The numbers of MDCs and PDCs are reduced in blood during HIV-1 infection (3
). Given that DCs are the only antigen-presenting cells with the capacity to stimulate naïve T cells, they are crucial to successful therapeutic vaccination with the induction of primary immune responses. It is therefore important to understand how HIV-1 infection affects DC numbers and function. This study characterized the infectibility and function of DCs after exposure to HIV-1. Previously, the role and function of DCs in HIV-1 infection have mainly been studied using in vitro-generated DCs (4
). However, comparative studies on autologous MDDCs and primary MDCs have shown that these myeloid cell populations do not respond in an identical manner with respect to maturation, cytokine production, and T-cell-stimulatory activity (23
). In addition, there is no in vitro system available for generating PDCs. Studies using pure preparations of isolated primary DCs to examine the effects of HIV-1 infection have been limited (17
). In this study, we isolated MDCs and PDCs from elutriated monocytes and thereby obtained relatively high numbers of DCs. Throughout the study, we used donor-matched MDCs and PDCs isolated from healthy donors. We could confirm that human PDCs and MDCs are susceptible to HIV-1 infection (15
). The presence of detectable p24+
DCs allowed us to extend prior findings with detailed studies of the effects of early HIV-1 infection on DC maturation and cytokine production.
Previous reports have described that blood-derived DCs are susceptible to HIV-1 in vitro by detecting proviral HIV-1 DNA in the DCs or monitoring the cell culture supernatant for reverse transcriptase activity or p24 content after 9 to 21 days of culture (15
). Here, we showed productive HIV-1 infection in MDCs and PDCs by applying intracellular p24 staining and flow cytometry. CD4+
T cells need stimulation to induce significant viral replication, while most reports point out that HIV-1 infection of DCs does not require prior activation of the cells (4
). Here, we found that MDCs and PDCs replicated HIV-1 and expressed significant amounts of p24 intracellularly without prior activation. Viral replication started early after HIV-1 exposure, and viral DNA transcripts could be detected as early as 3 h after exposure. HIV-1 p24+
cells appeared after 24 h of viral exposure, and the numbers increased over time. No viral replication was detected when the DCs were cultured in the presence of AZT. We found that MDCs were more effectively infected by HIV-1BaL
than donor-matched PDCs. MDCs were less susceptible to infection with HIV-1IIIB
. However, no significant difference was found in PDCs, although the expression of CCR5 was low and CXCR4 was dim/high on these cells. These differences in susceptibility to HIV-1 in PDCs and MDCs could potentially play a role in HIV-1 transmission and pathogenesis, as DCs are proposed to be important in the spread of virus to T cells. Although it is established that DCs have the capacity to bind and transfer HIV-1 to T cells in the absence of infection of the DCs (18
), transfer via productive infection of DCs may predominate in vivo. Turville and coworkers have proposed a two-phase model (42
) where early viral transfer from DCs to T cells is not dependent on infection of the DCs. However, at later time points, productive infection of the DCs is required for virus transfer to T cells to occur (42
). In that model, those authors proposed that the second phase of transfer (via productive infection of DCs) may predominate in vivo, as there is selective transmission and persistence of R5-using isolates in early HIV-1 infection. Viral transfer in the absence of infection should be nonselective for X4- and R5-using viruses, while productive infection of DCs should depend on the preferential infection of DCs with one or the other type of HIV-1 isolate. Still, the relative contribution of these different pathways in vivo remains to be determined and may not be the same in all DC subsets. As MDCs are more prevalent in mucosal tissues, they may contribute to the dominance of R5 virus isolates found in infected individuals. PDCs may primarily encounter virus that reaches lymphatic tissues and play a role in DC-mediated transfer of HIV-1 to T cells during antigen presentation at that site.
The reduction of DCs in blood in HIV-1-infected individuals can be an effect of infection and depletion of DCs. It may also be due to relocalization of DCs to secondary lymphoid tissues as a consequence of antigen uptake, migration, and/or maturation (17
). Several studies have shown that MDDCs do not alter their expression of costimulatory molecules such as CD40, CD80, CD83, CD86, or major histocompatibility complex class II after HIV-1 exposure (5
). Still, little is known about the maturation pattern after HIV-1 exposure of DCs directly isolated from blood. It has recently been shown that PDCs, but not MDCs, mature in response to HIV-1 exposure. However, the cytokines secreted by PDCs after 16 h of HIV-1 exposure induce maturation in bystander MDCs (17
). Important differences between that study and the current study are the dose of HIV-1 used to expose the DCs and the length of virus exposure. We exposed MDCs and PDCs to 1 μg p24/ml of HIV-1 for 72 h, which resulted in a p24+
population of DCs, without induction of cell death above background levels. The presence of detectable p24+
DCs allowed us to extend prior findings with detailed studies of the effects of early HIV-1 infection on DC maturation and cytokine production. However, the dose of 1 μg p24/ml also resulted in partial maturation of the MDCs and PDCs after 72 h of HIV-1 exposure. In the study performed by Fonteneau and colleagues, a lower dose, 0.3 μg p24/ml, was used to expose the DCs for 16 h, which resulted in upregulation of CD83 and CCR7 on the PDCs but not on the MDCs. Use of a similar dose of HIV-1 in our experimental system resulted in no or minimal maturation of the MDCs. Thus, the effect of HIV-1 exposure on DC maturation appears to be a consequence of virus dose. Importantly, the maturation induced after HIV-1 exposure was only partial, since the addition of R-848 to the DC cultures resulted in further upregulation of these molecules.
HIV-1-infected DCs were also able to upregulate costimulatory molecules and produce TNF-α after TLR7/8 stimulation, which may again suggest that HIV-1 infection does not induce major functional defects in DCs. Collectively, these results indicate that there is no major functional impairment in infected primary DCs early after HIV-1 infection, with respect to the parameters investigated here. In addition, we demonstrated that R-848 stimulation induced DC maturation without increased HIV-1 replication. This could indicate that the use of adjuvants that stimulate DCs via defined TLRs may be a way to augment HIV-1-specific immune responses without enhancing HIV-1 replication in DCs. However, we cannot exclude that TLR stimulation and maturation of DCs would lead to more efficient activation of CD4+ T cells and replication of HIV-1.
We previously reported a differential lack of IL-12p70 production but not TNF-α in HIV-1-infected p24+
). Here, we extended these findings by showing that HIV-1-infected primary MDCs and PDCs also produce TNF-α after stimulation. We have previously shown that R-848 stimulation induces IL-12p70 production in MDCs (28
). However, it has also been shown that blood-derived MDCs produce significantly less IL-12p70 than MDDCs in response to stimuli (23
). This could be one explanation as to why we failed to detect intracellular IL-12p70 in R-848-stimulated MDCs and PDCs (data not shown). These results show that certain functional capabilities of primary MDCs and PDCs are retained despite HIV-1 infection and that HIV-1 alone does not fully activate DCs.
Our data offer an increased understanding of the effects of early HIV-1 infection on the functional properties of blood-derived MDCs and PDCs. In conclusion, MDCs and PDCs are susceptible to HIV-1 infection but display no functional defects upon stimulation. Given the central role of DCs in generating primary immune responses, these data may help to address the issue of how to mount proper anti-HIV-1 immune responses in infected individuals.