JAIDS Journal of Acquired Immune Deficiency Syndromes:
Differentiation of Monocytes Into CD1a− Dendritic Cells Correlates With Disease Progression in HIV-Infected Patients
Sacchi, Alessandra PhD*; Cappelli, Giulia PhD†; Cairo, Cristiana PhD‡; Martino, Angelo PhD*; Sanarico, Nunzia PhD‡; D'Offizi, Gianpiero MD*; Pupillo, Leopoldo Paolo MD*; Chenal, Henri MD§; Libero, Gennaro De MD∥; Colizzi, Vittorio MD‡; Vendetti, Silvia PhD*¶
From the *National Institute for Infectious Disease “L. Spallanzani”-IRCCS, Rome, Italy; †Institute of Neurobiology and Molecular Medicine, National Research Council, Rome, Italy; ‡Department of Biology, University of Rome-Tor Vergata, Rome, Italy; §Centre Intégré de Recherches Biocliniques d'Abidjan, Abidjan, Ivory Coast; ∥Experimental Immunology, Department of Research, University Hospital, Basel, Switzerland; and the ¶Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy.
Received for publication February 16, 2007; accepted September 12, 2007.
The authors have no conflicting financial interests.
Correspondence to: Silvia Vendetti, PhD, Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena, 00161 Rome, Italy (e-mail: firstname.lastname@example.org).
Monocyte differentiation into dendritic cells (DCs) depends on microenvironmental conditions. In this study, the capacity of human monocytes to differentiate into mature DCs and their ability to induce an antiviral immune response was investigated in HIV-infected patients. In healthy subjects, monocytes differentiate into CD1a+ DCs in the presence of granulocyte macrophage colony-stimulating factor and interleukin (IL)-4 and matured in the presence of lipopolysaccharide. Here, we found that in 30% and 45% of HIV-infected white and African subjects, respectively, monocytes gave rise to a homogeneous CD1a− DC population. In the patients who gave rise only to the CD1a− DCs, this population spontaneously produced IL-10 but not IL-12, and induced a T helper 2-like immune response when cultured with human T cells isolated from cord blood mononuclear cells. In patients with monocytes differentiated into CD1a− DCs, a high percentage of HIV-specific CD4+ T cells producing IL-4 were seen in the peripheral blood. Furthermore, differentiation of monocytes into DCs with CD1a− phenotype correlated with low CD4+ T-cell counts and high viral loads in HIV-infected subjects. These results suggest that the differentiation of monocytes into CD1a− DCs may be a phenotypic marker associated with progression of the disease.
Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that play a dominant role in the initiation and regulation of immune responses.1 They are able to capture and present peptides2 and lipids and glycolipids3 to T cells, through major histocompatibility complex (MHC) class I and II molecules or through the nonclassic MHC-like CD1a-d molecules. Compared with other APCs, DCs have a unique capacity to stimulate naive T lymphocytes, driving them into distinct classes of effector cells.4
Two main populations of DCs have been described to date in human peripheral blood: CD11c+ cells (myeloid DCs) show monocyte-like morphology and express myeloid origin markers, whereas CD11c− cells (plasmacytoid DCs) display poor endocytic activity and lymphoplasmacytoid morphology.5 Peripheral blood monocytes are precursors of myeloid DCs in vitro6 and in vivo.7 In vivo, they migrate from the blood into inflammatory sites, where they differentiate into DCs if proinflammatory cytokines are present.8,9 Once in peripheral tissues, immature DCs capture antigens, undergo a maturation process, and migrate to the lymphoid organs, where mature DCs induce activation of naive and memory antigen-specific T lymphocytes, thus providing immediate protection against microbes.10,11 Microenvironmental conditions and several pathogens, or their components, can interact with monocytes to influence DC generation.12,13 During infection, pathogens can affect DC precursors directly but also indirectly by stimulating a cascade of inflammatory-associated factors released by the cells in response to infection. The pathophysiologic events affecting the capacity of monocytes to differentiate into DCs and the influence of their functions on the framework of local T-cell activation and differentiation have yet to be properly characterized. In this study, we evaluate the capacity of monocytes to differentiate into DCs ex vivo and the ability of these DCs to induce an antiviral immune response in HIV-infected patients.
The progression of HIV infection is associated with activation of the immune response as viral replication increases, followed by immunoregulatory defects that precede CD4 T-cell depletion.14 Increased production of type 2 cytokines (T helper [Th] 2), including interleukin (IL)-10, is detected in most patients during disease progression.15-17 Furthermore, the production by peripheral blood mononuclear cells (PBMCs) of IL-12, a strong inducer of the Th1-type response, is defective in HIV-infected patients.18,19 Functional impairment of APCs and a depletion of professional myeloid and plasmacytoid DCs are also associated with HIV disease progression.20-22
In the present work, we found that in HIV-infected white (30%) and African (45%) subjects, respectively, monocytes that were differentiated into DCs and matured with lipopolysaccharide (LPS) had a homogeneous CD1a− phenotype, produced IL-10 rather than IL-12, and induced a Th2-like immune response. In addition, we observed that the capacity of monocytes to differentiate into CD1a− monocyte-derived dendritic cells (MoDCs) correlated with low CD4 counts and high viral load in HIV-infected patients, suggesting that the differentiation of monocytes into CD1a− DCs could be a phenotypic marker associated with the progression of the disease.
MATERIALS AND METHODS
Fifty-seven blood samples of HIV-infected patients from the “Lazzaro Spallanzani” Hospital (Rome, Italy) and 61 HIV-infected patient samples from the Centre Intégré de Recherches Biocliniques d'Abidjan (CIRBA; Abidjan, Ivory Coast) were included in this study. Written informed consent was obtained from African subjects, whereas for Italian patients, anonymously and unlinked residual blood count samples were used. Approval of the local ethical committee was given. Buffy coats drawn from healthy donors were provided by the “La Sapienza” University Transfusion service. Cord blood cells were from a cord blood bank designated to the generation of stem cells. Among African and white patients, 64 were receiving antiviral therapy. Antiviral drugs consisted of nucleoside reverse transcriptase inhibitors (NRTIs) in association or not with protease inhibitors (PIs). CD4 T-cell counts ranged from 22 to 1091 cells/mm3, and viral loads ranged from 200 to 830.700 copies/mL. Blood from 50 African and white HIV-seronegative healthy donors was used as a control and processed using the same conditions as blood from HIV-infected subjects.
Dendritic Cell Preparation
PBMCs were isolated from peripheral blood by density gradient centrifugation using Lympholyte-H (CEDERLANE, Hornby, Ontario, Canada). Monocytes were positively separated by anti-CD14 magnetic beads (MACS; Milteny Biotec, Bergish Gladbach, Germany) according to manufacturer's instructions. Cells were then resuspended in RPMI 1640 (EuroClone, Paignton, United Kingdom) supplemented with 10% heat-inactivated defined fetal bovine serum (FBS; HyClone, Logan, UT), 2 mM of L-glutamine, 10 mM of N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) buffer, 0.1 mM of sodium pyruvate, 2 mM of penicillin, and 50 μg/mL of streptomycin (EuroClone). Monocytes were cultured for 5 days in fresh complete medium in the presence of 200 U/mL of granulocyte macrophage colony-stimulating factor (GM-CSF) and 10 ng/mL of IL-4 (EuroClone) to generate MoDCs. On day 5 of culture, cells were washed and starved for 8 hours; LPS (100 ng/mL) was then added, and the cells were cultured another 2 days to induce final maturation. Monocyte-depleted PBMCs were frozen and used in the antigen-presenting assays with peptide-pulsed MoDCs.
Immunophenotyping of DCs was accomplished using direct fluorochrome conjugates of anti-CD1a, -human leukocyte antigen (HLA) ABC, -HLA D-related (DR), -CD80, -CD86, -CD83, -CD11c, and -CD14 monoclonal antibodies (mAbs; Becton Dickinson Biosciences, San Jose, CA). CD4 and CD8 T-cell counts were performed by flow cytometry using directly conjugated anti-CD4 and anti-CD8 mAbs. Briefly, cells were incubated at 4°C for 20 minutes with 50 μL of antibodies and then washed and fixed with 1% paraformaldehyde. For intracellular staining, cells were collected and stained with fluorescein isothiocyanate (FITC)-labeled anti-CD4 mAbs and peridinin chlorophyll protein (PerCP)-labeled anti-CD8 mAbs (Becton Dickinson Biosciences); they were then washed, fixed with 4% paraformaldehyde, permeabilized, and stained with phycoerythrin (PE)-labeled anti-IL-4 and APC-labeled anti-interferon (IFN)-γ mAbs. Control for nonspecific staining was monitored by isotype-matched mAbs. Acquisition of 30.000 events in the lymphocyte-gated population and analysis were performed using Cell Quest software (Becton Dickinson Biosciences).
T-Cell Activation and Polarization Assay
To assess the APC function of MoDCs derived from HIV-infected patients, after 5 days of culture, cells (1 × 105) were washed, starved, and then pulsed with 5 μg/mL of a pool of HIV GAG-, NEF-, and TAT-derived peptides23 for 3 hours. Peptide-pulsed MoDCs were cultured for 1 hour with autologous T cells (5 × 105) and then treated with 10 μg/mL of brefeldin A for 12 hours. The percentage of IL-4-positive and IFNγ-positive cells was evaluated by intracellular staining.
The capacity of MoDCs to activate T cells was evaluated using a mixed lymphocyte reaction. MoDCs (from 0 to 2 × 104 cells/well) were cocultured with allogeneic PBMCs (1 × 105 cells/well) in 96-well U-bottom microplates (Corning-Costar, New York, NY) for 5 days. 3H-thymidine (1 μCi per well) was added 18 hours before the cells were harvested. Incorporation of 3H-thymidine was quantified using a β-counter (Beckman, Fullerton, CA).
The ability of HIV-infected patients' MoDCs to stimulate and polarize naive T cells was evaluated. Cord blood mononuclear cells (CBMCs) were isolated by Lympholyte-H, and monocytes were depleted by adherence. T cells were separated from CBMCs by magnetic beads (MACS) according to manufacturer's instructions. MoDCs (2 × 105) from HIV-infected patients or healthy donors were extensively washed, starved for 8 hours, and then cultured with T cells from CBMCs (1 × 106). Culture supernatants were harvested 8 days later, and IL-4, IL-10 and IFNγ levels were quantified by enzyme-linked immunosorbent assay (ELISA).
Supernatants of MoDCs (1 × 106 cells/mL) derived from HIV-infected patients were collected and stored at −80°C. IL-10 (limit of sensitivity <5 pg) and IL-12 (limit of sensitivity <5 pg) levels were determined by ELISA kits (Pierce Endogen, Rockford, IL) according to the manufacturer's instructions. Supernatants of T cells from CBMCs cultured with MoDCs from HIV-infected patients were tested for IL-4 (limit of sensitivity <2 pg) and IFNγ (limit of sensitivity <2 pg) using commercially available ELISA kits (Pierce Endogen). Results are expressed as picograms per milliliter and reported as means (+ standard error of the mean [SEM]).
Statistical analysis was conducted using a parametric unpaired Student t test. The P < 0.05 values were considered statistically significant. The parametric Pearson test was used to describe correlation. GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, CA) was used to perform the analysis.
CD1a− MoDCs From HIV-Infected Patients
To investigate the ability of HIV-infected patient monocytes to differentiate into fully competent MoDCs, we analyzed the phenotype of immature and LPS-maturated MoDCs from 57 white and 61 African HIV-infected patients and from 50 HIV-seronegative white and African healthy subjects. We found that monocytes from HIV-infected patients and healthy donors are all able to differentiate into MoDCs, downregulate CD14 molecules, and express similar levels of HLA class I and class II and costimulatory molecules (Fig. 1A). In healthy subjects, more than 95% of MoDCs are CD1a+. In HIV-positive subjects of white and African origin, 70% and 55% of MoDCs, respectively, show a heterogeneous expression level of CD1a ranging from 35% to 95% of CD1a+ MoDCs (CD1a±). Interestingly, in 30% and 45% of HIV-infected white and African subjects, respectively, MoDCs lack the expression of the CD1a molecule (see Fig. 1), giving rise to a homogeneous CD1a− MoDC population. The morphology of this subset is similar to that of MoDCs from healthy donors (data not shown). The homogeneous CD1a− population is maintained after maturation induced by LPS. Furthermore, on LPS treatment, the CD1a− and CD1a± MoDCs from HIV-infected patients have upregulated expression of CD83, HLA class I, HLA-DR, CD86, and CD80 costimulatory molecules (see Fig. 1), suggesting that the CD1a− MoDCs can proceed into the fully mature phenotype, ruling out the possibility of being blocked in an immature stage. The upregulation of CD83 molecules, described as being selectively expressed by mature DCs, excludes the possibility that there is a switch toward a macrophage-like population in patients who give rise to the CD1a− phenotype. The differentiation into a homogeneous CD1a− population of MoDCs is observed in patients untreated or treated with different antiretroviral agents, suggesting that the CD1a− phenotype is not an effect derived from pharmacologic treatment combinations.
T-Cell Stimulation by MoDCs From HIV-Infected Patients
To determine whether the phenotypic differences between CD1a± and CD1a− MoDCs have any effect on primary T-cell proliferation, we performed allogeneic mixed lymphocyte reactions. We found that CD1a± and CD1a− MoDCs are able to stimulate T-cell proliferation at comparable levels (data not shown), suggesting that CD1a− MoDCs are not impaired in their capacity to stimulate T-cell activation.
Similarly, a series of experiments was performed to evaluate the antigen-presenting capacity of CD1a− compared with CD1a± MoDCs by pulsing them with a pool of HIV-derived peptides and culturing with autologous PBMCs. After overnight culture, the percentage of IL-4- and IFNγ-producing T cells was evaluated by intracellular staining. Interestingly, we found that the percentage of HIV-specific CD4+ T cells producing IL-4 is higher in patients with homogeneous CD1a− MoDCs compared with that of a mixed CD1a± MoDC population (Figs. 2A, B). The percentage of CD4+ T cells producing IFNγ in patients with mixed CD1a± or CD1a− populations of MoDCs is similar in both phenotypes (see Figs. 2A, C). The percentage of HIV-specific CD8+ T cells producing IL-4 or IFNγ among patients with CD1a± or CD1a− MoDCs was also evaluated, but no significant differences were found between cells stimulated in the presence of CD1a± or CD1a− MoDCs (data not shown). PBMCs isolated from healthy donors and stimulated with autologous MoDCs pulsed with HIV peptide were included in the study as a control; they failed to produce any cytokines, as expected (see Figs. 2A, C). These data suggest a correlation between the differentiation of monocytes into CD1a− MoDCs and the presence of a high percentage of IL-4-producing CD4+ T lymphocytes, a link correlated with the progression of HIV disease.
Cytokine Production by CD1a± and CD1a− MoDCs Generated From HIV-Infected Patients
We next asked whether the CD1a± and CD1a− MoDCs from HIV-infected patients can produce regulatory cytokines. The accumulation of IL-10 and IL-12, which are pivotal cytokines in directing polarization of the immune response, was evaluated in the supernatants of CD1a± and CD1a− MoDCs from HIV-infected patients. We found that CD1a− MoDCs generated from HIV-infected patients produce higher levels of IL-10 compared with CD1a± MoDCs or MoDCs generated from healthy donors (Fig. 3A). Conversely, CD1a− MoDCs produce a significantly lower amount of IL-12 (p70) compared with CD1a± MoDCs (see Fig. 3B). Furthermore, by separating CD1a− and CD1a+ DCs using anti-CD1a magnetic beads (MACS), we found that CD1a− DCs separated from the mixed CD1a± population from the same subject produced IL-10, whereas CD1a+ did not (data not shown). The production of IL-12 (p70) by CD1a± MoDCs is not significantly different from that of MoDCs generated from healthy donors (see Fig. 3B). This result suggests that CD1a± and CD1a− MoDCs have different regulatory properties.
Polarization of Naive T Lymphocytes
DCs play a major role in directing the immune response against pathogens, having a unique capacity to stimulate naive T lymphocytes, driving them into distinct classes of effector cells. Because CD1a− MoDCs from HIV-infected patients spontaneously produce IL-10 but are unable to produce IL-12, we speculated that CD1a− and CD1a± MoDC subsets may differ in their capacity to polarize the immune response. To address this question, we cultured CD1a± or CD1a− MoDCs generated from distinct HIV-infected patients with T cells isolated from cord blood (CBMCs) for 8 days. The accumulation of IL-4 and IFNγ in the supernatants of the cocultures was analyzed by ELISA. We found that when T cells from CBMCs are cultured in the presence of CD1a− MoDCs, they differentiate into cells that produce significantly higher levels of IL-4 and IL-10 compared with T cells cultured with CD1a± MoDCs (Figs. 4A, B). T cells from CBMCs cultured in the presence of CD1a− MoDCs produce similar levels of IFNγ compared with the cells cultured with CD1a± MoDCs (see Fig. 4C). These data suggest that CD1a± and CD1a− MoDCs are able to direct the differentiation of naive T cells with different cytokine profiles. In particular, CD1a− MoDCs preferentially induce a Th2-like immune response considered dominant during HIV disease progression.
Correlation of the Differentiation Into CD1a− MoDCs With CD4 Cell Count and HIV Viral Load in HIV-Infected Patients
The progression of HIV infection is associated with reduced CD4 cell counts, increased HIV plasma viremia, and a progressive increase of type 2 cytokines (Th2) production, including IL-10.15-17 Because we observed that CD1a− MoDCs produced IL-10 and preferentially induced a Th2-like immune response, we studied how the differentiation into CD1a− MoDCs is correlated with CD4 cell counts and HIV viremia. CD4 T-cell counts in HIV-infected patients ranged from 22 to 1091 cells/mm3; viral loads from 200 to 830.700 copies/mL were included in this study and grouped on the basis of CD1a expression into CD1a− and CD1a± MoDCs. We found that patients with CD1a− MoDCs have lower CD4 cell counts compared with those who generate CD1a± MoDCs (P = 0.01) (Fig. 5A). Not all the patients with low CD4 cell counts display the CD1a− phenotype, however. In addition, we found that the differentiation into CD1a− MoDCs correlates with a higher HIV plasma viremia compared with those that give rise to CD1a± MoDC populations (P = 0.0006) (see Fig. 5B). Furthermore, to analyze possible associations between CD4 cell counts and viral loads with CD1a expression on MoDCs from HIV-infected patients, we performed correlation analyses. Because of the high variability in the sample, we transformed the CD1a mean fluorescence intensity (MFI) and viral loads in logarithmic values to have a normal distribution of them. The Pearson test that is used to evaluate correlation of variables approximately normally distributed was then applied. We found a positive correlation between CD4 cell counts and the expression of CD1a molecules on MoDCs (P < 0.05, r = 0.4; see Fig. 5C). An inverse correlation between viral loads and CD1a expression was also observed (P < 0.05, r = −0.37; see Fig. 5D). These findings suggest that the differentiation into a homogeneous CD1a− population of MoDCs correlates with the progression of HIV disease. In addition, Table 1 shows in more detail the immunologic and clinical parameters of white and African HIV-infected patients grouped into CD1a± and CD1a− MoDCs. Patients who received or did not receive antiretroviral therapy were grouped together.
It is well established that monocytes differentiate into DCs or macrophages depending on microenvironmental conditions.24-27 Accumulating evidence shows that interaction with microbes by means of pattern recognition receptors influences the commitment of monocytes toward DC differentiation.12,13,28,29 Alternatively, other cell types can respond to microbes by secreting different cytokines or tissue factors, interfering with DC differentiation and leading to distinct types of immune responses. The microenvironment derived from persistent pathologic conditions may also affect the capacity of monocytes to migrate from blood to tissue or the ability to differentiate into DCs. Therefore, we examined whether monocytes isolated from HIV-infected patients at different stages of the disease are able to differentiate into competent DCs. According to previous results,30 monocytes isolated from HIV-infected patients are able to differentiate into competent MoDCs expressing MHC class I, MHC class II, and costimulatory molecules and, upon maturation stimuli, can become fully mature DCs. Our study reveals that in white (30%) and African (45%) HIV-infected subjects, monocytes differentiate into a homogeneous MoDC population that fails to express the CD1a molecule on the membrane. The CD1a− phenotype is maintained after maturation, seeming to be a cell lineage marker. Upregulation by the CD1a− MoDCs of CD83 molecules, described as selectively expressed by mature DCs, indicates that there is not a switch toward a macrophage-like population in patients expressing the CD1a− phenotype. The differentiation into CD1a− MoDCs does not seem to be an effect derived from pharmacologic treatment, because the CD1a− phenotype was observed in untreated patients and in those treated with different antiviral agents. It is still not known if MoDCs from the 2 groups of patients reach different stages of maturation during the culture or, alternatively, if the starting monocytes are part of cell populations with distinct differentiation capabilities. Heterogeneity within the human monocyte population has been reported.31 In particular, the minor CD14+CD16+ circulating pool of monocytes, accounting for 10% of total monocytes in healthy donors, differentiates into DCs with a reduced expression of CD1a. Interestingly, the percentage of CD16+ monocytes is greatly increased in some pathologic conditions and has been associated with acute or chronic inflammation.32,33 During HIV infection and in patients with AIDS, they may represent up to 40% of all circulating monocytes.34 Whether the CD1a− MoDCs seen in HIV-infected patients derive from CD16+ monocyte subsets needs further investigation, however. Future studies are devoted to dissection of the phenotypically different monocytes in infected individuals; this could not only be a convenient marker associated with the disease progression but may give insight into the potential breakdown of normal APC differentiation.
Mixed CD1a± and CD1a− MoDCs derived from distinct HIV-infected patients induced an allogeneic response, indicating that CD1a− MoDCs are not defective in T-cell stimulation capacity. There are conflicting reports regarding the ability of DCs from HIV-infected patients to stimulate T-lymphocyte proliferation, but these differences may be related to the numerous protocols used in the generation or isolation of DCs.35,36 A previous study reported a functional impairment of circulating myeloid DCs from HIV-infected patients with low CD4 T-cell counts, although MoDCs from the same patients induced an allogeneic T-cell expansion.37 We observed that CD1a± and CD1a− MoDCs from distinct HIV-infected patients stimulated HIV-specific T cells. In fact, we found that the percentage of HIV-specific CD4+ T cells producing IL-4 was higher in patients expressing CD1a− MoDCs compared with those expressing CD1a± MoDCs. These data suggest a possible correlation between the differentiation of monocytes into CD1a− MoDCs and the presence of a high percentage of IL-4-producing CD4+ T lymphocytes, known to be correlated with progressive HIV disease.38 Taking into consideration that the in vitro methods of generating DCs may not truly reflect what exist in vivo, whether the CD1a− MoDCs differentiate in vivo, and whether they play a role in the expansion of IL-4-producing HIV-specific CD4+ T cells requires a more accurate study, however.
DCs produce immunoregulatory cytokines such as IL-10 and IL-12; therefore, the balance of the production of these cytokines plays a pivotal role by influencing the innate and acquired immune responses and determines polarization of T-cell precursors. We found that CD1a− MoDCs spontaneously produce IL-10 but not IL-12. HIV infection is associated with increased IL-10 production, which reportedly enhances the entry of HIV into target cells through upregulation of CD4 and CCR5.39 Moreover, IL-10 was shown to increase HIV infection of human monocytes and to stimulate viral replication directly in APCs.40
Alterations of DC number and functions have been observed in patients with other infective or cancerous diseases.41-43 We and others have previously shown that monocytes infected with different strains of Mycobacterium differentiate into CD1a−/IL-10-producing DCs.12,13,29 These findings suggest that the shift of balance toward a CD1a−/IL-10+ phenotype may be a common feature in some chronic infectious diseases.
The secretion of IL-10 and the lack of IL-12 by CD1a− MoDCs compared with CD1a± MoDCs may account for the varying capability of these cells to polarize the response of naive lymphocytes. Indeed, when T cells from CBMCs were cultured with CD1a± or CD1a− MoDCs from HIV-infected patients, homogeneous CD1a− MoDCs consistently induced a higher secretion of IL-4 and IL-10 by T lymphocytes than CD1a± MoDCs. The levels of IFNγ elicited by CD1a± and CD1a− MoDCs were similar. The differentiation of naive T cells toward the Th1 or Th2 phenotype is controlled by the activity of IL-12 produced by APCs and IFNγ or IL-4 secreted by the responding T cells.44 The greater induction of IL-4 and IL-10 that we found on lymphocytes stimulated by CD1a− MoDCs might be linked with the reduced ability of CD1a− MoDCs to produce IL-12. Conversely, the induction of T lymphocytes producing IFNγ may be attributable to the capacity of CD1a− MoDCs to produce other factors that support Th1 differentiation, such as IL-23 or IL-27.45 Furthermore, the capacity of CD1a− MoDCs to induce IL-10 during culture with CBMCs could result in tolerance induction. There is evidence that DCs play an important role in the control of peripheral tolerance through the induction and maintenance of IL-10-secreting type 1 regulatory T cells.46 CD1a− MoDCs may be involved in the induction of IL-10-producing regulatory T cells, possibly preventing an effective immune response. Whether or not IL-10-secreting T cells from CBMCs induced by CD1a− MoDCs have regulatory activity requires further investigation, however.
In HIV-infected subjects, impairment in the production of type 1 cytokines and increased secretion of type 2 cytokines, including IL-10 by T lymphocytes, was postulated to be associated with progression of the disease.38 In this study, we observed that the in vitro differentiation of a homogeneous CD1a− MoDC population induces a Th2-like immune response in HIV-infected patients. Interestingly, patients giving rise to CD1a− MoDCs have lower CD4 T-cell counts and higher HIV plasma viremia compared with those who generate CD1a± MoDCs. Moreover, the expression of CD1a molecules on MoDCs is positively correlated with CD4 cell counts and inversely correlated with viral loads. These data indicate that the capacity of monocytes to differentiate into a homogeneous CD1a− MoDCs population correlates with progression of HIV disease. Furthermore, we found that the percentage of patients who developed the homogeneous CD1a− subset of MoDCs was higher among infected African subjects than among infected white subjects. An abnormal activation of the immune system is thought to be involved in the pathogenesis of African HIV infection, and several studies confirmed that lymphocytes from African HIV-infected and uninfected individuals show functional and phenotypical signs of activation.17,47 Furthermore, it has been reported that immune activation in Africans is environmentally related and not genetically predetermined.48 This finding may be linked to the presence of many opportunistic infections and the quality of health care in developing countries. In particular, the Ivory Coast is a West African country with a high incidence of HIV and other sexually transmitted diseases, malaria, and tuberculosis.
In conclusion, we show that in a small but still substantial proportion of HIV-infected patients, monocytes can differentiate into CD1a− MoDCs that produce IL-10 rather then IL-12, driving a predominant Th2-like immune response. This phenomenon correlates with a high percentage of IL-4-producing T cells, with low CD4 T-cell counts and high viral load, suggesting that monocyte differentiation into CD1a− MoDCs could be a new phenotypic marker associated with progression of the disease. The relevance of the in vitro methods of generating DCs to determine what is truly going on in vivo is not at all clear. Furthermore, the complexity of DC differentiation pathways is complicated not only in vivo but ex vivo. It has recently been observed in ex vivo-derived DCs that the CD1a expression is biased by lipid environment and peroxisome proliferators activated receptor (PPARγ) expression.49 Therefore, differential lipid content in FBS could represent a key factor that drives CD1a expression ex vivo. In light of these observations, a key question would be what characteristic feature of monocytes from HIV-infected patients are (or are not) expressed, which leads them not to be influenced by FBS to express CD1a. The identification of these factor(s) would be a useful phenotypic marker associated with progression of the disease and would also avoid the need to culture monocytes in IL-4/GM-CSF to be detected. A more detailed understanding of monocyte/DC heterogeneity and its role in HIV infection should be useful in the regeneration of immune response to HIV and modulation of disease progression.
The authors thank Dr. R. Lindstedt, Dr. D. Horejsh, and Professor A. Cassone for helpful discussions and critical comments on the manuscript; Dr P. Piselli for statistical analysis; and Dr. M. Amicosante for kindly providing the HIV peptides.
1. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature. 1998;392:245-252.
2. Thery C, Amigorena S. The cell biology of antigen presentation in dendritic cells. Curr Opin Immunol. 2001;13:45-51.
3. Briken V, Jackman RM, Watts GF, et al. Human CD1b and CD1c isoforms survey different intracellular compartments for the presentation of microbial lipid antigens. J Exp Med. 2000;192:281-288.
4. Liu YJ, Kanzler H, Soumelis V, et al. Dendritic cell lineage, plasticity and cross-regulation. Nat Immunol. 2001;2:585-589.
5. Kadowaki N, Ho S, Antonenko S, et al. Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens. J Exp Med. 2001;194:863-869.
6. Sallusto F, Lanzavecchia A. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J Exp Med. 1994;179:1109-1118.
7. Randolph GJ, Beaulieu S, Lebecque S, et al. Differentiation of monocytes into dendritic cells in a model of transendothelial trafficking. Science. 1998;282:480-483.
8. Randolph GJ, Inaba K, Robbiani DF, et al. Differentiation of phagocytic monocytes into lymph node dendritic cells in vivo. Immunity. 1999;11:753-761.
9. Soruri A, Riggert J, Schlott T, et al. Anaphylatoxin C5a induces monocyte recruitment and differentiation into dendritic cells by TNF-alpha and prostaglandin E2-dependent mechanisms. J Immunol. 2003;171:2631-2636.
10. Lanzavecchia A, Sallusto F. Regulation of T cell immunity by dendritic cells. Cell. 2001;106:263-266.
11. Palucka K, Banchereau J. How dendritic cells and microbes interact to elicit or subvert protective immune response. Curr Opin Immunol. 2002;14:420-431.
12. Martino A, Sacchi A, Sanarico N, et al. Dendritic cells derived from BCG-infected precursors induce Th-2 T cell differentiation. J Leukoc Biol. 2004;76:827-834.
13. Martino A, Sacchi A, Volpe E, et al. Non-pathogenic Mycobacterium smegmatis induces the differentiation of human monocytes directly into fully mature dendritic cells. J Clin Immunol. 2005;25:365-375.
14. Fauci AS. Immunopathogenesis of HIV infection. J Acquir Immune Defic Syndr Hum Retrovirol. 1993;6:655-662.
15. Clerici M, Wynn TA, Berzofsky JA, et al. Role of interleukin-10 (IL-10) in T helper cell dysfunction in asymptomatic individuals infected with human immunodeficiency virus (HIV-1). J Clin Invest. 1994;93:768-775.
16. Klein SA, Dobmeyer JM, Dobmeyer TS, et al. Demonstration of the Th1 to Th2 cytokine shift during the course of HIV-1 infection using cytoplasmic cytokine detection on single cell level by flow cytometry. AIDS. 1997;11:1111-1118.
17. Rizzardini G, Piconi S, Ruzzante S, et al. Immunological activation markers in the serum of African and European HIV-seropositive and seronegative individuals. AIDS. 1997;10:1535-1542.
18. Marshall JD, Chehimi J, Gri G, et al. The interleukin-12-mediated pathway of immune events is dysfunctional in human immunodeficiency virus-infected individuals. Blood. 1999;94:1003-1011.
19. Clerici M, Lucey DR, Berzofsky JA, et al. Restoration of HIV-specific cell mediated immune responses by interleukin-12 in vitro. Science. 1993;262:1721-1724.
20. Donaghy H, Pozniak A, Gazzard B, et al. Loss of blood CD11c(+) myeloid and CD11c(−) plasmacytoid dendritic cells in patients with HIV-1 infection correlates with HIV-1 RNA virus load. Blood. 2001;98:2574-2576.
21. Pacanowski J, Kahi S, Baillet M, et al. Reduced blood CD123+ (lymphoid) and CD11c+ (myeloid) dendritic cell numbers in primary HIV-1 infection. Blood. 2001;98:3016-3021.
22. Barron MA, Blyveis N, Palmer BE, et al. Influence of plasma viremia on defects in number and immunophenotype of blood dendritic cell subsets in human immunodeficiency virus 1-infected individuals. J Infect Dis. 2003;187:26-37.
23. Amicosante M, Gioia C, Montesano C, et al. Computer-based design of an HLA-haplotype and HIV-clade independent cytotoxic T-lymphocyte assay for monitoring HIV-specific immunity. Mol Med. 2002;8:798-807.
24. Palucka KA, Taquet N, Sanchez-Chapuis F, et al. Dendritic cells as the terminal stage of monocyte differentiation. J Immunol. 1998;160:4587-4595.
25. Delneste Y, Charbonnier P, Herbault N, et al. Interferon-gamma switches monocyte differentiation from dendritic cells to macrophages. Blood. 2003;101:143-150.
26. Chomorat P, Dantin C, Bennett L, et al. TNF skews monocyte differentiation from macrophages to dendritic cells. J Immunol. 2003;171:2262-2269.
27. Chomorat P, Banchereau J, Davoust J, et al. IL-6 switches the differentiation of monocytes from dendritic cells to macrophages. Nat Immunol. 2000;1:510-514.
28. Underhill DM, Ozinsky A. Toll-like receptors: key mediators of microbe detection. Curr Opin Immunol. 2002;14:103-110.
29. Mariotti S, Teloni R, Iona E, et al. Mycobacterium tuberculosis subverts the differentiation of human monocytes into dendritic cells. Eur J Immunol. 2002;32:3050-3058.
30. Chougnet C, Cohen SS, Kawamura T, et al. Normal immune function of monocyte-derived dendritic cells from HIV-infected individuals: implications for immunotherapy. J Immunol. 1999;163:1666-1673.
31. Sanchez-Torres C, Garcia-Romo GS, Cornejo-Cortes MA, et al. CD16+ and CD16− human blood monocyte subsets differentiate in vitro to dendritic cells with different abilities to stimulate CD4+ T cells. Int Immunol. 2001;13:1571-1581.
32. Blumenstein M, Boekstegers P, Fraunberger P, et al. Cytokine production precedes the expansion of CD14+CD16+ monocytes in human sepsis: a case report of a patient with self-induced septicemia. Shock. 2001;8:73-75.
33. Saleh MN, Goldman SJ, LoBuglio AF, et al. CD16+ monocytes in patients with cancer: spontaneous elevation and pharmacologic induction by recombinant human macrophage colony-stimulating factor. Blood. 1995;85:2910-2917.
34. Thieblemont N, Weiss L, Sadeghi HM, et al. CD14lowCD16high: a cytokine-producing monocyte subset which expands during human immunodeficiency virus infection. Eur J Immunol. 1995;25:3418-3424.
35. Donaghy H, Gazzard B, Gotch F, et al. Dysfunction and infection of freshly isolated blood myeloid and plasmacytoid dendritic cells in patients infected with HIV-1. Blood. 2003;101:4505-4511.
36. Sapp M, Engelmayer J, Larsson M, et al. Dendritic cells generated from blood monocytes of HIV-1 patients are not infected and act as competent antigen presenting cells eliciting potent T-cell responses. Immunol Lett. 1999;66:121-128.
37. Hsieh SM, Pan SC, Hung CC, et al. Differential impact of late-stage HIV-1 infection on in vitro and in vivo maturation of myeloid dendritic cells. J Acquir Immune Defic Syndr. 2003;33:413-419.
38. Clerici M, Shearer GM. The Th 1-Th2 hypothesis of HIV infection: new insights. Immunol Today. 1994;15:575-581.
39. Wang J, Crawford K, Yuan M, et al. Regulation of CC chemokine receptor 5 and CD4 expression and human immunodeficiency virus type 1 replication in human macrophages and microglia by T helper type 2 cytokines. J Infect Dis. 2002;185:885-897.
40. Kedzierska K, Crowe SM, Turville S, et al. The influence of cytokines, chemokines and their receptors on HIV-1 replication in monocytes and macrophages. Rev Med Virol. 2003;13:39-56.
41. Szabo G, Dolganiuc A. Subversion of plasmacytoid and myeloid dendritic cell functions in chronic HCV infection. Immunobiology. 2005;210:237-247.
42. Yang L, Carbone DP. Tumor-host immune interactions and dendritic cell dysfunction. Adv Cancer Res. 2004;92:13-27.
43. Banchereau J, Palucka AK. Dendritic cells as therapeutic vaccines against cancer. Nat Rev Immunol. 2005;5:296-306.
44. Trinchieri G. Proinflammatory and immunoregulatory functions of interleukin-12. Int Rev Immunol. 1998;16:365-396.
45. Trinchieri G. Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat Rev Immunol. 2003;3:133-146.
46. Levings MK, Gregari S, Tresoldi E, et al. Differentiation of Tr1 cells by immature dendritic cells requires IL-10 but not CD25+CD4+ Tr cells. Blood. 2005;105:1162-1169.
47. Rizzardini G, Trabattoni D, Saresella M, et al. Immune activation in HIV-infected African individuals. Italian-Ugandan AIDS Cooperation Program. AIDS. 1998;12:2387-2396.
48. Clerici M, Butto S, Lukwiya M, et al. Immune activation in Africa is environmentally-driven and is associated with upregulation of CCR5. Italian-Ugandan AIDS Project. AIDS. 2000;14:2083-2092.
49. Gogolak P, Rethi B, Szatmari I, et al. Differentiation of CD1a− and CD1a+ monocyte-derived dendritic cells is biased by lipid environment and PPARγ. Blood. 2007;109:643-652.
This article has been cited 2 time(s).
Journal of Infectious DiseasesMonocyte-Derived Dendritic Cells from HIV Type 1-Infected Individuals Show Reduced Ability to Stimulate T Cells and Have Altered Production of Interleukin (IL)-12 and IL-10Journal of Infectious Diseases
Advances in Immunology, Vol 102Antigen Presentation by CD1: Lipids, T Cells, and NKT Celts in Microbial ImmunityAdvances in Immunology, Vol 102
AIDS; cell differentiation; dendritic cells; human; Th1/Th2 cells
© 2007 Lippincott Williams & Wilkins, Inc.
Highlight selected keywords in the article text.