Jihed Chehimi

Constitutive and induced expression of DC-SIGN on dendritic cell and macrophage subpopulations in situ and in vitro

DC‐SIGN is a C‐type lectin, highly expressed on the surface ofimmature dendritic cells (DCs), that mediates efficient infection of Tcells in trans by its ability to bind HIV‐1, HIV‐2, and SIV. Inaddition, the ability of DC‐SIGN to bind adhesion molecules on surfacesof naïve T cells and endothelium also suggests its involvementin T‐cell activation and DC trafficking. To gain further insights intothe range of expression and potential functions of DC‐SIGN, weperformed a detailed analysis of DC‐SIGN expression in adult and fetaltissues and also analyzed its regulated expression on cultured DCs andmacrophages. First, we show that DC‐SIGN expression is restricted tosubsets of immature DCs in tissues and on specialized macrophages inthe placenta and lung. There were no overt differences between DC‐SIGNexpression in adult and fetal tissues except that DC‐SIGN expression inalveolar macrophages was only present after birth. Similarly, intissues, DC‐SIGN was observed primarily on immature (CD83‐negative)DCs. Secondly, in the peripheral blood, we found expression of DC‐SIGNon a small subset of BDCA‐2+ plasmacytoid DC precursors (pDC2),concordant with our finding of large numbers of DC‐SIGN‐positive cellsin allergic nasal polyps (previously shown to be infiltrated by DC2).Triple‐label confocal microscopy indicated that DC‐SIGN was colocalizedwith BDCA‐2 and CD123 on DCs in nasal polyp tissue. Consistent withthis finding is our observation that DC‐SIGN can be up‐regulated onmonocyte‐derived macrophages upon exposure to the Th2 cytokine, IL‐13. In summary, our data demonstrate the relevant populations of DC andmacrophages that express DC‐SIGN in vivo where it may impact theefficiency of virus infection and indicate that DC‐SIGN expression maybe involved in the Th2 axis of immunity.

Persistent decreases in blood plasmacytoid dendritic cell number and function despite effective highly active antiretroviral therapy and increased blood myeloid dendritic cells in HIV-infected individuals.

Dendritic cells (DC) have an instrumental role in the activation and function of both innate and adaptive immune responses. In humans, at least two distinct DC subsets have been characterized based on phenotypic markers: the myeloid DC (MDC) and the plasmacytoid DC (PDC). Both subsets are critical producers of cytokines (IL-12 for MDC and type I/II IFNs for PDC) and are functionally different. We show in this study that HIV+ individuals have a significant decrease in the number of the Lin−HLA-DR+CD123+ and BDCA-2+ PDC compared with uninfected donors (p = 0.0001). HIV+ individuals also have a sustained impairment in viral-induced IFN-α production (p < 0.0001). The decrease of the PDC subsets did not correlate with CD4 count or viral load and was not reversed in subjects under virally suppressive treatment, suggesting an irreversible change after infection. By contrast, the absolute number and median frequency of MDC in HIV-infected individuals were similar to those observed in uninfected controls, while a significant decrease was present in subjects with >5000 HIV-1 copies/ml. The inverse association with viral load of the MDC number, but not of IFN-α secretion or the number of PDC, suggests a role for MDC in viral control. Our data suggest that DC subsets are differentially reconstituted during the immune recovery associated with antiviral therapy. The persistent impairment of certain DC subsets may result in a sustained defect in DC-mediated innate immune functions despite an effective treatment regimen.

Interleukin-12: a bridge between innate resistance and adaptive immunity with a role in infection and acquired immunodeficiency.

Interleukin-12 (IL-12) is a disulfide-linked heterodimeric cytokine originally identified as a product of EBV-transformed B cell lines. Monocyte/macrophages are the physiologically most relevant producers of IL-12, in response to both Gram-positive and -negative bacteria, bacterial products, and intracellular parasites. Although IL-12 has an enhancing effect on the survival and growth of early hematopoietic progenitor cells, most of the IL-12 biological activity has been described on T and NK cells, on which it induces production of lymphokines, primarily IFN-γ, enhances cytotoxic activity, and, in cooperation with other stimuli, increases proliferation. IL-12 is an inducer of development of T helper type 1 (Th-1) cells and the equilibrium between IL-12 and IL-4 is probably important for the balancein vivo between Th-1 and Th-2 responses. IL-12 has an important role in the host resistance to infection, in particular to intracellular pathogens, by activating macrophages through induction of IFN-γ from NK and T cells and by enhancing cell-mediated immune responses, dependent on Th-1 cell development. Peripheral blood mononuclear cells from HIV-seropositive individuals are impaired in their ability to produce IL-12 in response to bacterial stimulation, and IL-12 restoresin vitro some of the depressed immunological functions, suggesting that a defect in IL-12 production may have a pathogenic role in the immunodeficiency of HIV-infected individuals. Natural IL-12 appears to provide a regulatory link between innate resistance and the development of the antigen-specific adaptive immune response and the recombinant protein has therapeutic potential because of its activity against tumors and infections and its effectiveness as an adjuvant enhancing cell-mediated immunity in vaccination.

Natural Killer cell Stimulatory Factor (NKSF) or interleukin-12 is a key regulator of immune response and inflammation

Natural Killer cell Stimulatory Factor (NKSF) or interleukin-12 (IL-12) is a heterodimeric cytokine of 70 kDa formed by a heavy chain of 40 kDa (p40) and a light chain of 35 kDa (p35). Although it was originally identified and purified from the supernatant of Epstein-Barr virus-transformed B cell lines, it has been shown that among peripheral blood cells NKSF/IL-12 is predominantly produced by monocytes, with lower production by B cells and other accessory cells. The most powerful inducers of NKSF/IL-12 production are bacteria, bacterial products and parasites. In addition to the biologically active p70 heterodimer, the cells producing NKSF/IL-12 also secrete a large excess of monomeric p40, a molecule with no demonstrable biological activity. NKSF/IL-12 is active on T lymphocytes and NK cells on which it induces production of lymphokines, enhancement of cytotoxic activity and mitogenic effects. NKSF/IL-12 induces T and NK cells to produce IFN-gamma and synergizes with other IFN-gamma inducers in this effect. In vitro, and probably in vivo, NKSF/IL-12 is required for optimal IFN-gamma production. When human lymphocytes are stimulated with antigens in vitro, addition of exogenous NKSF/IL-12 to the culture induces differentiation of T helper type 1 (Th1) cells, whereas neutralization of endogenous NKSF/IL-12 with antibodies favors differentiation of Th2 cells. IFN-gamma, a product of Th1 cells, enhances NKSF/IL-12 production by mononuclear cells, whereas IL-10 and IL-4, products of Th2 cells, efficiently inhibit it. Therefore, NKSF/IL-12 appears to be an important inducer of Th1 responses produced by accessory cells during early antigenic stimulation and its production is regulated by a positive feedback mechanism mediated by Th1 cells through IFN-gamma and a negative one by Th2 cells through IL-10 and IL-4. The balance of IL-12 production versus IL-10 and IL-4 production early during an immune response might therefore be instrumental in determining Th1-type versus Th2-type immune responses. Because of this potential role of IL-12 during immune responses, our results demonstrating the impaired ability of HIV seropositive patients to produce NKSF/IL-12 in response to bacterial stimulation suggest that this defect in NKSF/IL-12 production might be a factor contributing to their immune depression.

Sustained Impairment of IFN-γ Secretion in Suppressed HIV-Infected Patients Despite Mature NK Cell Recovery: Evidence for a Defective Reconstitution of Innate Immunity

The impairment of NK cell functions in the course of HIV infection contributes to a decreased resistance against HIV and other pathogens. We analyzed the proportion of mature and immature NK cell subsets, and measured subsets of IFN-γ and TNF-α-producing NK and T cells in viremic or therapy-suppressed HIV-infected subjects, and noninfected control donors. Viremic HIV+ individuals had significantly lower proportions of mature CD3−/CD161+/CD56+ NK cells and of IFN-γ-producing NK cells compared with noninfected donors, independent of CD4+ T cell counts. HIV-infected subjects with undetectable viral load recovered mature CD3−/CD161+/CD56+ NK cells and cytotoxicity against tumor (K562) and HSV-infected target cells to percentages comparable with those of uninfected individuals, but their NK cells remained impaired in their ability to produce IFN-γ. In parallel to these ex vivo findings, in vitro NK cell differentiation of CD34-positive cord blood precursors in the presence of R5 or X4 HIV-1 resulted in the production of NK cells with a normal mature phenotype, but lacking the ability to produce IFN-γ, whereas coculture of uninfected PBMC with HIV failed to affect mature NK cell properties or IFN-γ secretion. Altogether, our findings support the hypothesis that mature NK cell phenotype may be uncoupled from some mature functions following highly active antiretroviral therapy-mediated suppression of HIV-1, and indicate that relevant innate immune functions of NK cell subsets may remain altered despite effective viral suppression following antiretroviral treatment.

Baseline Viral Load and Immune Activation Determine the Extent of Reconstitution of Innate Immune Effectors in HIV-1-Infected Subjects Undergoing Antiretroviral Treatment

We analyzed dendritic cell (DC) and NK cell compartments in relation to CD4 recovery in 21 HIV-infected subjects followed to <50 copies/ml once starting antiretroviral therapy (ART) and observed for 52 wk of sustained suppression. Although CD4 counts increased in all subjects in response to ART, we observed a restoration of functional plasmacytoid DC (PDC) after 52 wk of sustained suppression under ART (from 1850 cells/ml to 4550 cells/ml) to levels comparable to controls (5120 cells/ml) only in subjects with a low baseline viral load, which also rapidly suppressed to <50 copies/ml upon ≤60 days from ART initiation. Recovery of PDC at week 52 correlates with level of CD95 expression on CD8 T cells and PDC frequency following first ART suppression. NK cytotoxic activity increased rapidly upon viral suppression (VS) and correlated with PDC function at week 52. However, restoration of total NK cells was incomplete even after 52 wk on ART (73 cells/μl vs 122 cells/μl in controls). Direct reconstitution experiments indicate that NK cytotoxic activity against virally infected target cells requires DC/NK cooperation, and can be recovered upon sustained VS and recovery of functional PDC (but not myeloid DC) from ART-suppressed subjects. Our data indicate that viremic HIV-infected subjects may have different levels of reconstitution of DC and NK-mediated function following ART, with subjects with lower initial viremia and the greatest reduction of baseline immune activation at VS achieving the greatest level of innate effector cell reconstitution.

HIV-1 transmission and cytokine-induced expression of DC-SIGN in human monocyte-derived macrophages

Dendritic cell‐specific intercellular adhesion molecule‐3‐grabbing nonintegrin (DC‐SIGN) has been described as an attachment molecule for human immunodeficiency virus type 1 (HIV‐1) with the potential to mediate its transmission. We examined DC‐SIGN expression in monocyte‐derived macrophages (MDM) and its role in viral transmission when MDM were exposed to interleukin (IL)‐13, IL‐4, or interferon‐γ (IFN‐γ). We show that IL‐13 and IL‐4 increase transcripts, total protein, and cell‐surface expression of DC‐SIGN in all MDM tested, IFN‐γ results ranged from no change to up‐regulation of surface expression, and message and total protein were, respectively, induced in all and 86% of donors tested. Transmission experiments of HIV‐1 X4 between cytokine‐treated MDM to Sup‐T1 cells showed no association between total transmission and DC‐SIGN up‐regulation. IL‐4 but not IL‐13 resulted in a less than twofold increase in MDM viral transmission to CD4+ T cells in spite of a fourfold up‐regulation in DC‐SIGN expression by either cytokine. In contrast, IFN‐γ treatment induced a decrease in total transmission by at least two‐thirds, despite its induction of DC‐SIGN. Soluble mannan resulted in a greater inhibition of viral transmission to CD4+ T cells than neutralizing anti‐DC‐SIGN monoclonal antibody (67–75% vs. 39–48%), supporting the role of mannose‐binding receptors in viral transmission. Taken together, results show that DC‐SIGN regulation in MDM does not singly predict the transmission potential of this cell type.

Dendritic and Natural Killer Cell Subsets Associated with Stable or Declining CD4+ Cell Counts in Treated HIV-1–Infected Children

BACKGROUND Natural killer (NK) cells and plasmacytoid and myeloid dendritic cells (DCs) are depleted, and their function impaired, in advanced adult human immunodeficiency virus (HIV)-1 infection. Studies in perinatally infected children are lacking. METHODS Percentages of NK cells and plasmacytoid and myeloid DCs were evaluated by flow cytometry. Forty children with perinatal HIV-1 infection were compared with 11 age-matched, uninfected children. Plasmacytoid and myeloid DC function was evaluated by activation-induced cytokine secretion. RESULTS Virally suppressed children had normal levels of circulating plasmacytoid and myeloid DCs and total NK cells but had sustained depletion of a mature (CD3-/161+/56+/16+) NK cell subset and decreased interferon- alpha secretion by plasmacytoid DCs. Despite similar viral loads, percentages of myeloid and plasmacytoid DCs and mature NK cells were significantly lower in viremic children with a history of decreasing CD4+ cell percentages, compared with children with stable CD4+ cell counts. CONCLUSIONS Children achieve partial reconstitution of myeloid and plasmacytoid DCs and NK cells during viral suppression; irrespective of viral load, a clinical history of decreasing CD4+ cell percentage is associated with greater depletion of these subsets. We hypothesize that the evaluation of selected innate-immunity effector cells may serve as a marker of CD4+ cell loss in pediatric HIV-1 infection.

Inability of plasmacytoid dendritic cells to directly lyse HIV-infected autologous CD4+ T cells despite induction of tumor necrosis factor-related apoptosis-inducing ligand.

ABSTRACT The function of plasmacytoid dendritic cells (PDC) in chronic human immunodeficiency virus type 1 (HIV-1) infection remains controversial with regard to its potential for sustained alpha interferon (IFN-α) production and induction of PDC-dependent tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-mediated cytotoxicity of HIV-infected cells. We address these areas by a study of chronically HIV-1-infected subjects followed through antiretroviral therapy (ART) interruption and by testing PDC cytolytic function against autologous HIV-infected CD4+ T cells. Rebound in viremia induced by therapy interruption showed a positive association between TRAIL and viral load or T-cell activation, but comparable levels of plasma IFN-α/β were found in viremic ART-treated and control subjects. While PDC from HIV-infected subjects expressed less interferon regulator factor 7 (IRF-7) and produced significantly less IFN-α upon Toll-like receptor 7/9 (TLR7/9) engagement than controls, membrane TRAIL expression in PDC from HIV+ subjects was increased. Moreover, no significant increase in death receptor 5 (DR5) expression was seen in CD4+ T cells from viremic HIV+ subjects compared to controls or following in vitro infection/exposure to infectious and noninfectious virus or exogenous IFN-α, respectively. Although activated PDC killed the DR5-expressing HIV-infected Sup-T1 cell line, PDC did not lyse primary autologous HIV+ CD4+ T cells yet could provide accessory help for NK cells in killing HIV-infected autologous CD4+ T cells. Taken together, our data show a lack of sustained high levels of soluble IFN-α in chronic HIV-1 infection in vivo and document a lack of direct PDC cytolytic activity against autologous infected or uninfected CD4+ T cells.

NK Cell Lysis of HIV-1-Infected Autologous CD4 Primary T Cells: Requirement for IFN-Mediated NK Activation by Plasmacytoid Dendritic Cells

In vivo, several mechanisms have been postulated to protect HIV-1-infected cells from NK surveillance. In vitro, previous research indicates HIV-1-infected autologous CD4+ primary T cells are resistant to NK lysis. We hypothesized that NK lysis of HIV-1-infected target cells would be augmented by the presence of accessory cells and/or accessory cell factors. In this study, we show that stimulation of plasmacytoid dendritic cells (PDC) with the TLR9 agonist, CpG ODN 2216, triggered NK lysis of HIV-1-infected autologous CD4+ primary T cells. PDC-stimulated NK lysis was dependent upon MHC class I (MHC-I) down-regulation on infected cells, and primary HIV-1 isolates that exhibited enhanced MHC-I down-regulation were more susceptible to NK-mediated lysis. PDC-stimulated NK lysis of HIV-1-infected autologous CD4+ primary T cells was blocked by neutralizing Abs to type 1 IFN and was perforin/granzyme dependent. Overall, our data suggest that HIV-infected cells are not innately resistant to NK lysis, and that exogenous NK stimulation derived from PDC can trigger NK cytotoxicity against HIV-1-infected autologous CD4+ primary T cells.