patients, with the highest concentrations in patients with clinical complications 
. Moreover, exogenous HMGB1 was reported to induce in vitro
the reactivation of HIV-1 in PBMCs from HIV-1-infected patients under antiretroviral therapy 
Secreted HMGB1 is necessary for proliferation, survival, and polarization of naïve CD4 T cells after activation by allogeneic DCs, and these effects involve RAGE expressed by DCs 
. Here, we show that, in syngeneic conditions, HMGB1 was not able by itself to induce Th1 polarization. Indeed, no Th1 response was induced in the presence of HIV-1-infected DCs, though they continued to produce normal levels of HMGB1, while being inhibited in the release of IL-12 and IL-18. Recent studies highlighted the essential role of NK cells in the modulation of Th1 polarization, suggesting that they trigger IL-12 and IL-18 release by DCs, promoting the production of IFN-γ by NK cells that in turn trigger the differentiation of T cells towards Th1 cells 
. The essential role of IL-12 and IL-18 on Th1 differentiation is confirmed here, since the defect of HIV-1 infected DCs to produce increased amount of IL-12 and IL-18 in response to NK cell activation was associated with a defective Th1 polarization. This defect was directly linked to HIV-1 replication in DCs, as shown by the positive effect of the HIV inhibitor AZT. These observations suggest that some of the functional alterations reported in DCs from HIV-infected patients 
, such as a decreased secretion of several cytokines, including IL-12, and an impaired ability to prime autologous CD4 T cells, may be linked to a defective NK-DC cross-talk, as suggested recently 
. Alteration of NK
DC cross may also lead to a defect in NK-cell elimination of iDCs, as reported in chronically HIV-infected patients 
and associated with the resistance of HIV-infected DCs to apoptosis induced by NK cells (Melki MT, Saïdi H, Gougeon ML, submitted to publication).
In summary, we have shown that activation of HIV-1 replication and the possible establishment of viral reservoirs in HIV-1-infected DCs is dependent on a cross-talk between aNK cells and autologous DCs. We have identified the pivotal role of HMGB1 in this process, produced both by NK cells and DCs during their cross-talk, and showed that NK-dependent triggering of HIV replication in DCs is completely abrogated by Glycyrrhizin, which binds specifically to HMGB1, or blocking anti-HMGB1 antibodies. In addition, our data indicate a strong impairment of HIV-1-infected DCs to induce Th1 polarization following their cross-talk with NK cells. These observations provide evidence of the crucial role of NK-DC cross-talk in promoting viral dissemination, and challenges the question of the in vivo involvement of HMGB1 in the triggering of viral replication and replenishment of viral reservoirs.