HIV infection is characterized by a slow and continuous depletion of CD4+ T cells and severe immunodeficiency symptoms with disease progression.1,2 At later stages of HIV disease, due to a weakened immune system, seropositive individuals develop opportunistic infections normally harmless in healthy individuals. Among the infections observed in patients with AIDS, oral candidiasis and candidemia caused by the Candida family, particularly Candida albicans (Candida), are the most frequent.3-6 In the absence of antiviral treatment, 90% of HIV-infected persons will develop at least one episode of candidiasis with disease progression.3,7 Although susceptibility to candidiasis can be determined by defective T-cell-mediated immune responses,8,9 other immune defects, in particular impairment of dendritic cell (DC) functions, may contribute to the loss of control of this commensal organism.10,11 DCs are present as sentinels throughout the body, and they play an important role in processing and presenting pathogens captured in periphery to initiate immune responses.12,13 HIV targets DCs in vitro14-17 and in vivo.10,18,19 DCs can entrap and internalize HIV very efficiently,15,20-22 but the frequency of HIV-infected DCs is generally lower than what is observed in T cells.23-25 DCs also drive virus amplification in T cells by transferring captured or newly produced virions,15,23,26-28 underscoring the importance of DCs in augmenting HIV dissemination during acute and chronic infection.
Previous studies have emphasized defects observed in DCs after HIV infection.10,11,29,30 DCs purified from blood of HIV-infected donors showed impaired capacity to stimulate allogeneic T-cell proliferation.10,31 Exposure of immature DCs to various HIV proteins (gp120, Nef, and Tat) has also been shown to induce changes in DCs, altering their responsiveness to stimuli and rendering them less able to induce potent T-cell responses.32-35 Dysfunctional DCs, generated after HIV exposure, likely induce inappropriate immune responses and contribute to the loss of control of HIV and of other pathogens.
In an attempt to assess how DC responses to a common commensal organism might influence the fate of HIV and vice versa, we studied the impact of Candida on immature monocyte-derived DCs that had captured or were infected with HIV. We provide the first evidence that HIV takes advantage of the normal DC-driven responses to Candida to further amplify HIV spread, potentially destroying the T cells needed to control Candida. These observations have important implications for the immunopathogenesis of HIV infection and identify another way that HIV capitalizes on DC biology to target other pathogen-specific T cells.
MATERIALS AND METHODS
Cell Isolation and DC Generation
Peripheral blood mononuclear cells were isolated from HIV-seronegative leukopacks (NY City Blood Center), and CD14+ monocytes were purified using the CD14 magnetic cell sorting system (Miltenyi Biotec, Auburn, CA). Immature monocyte-derived DCs were generated in complete medium (RPMI 1640 Cellgro; Fisher Scientific, Springfield, NJ) with 1% human plasma (Innovative research, Southfield, MI) containing 100 U/mL of interleukin-4 (R&D Systems, Minneapolis, MN) and 1000 U/mL of granulocyte-macrophage colony stimulating factor (GM-CSF) (Berlex Laboratories, Montville, NJ) as previously described.20 The CD14− fractions were cultured alongside in complete medium at 107 cells per milliliter. Rare DC preparations did not respond to Candida, and these donors have been excluded from our analyses. CD14− cells were depleted of CD11b+ and human leukocyte antigen type-DR+ cells by magnetic cell sorting system (Miltenyi) to provide enriched T-cell populations. The purity and phenotype of each T cell and DC preparation were determined by fluorescence-activated cell sorter (FACS) analysis. T cells were stained with fluorescein iothiocyanate-conjugated anti-CD3 (BD/Pharmingen, San Jose, CA) vs phycoerythrin(PE)-conjugated anti-HLA-DR (BD/Pharmingen) and -CD11b (Exalpha, Boston, MA). DCs were stained using FITC-conjugated anti-HLA-DR combined with PE-conjugated anti-CD25, -CD80, -CD86 (BD/Pharmingen), -CD83 (Immunotech-Beckman-Coulter, Marseille, France), or -CD209 (R&D Systems). Samples were acquired on a FACScalibur flow cytometer (BD/Pharmingen), and data were analyzed using FlowJo software (Tree Star, Ashland, OR).
DC Loading and Infection With HIV
HIVBaL (HIV, lots P3953 and P4143) and the no virus microvesicle (MV) control (prepared from the cells used to generate the HIV stocks, lot P3826) were provided by the AIDS Vaccine Program, Science Applications International Corporation-Frederick (National Cancer Institute at Frederick, MD). All stocks were sucrose density gradient purified.36 Infectious titers were verified by titration on TZM.bl cells.14 DCs were pulsed with HIV (5.4 × 104 TCID50/106 DCs) or the MV control (normalized to the equivalent amount of total protein in the HIV pulse) for 1 hour at 37°C in a 15-mL conical tube (adapted from Frank et al 20). After 1 hour, cells were washed 3 times with Dulbecco's Phosphate Buffered Saline (Cellgro). DCs were then used immediately or after 48 hours of re-culture in GM-CSF and IL-4 (2 × 106 DCs/well, 6-well plate). DCs cultured for the additional 48 hours to allow HIV infection were collected and washed before exposure to Candida. Virus capture (at 0 hour) was verified for each experiment by flow cytometry. Cells were fixed with Cytofix/Cytoperm (BD Bioscience) according to the manufacturer's protocol and stained with rhodamide-labeled anti-p24 (KC57; Beckman Coulter, San Diego, CA). Aliquots of DCs were also monitored for the level of HIV infection after 5 days of culture in GM-CSF/IL-4. Quantitative polymerase chain reaction (QPCR) for HIV gag DNA was performed with the primers and methods previously published1 using an ABIprism 7000 real-time polymerase chain reaction instrument (PerkinElmer Life and Analytical Sciences, Boston, MA).
Candida albicans and DC Exposure
Candida albicans (strain SC5314, obtained from the American Type Culture Collection) was maintained at room temperature on yeast-peptone-dextrose agar plates (Sigma Chemical Company, St Louis, MO), and Candida blastoconidia were amplified in Sabouraud dextrose broth (Sigma) overnight at 30°C. Viable blastoconidia were counted by trypan blue exclusion. For FITC labeling, 5 × 107 blastoconidia were resuspended in 1 mL of 50 mM carbonate/bicarbonate buffer pH 9.5 along with 0.1 mg/mL FITC solution (Sigma). Cells were incubated at room temperature for 1.5 hours in the dark and washed 3 times in carbonate/bicarbonate buffer before recounting. DCs (5 × 105) were cocultured with Candida (1:1 ratio) in 48-well plate in 250 μL of complete media containing GM-CSF/IL-4. Amphotericin B (5 μg/mL, Sigma) was added in all conditions to limit Candida overgrowth. Initial studies comparing untreated DCs with MV-loaded DCs confirmed that there were negligible responses to the MVs and no impact of MV on DC responses to Candida (data not shown), allowing the direct comparison of MV to HIV-loaded cells for the extensive studies.
Luminex Analysis of Cytokines and Chemokines
Cytokines and chemokines were detected in 50 μL of cell-free supernatants using a Beadlyte 24-Plex Detection System according to the manufacturer's instructions (Upstate, Lake Placid, NY). The kit detects the following cytokines/chemokines: IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, interferon-α, interferon-γ, GM-CSF, tumor necrosis factor-α, CXCL8, CXCL10, CCL2, CCL3, CCL4, CCL5, and CCL11. The plates were washed between steps using a MultiScreen Vacuum Manifold (Millipore, Billerica, MA) and read on a Luminex 100 instrument (Luminex Corporation, Austin, TX). Data were analyzed using the STarStation software (Applied Cytometry System, Inc, Sacramento, CA).
Decreasing numbers of DCs were mixed with 2 × 105 autologous T cells (in triplicate) in 96-well flat-bottomed plates (200 μL/well) with or without Candida in complete medium containing 10 U/mL of IL-4 and 100 U/mL of GM-CSF. Controls were DCs (highest dose) and T cells cultured alone (±Candida). AZT (10 μM) was added to limit HIV amplification in the cocultures. Plates were incubated for 3-5 days and then pulsed with tritiated thymidine (3H-TdR; 1 μCi/well) for 8 hours before being harvested using a cell harvester (Brander, Gaithesburg, MD). The amount of 3H-TdR incorporated by the cells was measured using a liquid scintillation counter, 1450 microbeta Wallac jet (PerkinElmer). Alternatively, proliferation was monitored by carboxyfluorescein diacetate succinimidyl ester (CFSE) staining.37 Autologous enriched T cells were loaded with CFSE (2 μM/mL) according to the manufacturer's protocol (Invitrogen, Carlsbad, CA) and cocultured with DCs at the 1:10 ratio as described above (±AZT). After 5 days, cells were stained with PE-conjugated anti-CD8, PerCY-conjugated anti-CD4, or allophycocyanin-conjugated anti-CD25 (BD/Pharmingen). The divided CFSElow cells were enumerated and characterized.
Regulatory T-Cell Staining
Autologous DC-T-cell cocultures were set up in a 48-well plate (2 × 106 T cells, 2 × 105 DCs in 1 mL) with or without Candida (±AZT) in complete medium containing 10 U/mL of IL-4 and 100 U/mL of GM-CSF. Cells were incubated for 2 or 7 days at 37°C before being fixed and permeabilized using a T-regulatory cell (Treg) commercial kit (eBioscience, San Diego, CA). The Treg population was identified using FITC-conjugated anti-FoxP3 (eBioscience), PerCY-conjugated anti-CD4 (BD/Pharmingen), and APC-conjugated anti-CD25 (BD/Pharmingen). Tregs were defined as CD4+CD25+FoxP3+.
DC Transfer of HIV to Permissive Targets
Transfer of infection from HIV-loaded or -infected DCs was performed by mixing 2 × 105 DCs with 2 × 106 SupT1-CCR5 in 1 mL of medium with 10% fetal calf serum (Cellgro) in a 24-well plate (±Candida). Cell-free supernatants were collected at 3, 7, 10, and 14 days of culture for p24 determination by enzyme linked immunosorbent assay (ELISA). Samples were inactivated in 1% Empigen detergent (Calbiochem, Los Angeles, CA) for 1 hour at room temperature. Anti-p24 ELISA was performed on the inactivated supernatants as previously described.38 Aliquots of the cultures were also harvested for HIV QPCR analysis (above).
Statistical analyses were performed using the nonparametric Wilcoxon matched pair test unless indicated otherwise. Data were analyzed using SPSS software (Chicago, IL). Differences were considered significant when P < 0.05.
HIV Exposure Does Not Impair DC Capture of or Activation by Candida albicans
To investigate the impact of HIV on the ability of immature DCs to capture and respond to Candida, immature DCs were loaded with HIV (vs the MV control) and then mixed with Candida immediately (0 hour) or 48 hours later (to monitor infected DCs). Homogenous capture of HIV by almost the entire population was observed (Fig. 1A), supporting earlier observations.15,20 The mean fluorescence intensity (MFI) of p24 detected on the HIV-pulsed cells was significantly above the background staining of MV-treated cells (Fig. 1B). Typical low-level infection of immature DCs (4.49 ± 1.96 HIV copies/102 DCs) was also verified by measuring the amount of HIV gag DNA by QPCR (Fig. 1C). As expected, little or no p24 expression as measured by ELISA or flow cytometry was detected at this time (data not shown).15 The low frequency of infection underscores that the 48-hour HIV-infected DC population, referred to as infected DCs for simplicity throughout, contains a mixture of infected and noninfected cells.
To monitor whether HIV-exposed DCs still captured Candida, DCs were incubated with FITC-labeled Candida immediately or 48 hours after the virus pulse (Fig. 2A). The majority of the capture occurred during the first hour and was maintained at 24 hours reaching 40%-46% of the DCs carrying Candida. Less Candida was initially captured by 48-hour-treated DCs, but the presence of HIV did not affect Candida capture. FACS analysis of virus-pulsed cells also confirmed that p24+ DCs captured FITC-labeled Candida similarly to Candida-negative DCs (data not shown).
As previously reported,39 Candida activates DCs, upregulating CD25, CD80, CD83, and CD86 and downregulating CD209 expression (Fig. 2B). There was little or no impact on the already high levels of HLA-DR. The effect of HIV on the Candida-induced DC maturation was restricted to the statistically significant enhancement of CD80 and CD83 expression after HIV exposure but not after HIV infection (P = 0.03 and 0.04, respectively, when comparing MFIs). The average fold increases in CD80 and CD83 expression by HIV-pulsed DCs were 1.56 ± 0.16 (vs 1.42 ± 0.11 for MV, not statistically significant) and 5.32 ± 1.62 (vs 3.27 ± 0.84 for MV, P = 0.014), respectively. All other Candida-induced changes in the DC membrane phenotype were unaffected by HIV.
Using FITC-labeled Candida, we evaluated if activation was more pronounced in DCs capturing the fungus and if the impact of HIV was similar in these cells. Results are expressed as fold increases in MFIs above those of control cells not exposed to Candida (set as 1), to more accurately assess the differences between the DC subsets that actually capture or not capture Candida after exposure. Comparable increases in CD25, CD80, CD83, and CD86 and the decrease in CD209 were detected in the Candida-negative and -positive DCs within the Candida-pulsed cells (Fig. 2C). CD209 downregulation in response to Candida was less pronounced in the cells bearing Candida, but this was only significant in the cells cultured for the additional 48 hours (independent of HIV, P = 0.04). Although higher in the cells exposed to HIV, CD25, CD80, and CD83 upregulation were not significant (P = 0.07 for CD83) when we examined the Candida-positive vs -negative populations.
Cytokine and Chemokine Responses Elicited by Candida albicans Exposure are Minimally Affected by HIV
Innate responses of immature DCs play important roles in the host's immediate and subsequent adaptive responses to a pathogen. To ascertain whether HIV alters the DC's innate responses to Candida, the supernatants of DCs exposed to (Fig. 3A) or infected with (Fig. 3B) HIV were analyzed 4 or 24 hours after exposure to Candida. Rapid Candida-induced secretion of IL-6, IL-10, TNF-α, and CCL3 was detected within 4 hours, whereas IL-1β, IL-12p40/70, CCL5, and CXCL10 were highest after 24 hours (Fig. 3). Overall, HIV had only subtle influence on the innate responses of the immature DCs to Candida. DCs that had captured HIV released slightly higher levels of IL-6, IL-10, and TNF-α in response to Candida, but these differences did not reach statistical significance (P = 0.06-0.07). Although the responses were low, HIV-infected DCs produced significantly more IL-10 after 4 hours (P = 0.04) independent of Candida and significantly greater IL-1β immediately after exposure to Candida (P = 0.03) (Fig. 3B, asterisks).
HIV-Infected DCs Induce Candida albicans-Specific T-Cell Proliferation
To determine if the subtle effects of HIV on innate DC biology translated into alterations in their ability to stimulate Candida-specific T cells, infected and noninfected DCs were cocultured with autologous T cells with or without Candida. Candida-specific T-cell proliferation was induced in a dose-dependent manner by the DCs, and this was unaffected by prior infection of the DCs with HIV (Fig. 4A). The slightly lower Candida-specific response induced by HIV-infected DCs at the highest DC:T-cell ratio after 5 days of culture was not significantly different from that induced by the uninfected cells. After 3 days of culture, there was absolutely no difference between the responses induced by the infected vs uninfected DCs, indicating that the responses induced by HIV-infected DCs exhibited similar kinetics to those induced by uninfected DCs.
CFSE labeling was used to analyze the cells responding to Candida when stimulated by uninfected vs HIV-infected DCs. A representative FACS plot showing the gated CFSElow cells is provided in Figure 4B. Both CD4+ and CD8+ T cells proliferated in response to Candida, and the bulk of these proliferating cells expressed CD25 (Figs. 4B, C). The percentages of each subset were comparable when stimulated by HIV-infected or uninfected DCs (Fig. 4C). This was not altered if AZT was excluded from the cultures (no AZT), although the percentages of CFSElowCD4+ and CFSElowCD25+ cells were decreased when AZT was not included (not statistically significant).
HIV-Infected DCs Favor Early Treg Expansion and Increased IL-10 Secretion When Responding to Candida
HIV infection in immature DCs has been reported to stimulate IL-10 secretion and enhance T-cell regulation in vitro.31 Moreover, early expansion of Tregs after establishment of Simian Immunodeficiency Virus (SIV) infection was demonstrated in macaques.40 In an attempt to further analyze the Candida-specific T cells activated by HIV-infected DCs, the levels of CD25 expression and the presence of CD4+CD25+FoxP3+ Tregs were measured. The initial upregulation of CD25 by the Candida-responsive T cells was evident within 2 days of culture and increased over time (Fig. 5A). In addition to the percentage of CD25+ T cells being lower in cultures stimulated by HIV-infected DCs in the absence of AZT (Fig. 4C), CD25 MFIs of the Candida-stimulated cultures were also slightly lower (though not statistically significant) than those detected in cultures stimulated by uninfected DCs (Fig. 5A, no AZT).
As already described in the mouse model,41,42 Candida induced the expansion of CD4+CD25+FoxP3+ Tregs in the DC-T-cell mixtures, with 3.4%-6.1% detected within 2 days and 5.9%-7.7% by 7 days of culture (Fig. 5B). When AZT was used to limit HIV spread, HIV-infected DCs significantly increased the initial Candida-driven expansion of CD4+CD25+FoxP3+ Tregs (P = 0.017, asterisk), but the difference was not significant after 7 days of culture (P = 0.13). In the absence of AZT, addition of Candida enhanced the expansion of CD4+CD25+FoxP3+ Tregs. Although the percentages detected in the HIV-infected DC-stimulated cultures were lower at both time points when HIV spread was not prevented with AZT, the differences did not reach significance.
Numerous cytokines and chemokines were secreted in the Candida-stimulated cocultures (Fig. 6). Comparable data were obtained for cultures set up with or without AZT (P > 0.05 using Mann-Whitney test when comparing the responses induced in the presence of HIV ± AZT), so these data have been pooled here for better statistical analyses. Overall, HIV infection of the DCs had minimal impact on the level of factors produced over time. Of note, coinciding with the elevated Treg percentages (Fig. 5B), there were significantly higher amounts of IL-10 secreted initially in Candida-exposed cocultures stimulated by HIV-infected DCs (P = 0.02, asterisk). However, this was not maintained over time. Lower interferon-γ release in Candida-responding cultures stimulated by HIV-infected DCs corresponded with the lower proliferative responses detected (Fig. 4A), but was not significant.
Persistence of HIV Infection in Candida-Matured DCs, and Increased Amplification in Candida-Activated DC-T-Cell Mixtures
Knowing that Candida induces DC maturation, we sought to define the impact it might have on DC infection with HIV and on the ability of DCs to transmit the virus. DCs were loaded with HIV and immediately or 48 hours later exposed to Candida for an additional 5 days, before infection was measured by QPCR for HIV gag (Fig. 7A). Characteristic low-level infections were detected in the immature DCs exposed to HIV. The infection levels were lower in the 48-hour samples because the cells were washed after the initial 48-hour period before re-culture with or without Candida, thereby reducing the amount of virus available to amplify within the cultures. When Candida was added immediately after HIV exposure, HIV infection was reduced by an average of 2.6-fold (0 hour, P = 0.0005, asterisks), but infection was never completely shut down. Interestingly, when Candida was added to already infected DCs (48 hours), there was limited impact on the low-level DC infection (P = 0.71). Thus, despite exhibiting typical mature DC characteristics (Figs. 2, 3), Candida-activated DCs were still able to support HIV replication.
Because DCs are also known to efficiently transfer captured or newly synthesized virus to permissive T cells,15,43,44 we evaluated the virus transfer from HIV-pulsed or -infected DCs to SupT1-CCR5 cells in presence or absence of the fungus. SupT1-CCR5 cells provided a sensitive readout for the detection of even small amounts of infectious virus45 and also allowed us to measure the impact of Candida on HIV transfer in the absence of Candida-mediated T-cell activation. Despite the Candida-induced reduction of HIV replication in DCs (Fig. 7A), HIV was still amplified upon addition of SupT1-CCR5 cells as determined by the release of p24 into the supernatants (Fig. 7B). The lower levels of infection detected in cocultures containing infected DCs coincide with the reduced level of virus present (2-4 times less HIV copies/cell in 48 hours compared with 0 hour DCs) in the former (Fig. 7A). The presence of Candida enhanced HIV infection in the cocultures containing HIV-loaded or -infected DCs. This was apparent at the level of more rapid kinetics of infection driven by HIV-loaded DCs, which produced significantly larger amounts of p24 after 7 days (P = 0.05) and 10 days (P = 0.0015) of culture (Fig. 7B, left panel). Addition of Candida to cultures containing infected DCs did not augment the kinetics but enhanced the overall production of virus at the p24 level at 14 days (P = 0.02; Fig. 7B, right panel), and this was verified by gag QPCR (P = 0.03; Fig. 7C). The enhanced amplification by Candida in the DC-SupT1-CCR5 mixtures was not due to direct effects of Candida on the SupT1-CCR5 cells because addition of Candida did not alter virus replication in cultures of SupT1-CCR5 cells (Fig. 7D).
Understanding how common copathogens and the immune responses to those pathogens might contribute to HIV pathogenesis (and vice versa) is important to advance strategies to control HIV. DCs are central to the activation and maintenance of immunity, but HIV exploits DCs to facilitate HIV infection and spread. The potential influence of other pathogens on DC-driven HIV infection has not been extensively studied however. Moreover, HIV-induced immune dysfunction contributes to the loss of control of normally commensal organisms like Candida and may involve changes in DC function. Using Candida as a model copathogen commonly found at the body surfaces, we provide the first evidence that although HIV has limited overall impact on Candida-induced stimulation of DCs, HIV takes advantage of DC-Candida interplay to foster HIV amplification and dissemination.
Despite HIV infection or exposure, DCs were able to mount solid responses against Candida and mature similarly to cells not exposed to HIV. HIV infection of DCs correlated with somewhat elevated production of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) immediately after contact with Candida. Similar results have been reported for lipopolysaccharide-stimulated HIV-infected DCs,46 although the responses were less pronounced herein probably due to lower virus inoculums used. Such proinflammatory cytokines promote DC maturation47,48 and likely contribute to the Candida-induced maturation of bystander and Candida-positive DCs. Activation by Candida also induced the production of chemokines (CCL3, CCL4, and CCL5), which would facilitate the recruitment of additional cell targets susceptible to HIV infection in vivo, to exacerbate DC-driven cell-to-cell spread.49,50
In general, Candida-induced DC maturation would encourage DC-T-cell conjugate formation,51,52 thereby facilitating transfer and amplification of virus within the T cells.23,53 The upregulation of CD80 and CD83 triggered by Candida was significantly greater in DCs that had just captured HIV. This observation complements some recently published data where similar upregulation of CD80 and CD83 was reported in immature DCs infected with high doses of HIV,54 but this was not observed in our low-level infected DCs herein. CD83 has been shown to be important in DC-mediated T-cell communication.55,56 Therefore, the greater elevation of CD83 expression by HIV-bearing DCs responding to Candida would further promote DC-T-cell interactions to facilitate HIV spread. In agreement with this, the significantly enhanced CD83 expression by HIV-loaded DCs exposed to Candida paralleled the more rapid amplification of virus when these cells were added to the permissive SupT1-CCR5 cells. In contrast, HIV-infected DCs did not express significantly higher CD83 in response to Candida, coinciding with the kinetics of HIV infection in the cocultures containing infected DCs not being enhanced. Virus production in these cocultures was ultimately amplified in the presence of Candida however. Candida-stimulated HIV-infected DCs did not downmodulate CD209 as much as uninfected DCs. This suggests that the persisting higher levels of CD209 on HIV-infected DCs carrying Candida could promote interactions with Candida-specific T cells to further encourage transmission of the newly produced virus to those cells.57,58
Because antigen-responsive T cells are particularly susceptible to HIV infection,43,59 HIV amplification within the DC-T-cell milieu would likely be significantly augmented as the DCs stimulate Candida-specific CD4+ T-cell activation. However, in addition to innate and effector responses that are needed to keep Candida in check,8,60,61 murine studies have highlighted that Tregs are an important part of the responses to Candida infection keeping a balance on immunopathogenesis.41,42 We confirmed that Candida induced Tregs in human DC-T-cell mixtures, even in the presence of HIV. Because Tregs may have increased sensitivity to HIV,62 it is possible that Candida-activated Tregs would further increase the amplification of HIV provided by the DCs. Interestingly, IL-10 production and Treg expansion in the Candida-exposed DC-T-cell mixtures were significantly increased early in cultures containing HIV-infected DCs. This supports earlier studies looking at the effect of HIV infection on autologous DC-T-cell mixtures.31 HIV-driven IL-10 production could further dampen DC function,63 thereby favoring improper T-cell activation. The initial increased Treg levels were only apparent when AZT was present, suggesting that replicating HIV could balance Treg expansion by destroying these activated targets. Thus, this milieu would be especially permissive to HIV spread.
In addition to amplifying virus in combination with permissive T cells, immature DCs are able to replicate R5 HIV, and even undetectable amounts of HIV in DCs are revealed upon T-cell contact.15,64 Typically, mature DCs are less susceptible to HIV infection15,16 in part due to a decline of virus fusion.65 However, little research has been performed on the fate of virus in immature DCs that receive a pathogen-driven maturation signal after virus exposure. Viruses captured by immature DCs sequester virus in small compartments at the periphery of the cell,20 and upon receiving a maturation signal, the captured virus moves deeper within the cell, securing it much like mature DCs.15 Even though Candida induced solid maturation of the DCs, this did not shut down their ability to replicate HIV, although it was reduced when Candida was added to HIV-pulsed DCs. Maturation of virus-bearing immature DCs with single stranded RNA or a cocktail of cytokines20 also only partially shuts down HIV replication in DCs, whereas exposure to poly(I:C) completely blocks virus replication in the DCs (Susanna Trapp, PhD and Melissa Robbiani, PhD, submitted 2008). These data highlight how DCs respond uniquely to different stimuli and that this has important implications for HIV spread.
This study provides the first data on how HIV can undermine normal DC responses to Candida to maintain and expand HIV infection. HIV is able to circumvent Candida-induced maturation of the DCs and continues to replicate to low levels within the DCs. Thus, in addition to initially captured viruses being transmitted to T cells, persistent low-level replication within the Candida-stimulated DCs provides an additional source of newly produced virus that would continue to promote virus spread. Sustained DC-driven amplification of HIV under these conditions would also ultimately destroy the cells needed to maintain the control of commensal Candida and likely contributes to the loss of immune integrity seen with HIV disease progression.
The assistance and use of the Population Council's Flow Cytometry Facility is gratefully acknowledged.
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