Understanding HIV-host cell interactions and defining the mechanisms of DC-mediated virus transmission are essential for developing effective strategies to combat HIV infection (54
). Here we have compared the efficiency and mechanisms of HIV transmission mediated by iDCs and mDCs by using single-cycle HIV quantification assays. We have found that mDCs significantly facilitate HIV endocytosis and efficiently concentrate HIV at virological synapses, which is likely to contribute to mDC-enhanced HIV transmission, at least in part. mDCs were more efficient than iDCs in transmitting HIV to various types of target cells independently of C-type lectins. Moreover, DC-target cell contact was required for efficient HIV-1 transmission mediated by iDCs and mDCs. These results suggest that HIV may exploit mDCs to efficiently spread viral infection in lymphoid tissues, which are the major resources for HIV replication (17
The enhanced efficiency of HIV transmission by LPS-induced mDCs has potential clinical implications for HIV pathogenesis. A recent finding indicated that significantly increased plasma LPS levels in HIV-infected humans correlate with AIDS progression and systemic immune activation. The increased plasma LPS levels may result from microbial translocation through a breach in the integrity of the mucosal barrier in the gut (7
). Indeed, LPS can induce mouse DC maturation in vivo (16
). Although the LPS concentration that we used (10 ng/ml) for in vitro DC maturation is about 50-fold higher than that found in the plasma of HIV-infected patients (7
), it is conceivable that increased LPS in HIV-infected individuals may induce DC maturation and potently stimulate HIV dissemination in vivo. In addition, HIV coinfection with other sexually transmitted pathogens can increase inflammatory stimulations at the mucosae (44
), which may directly activate DCs in vivo and promote HIV spread. Further studies using myeloid DCs, plasmacytoid DCs, or Langerhans cells from HIV-infected individuals may be required to test this hypothesis.
Our data suggest that both cell surface-bound and internalized HIV contributes to DC-mediated viral transmission. In contrast, a recent study indicates that DC-mediated HIV trans
-infection mainly derives from DC surface-bound virions (11
). Although it is difficult to directly compare these results owing to the different approaches used in the studies, the dynamic recycling of internalized HIV to DC surfaces may also mediate HIV trans
-infection, which should be an important consideration. It has been shown that HIV trafficking to the infectious synapse between LPS-induced mDCs and CD4+
T cells occurs via a tetraspanin-sorting pathway (21
). HIV is internalized into endocytic compartments in LPS-induced mDCs, which are nonconventional, nonlysosomal vesicles (21
). Upon contact with T cells, internalized HIV in mDCs redistributes to form infectious synapses (21
). Together, these results support a model in which intracellular HIV trafficking contributes to HIV transmission mediated by DCs, particularly to mDC-enhanced viral transmission. Although endocytosis of HIV by DCs may not occur at 4°C, we have observed that the invaginations of mDC plasma membranes were strongly labeled with RR at 4°C. We observed that trypsin treatment only slightly decreased mDC-bound HIV at 4°C, by 13%, which might be due to viral protection by the invaginations of mDC plasma membranes.
We found that monensin, an intracellular trafficking inhibitor, significantly blocked iDC- and mDC-mediated HIV transmission to CD4+
T cells. In addition to inhibiting vesicular transport in eukaryotic cells, monensin can also disrupt the structure of the Golgi apparatus and glycoprotein synthesis (18
). In our experiments, monensin was washed away after the 2.5-h incubation with DCs, and no significant cytotoxic effects on DCs were observed after 3 days in culture. Therefore, it is unlikely that the reduced HIV transmission by monensin was mainly due to disrupted protein synthesis, although the possibility cannot be ruled out. Monensin is used as an antiprotozoal, antibacterial, or antifungal agent and as a growth promoter in veterinary medicine (9
). It might be interesting to further explore whether monensin can be used as an antiviral agent against HIV transmission in vivo.
Previous results (34
) and the present study indicate that DC-T-cell contact is required for efficient HIV trans
-infection mediated by iDCs and mDCs. The exocytosis of HIV-associated exosomes also can play a role in iDC-mediated HIV trans
), but it may not be an efficient pathway in mDC-enhanced HIV transmission given that iDCs produce more exosomes than do mDCs (45
). Although cell-free supernatants from single-cycle HIV-pulsed mDCs were positive for HIV Gag p24, they failed to initiate HIV infection in GHOST/R5 cells or Hut/CCR5 cells (data not shown). Nevertheless, the efficiency of exosome-mediated trans
-infection by mDCs remains to be confirmed with replication-competent HIV.
Increased ICAM-1 expression on mDCs has been shown to correlate with mDC-enhanced HIV transmission (42
). This is possibly due to stronger DC-T-cell interactions through ICAM-1 binding to T-cell-expressed LFA-1 (for “leukocyte function-associated molecule 1”) (25
). Despite the lack of expression of any identified ICAM ligands, such as LFA-1, CD11b/CD18, and CD11c, GHOST/R5 cells efficiently supported mDC-enhanced HIV transmission (Fig. and data not shown). Moreover, ICAM-1 MAb blockade of DCs, GHOST/R5 cells, or both did not significantly affect HIV transmission mediated by iDCs or mDCs (data not shown). Therefore, ICAM-1 may not be the only cellular factor that contributes to mDC-enhanced efficiency of HIV trans
-infection. Cell-type-dependent HIV trafficking may play a role in mDC-enhanced viral transmission, at least in part.
Our results indicate that HIV capture by iDCs is less efficient than that by mDCs; thus, the differences in viral transmission efficiencies and virological synapses between iDCs and mDCs may only reflect the low levels of viral capture by iDCs. HIV entry in DCs can occur through endocytosis and viral receptor-mediated fusion, while productive HIV replication requires viral fusion (8
). To visualize viral interaction with DCs, high concentrations of AT-2-inactivated HIV were used in a previous study (2 to 3 μg of p24/106
) and in our electron microscopy assays (2 μg of p24/6 × 105
DCs). Given that AT-2-inactivated HIV can mediate viral fusion with cell membranes (41
), the majority of iDC-associated HIV particles may undergo fusion, uncoating, or degradation processes in iDCs or in cocultured T cells. Therefore, intact HIV particles could not be easily observed in iDC-T-cell cocultures by electron microscopy (Fig. and data not shown).
It has been shown that HIV fusion to DCs decreases as cells mature (10
). The entry of HIV into LPS-induced mDCs seemed to be primarily through endocytosis. The large intracellular compartments that confined numerous HIV particles in mDCs (Fig. ) appeared morphologically similar to macropinocytosis-mediated HIV entry in macrophages and brain microvascular endothelia (30
). Activation of DCs can trigger extensive and prolonged macropinocytic activity, enabling DCs to sample large volumes of the extracellular milieu for immune surveillance (13
). Although mDC-associated HIV was rapidly degraded, by 72%, after 1 day, about 14% and 9% of HIV p24 remained at day 2 and 3 in mDCs, respectively (Fig. ). Due to the high capacity in enhancing HIV trans
-infection by mDCs, these intracellularly retained viruses could represent an important HIV reservoir in vivo.
Cellular restriction factors that block productive HIV infection in DCs may reflect the intrinsic antiviral immunity of the antigen-presenting cells. It has been suggested that reduced viral replication in mDCs is due to a block in reverse transcription (23
), postintegration blocks at the transcriptional level (3
), and decreased viral fusion (10
). It has been recently reported that APOBEC3G and APOBEC3F (for “apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G and 3F”) mediate the postentry block to HIV replication in DCs (40
). However, when the efficiency and mechanisms of HIV infection and transmission between different subsets of DCs are compared, it is extremely important to consider different approaches to DC generation and different stimuli for DC maturation (54
). Using replication-competent and single-cycle HIV, we have found that HIV infection and transmission are functionally distinct from different subsets of mDCs induced by various stimuli (Dong et al. and L. Wu, unpublished results). Further understanding of the regulation of antiretroviral immunity in DCs may provide new insights into more effective interventions against HIV infection and dissemination mediated by DCs.