Luis J. Montaner

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.

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.

IL-12 Suppression During Experimental Endotoxin Tolerance: Dendritic Cell Loss and Macrophage Hyporesponsiveness

Endotoxin tolerance, the transient, secondary down-regulation of a subset of endotoxin-driven responses after exposure to bacterial products, is thought to be an adaptive response providing protection from pathological hyperactivation of the innate immune system during bacterial infection. However, although protecting from the development of sepsis, endotoxin tolerance also can lead to fatal blunting of immunological responses to subsequent infections in survivors of septic shock. Despite considerable experimental effort aimed at characterizing the molecular mechanisms responsible for a variety of endotoxin tolerance-related phenomena, no consensus has been achieved yet. IL-12 is a macrophage- and dendritic cell (DC)-derived cytokine that plays a key role in pathological responses to endotoxin as well as in the induction of protective responses to pathogens. It recently has been shown that IL-12 production is suppressed in endotoxin tolerance, providing a likely partial mechanism for the increased risk of secondary infections in sepsis survivors. We examined the development of IL-12 suppression during endotoxin tolerance in mice. Decreased IL-12 production in vivo is clearly multifactorial, involving both loss of CD11chigh DCs as well as alterations in the responsiveness of macrophages and remaining splenic DCs. We find no demonstrable mechanistic role for B or T lymphocytes, the soluble mediators IL-10, TNF-α, IFN-αβ, or nitric oxide, or the NF-κB family members p50, p52, or RelB.

Pegylated Interferon Alfa-2a Monotherapy Results in Suppression of HIV Type 1 Replication and Decreased Cell-Associated HIV DNA Integration

BACKGROUND Antiretroviral therapy (ART)-mediated immune reconstitution fails to restore the capacity of the immune system to spontaneously control human immunodeficiency virus (HIV) replication. METHODS A total of 23 HIV type 1 (HIV-1)-infected, virologically suppressed subjects receiving ART (CD4(+) T-cell count, >450 cells/μL) were randomly assigned to have 180 μg/week (for arm A) or 90 μg/week (for arm B) of pegylated (Peg) interferon alfa-2a added to their current ART regimen. After 5 weeks, ART was interrupted, and Peg-interferon alfa-2a was continued for up to 12 weeks (the primary end point), with an option to continue to 24 weeks. End points included virologic failure (viral load, ≥ 400 copies/mL) and adverse events. Residual viral load and HIV-1 DNA integration were also assessed. RESULTS At week 12 of Peg-interferon alfa-2a monotherapy, viral suppression was observed in 9 of 20 subjects (45%), a significantly greater proportion than expected (arm A, P = .0088; arm B, P = .0010; combined arms, P < .0001). Over 24 weeks, both arms had lower proportions of subjects who had viral load, compared with the proportion of subjects in a historical control group (arm A, P = .0046; arm B, P = .0011). Subjects who had a sustained viral load of <400 copies/mL had decreased levels of integrated HIV DNA (P = .0313) but increased residual viral loads (P = .0078), compared with subjects who experienced end-point failure. CONCLUSIONS Peg-interferon alfa-2a immunotherapy resulted in control of HIV replication and decreased HIV-1 integration, supporting a role for immunomediated approaches in HIV suppression and/or eradication. CLINICAL TRIALS REGISTRATION NCT00594880.

Enhancement of Human Immunodeficiency Virus Type 1—Specific CD4 and CD8 T Cell Responses in Chronically Infected Persons after Temporary Treatment Interruption

Immunologic and virologic outcomes of treatment interruption were compared for 5 chronically human immunodeficiency virus (HIV)-infected persons who have maintained antiretroviral therapy-mediated virus suppression, as compared with 5 untreated controls. After a median interruption of 55 days of therapy accompanied by rebound of virus, reinitiated therapy in 4 of 5 subjects resulted in suppression of 98.86% of plasma virus load by 21-33 days and no significant decrease in CD4 T cell percentage from baseline. Increased T helper responses against HIV-1 p24 antigen (P=. 014) and interferon-gamma-secreting CD8 T cell responses against HIV-1 Env (P=.004) were present during interruption of therapy and after reinitiation of treatment. The remaining subject whose treatment was interrupted did not resume treatment and continued to have a low virus load (<1080 HIV-1 RNA copies/mL) and persistent antiviral cell-mediated responses. In summary, cellular immunity against autologous HIV-1 has the potential to be acutely augmented in association with temporary treatment interruption in chronically infected persons.

Inhibition of IL-12 Production in Human Monocyte-Derived Macrophages by TNF

IL-12 is a pivotal cytokine that links the innate and adaptive immune responses. TNF-α also plays a key role in orchestrating inflammation and immunity. The reciprocal influence of these two inflammatory mediators on each other may have significant impact on the cytokine balance that shapes the type and extent of immune responses. To investigate the relationship between TNF-α and IL-12 production, we analyzed the effects of exposure of human monocyte-derived macrophages to TNF-α on LPS- or Staphylococcus aureus-induced IL-12 production in the presence or absence of IFN-γ. TNF-α is a potent inhibitor of IL-12 p40 and p70 secretion from human macrophages induced by LPS or S. aureus. IL-10 is not responsible for the TNF-α-mediated inhibition of IL-12. TNF-α selectively inhibits IL-12 p40 steady-state mRNA, but not those of IL-12 p35, IL-1α, IL-1β, or IL-6. Nuclear run-on analysis identified this specific inhibitory effect at the transcriptional level for IL-12 p40 without down-regulation of the IL-12 p35 gene. The major transcriptional factors identified to be involved in the regulation of IL-12 p40 gene expression by LPS and IFN-γ, i.e., c-Rel, NF-κB p50 and p65, IFN regulatory factor-1, and ets-2, were not affected by TNF-α when examined by nuclear translocation and DNA binding. These data demonstrate a selective negative regulation on IL-12 by TNF-α, identifying a direct negative feedback mechanism for inflammation-induced suppression of IL-12 gene expression.

Circulating Monocytes in HIV-1-Infected Viremic Subjects Exhibit an Antiapoptosis Gene Signature and Virus- and Host-Mediated Apoptosis Resistance

Mechanisms that may allow circulating monocytes to persist as CD4 T cells diminish in HIV-1 infection have not been investigated. We have characterized steady-state gene expression signatures in circulating monocytes from HIV-infected subjects and have identified a stable antiapoptosis gene signature comprised of 38 genes associated with p53, CD40L, TNF, and MAPK signaling networks. The significance of this gene signature is indicated by our demonstration of cadmium chloride- or Fas ligand-induced apoptosis resistance in circulating monocytes in contrast to increasing apoptosis in CD4 T cells from the same infected subjects. As potential mechanisms in vivo, we show that monocyte CCR5 binding by HIV-1 virus or agonist chemokines serves as independent viral and host modulators resulting in increased monocyte apoptosis resistance in vitro. We also show evidence for concordance between circulating monocyte apoptosis-related gene expression in HIV-1 infection in vivo and available datasets following viral infection or envelope exposure in monocyte-derived macrophages in vitro. The identification of in vivo gene expression associated with monocyte resistance to apoptosis is of relevance to AIDS pathogenesis since it would contribute to: 1) maintaining viability of infection targets and long-term reservoirs of HIV-1 infection in the monocyte/macrophage populations, and 2) protecting a cell subset critical to host survival despite sustained high viral replication.

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.

Evidence for the innate immune response as a correlate of protection in human immunodeficiency virus (HIV)-1 highly exposed seronegative subjects (HESN)

The description of highly exposed individuals who remain seronegative (HESN) despite repeated exposure to human immunodeficiency virus (HIV)‐1 has heightened interest in identifying potential mechanisms of HIV‐1 resistance. HIV‐specific humoral and T cell‐mediated responses have been identified routinely in HESN subjects, although it remains unknown if these responses are a definitive cause of protection or merely a marker for exposure. Approximately half of HESN lack any detectible HIV‐specific adaptive immune responses, suggesting that other mechanisms of protection from HIV‐1 infection also probably exist. In support of the innate immune response as a mechanism of resistance, increased natural killer (NK) cell activity has been correlated with protection from infection in several high‐risk cohorts of HESN subjects, including intravenous drug users, HIV‐1 discordant couples and perinatally exposed infants. Inheritance of protective NK KIR3DL1high and KIR3DS1 receptor alleles have also been observed to be over‐represented in a high‐risk cohort of HESN intravenous drug users and HESN partners of HIV‐1‐infected subjects. Other intrinsic mechanisms of innate immune protection correlated with resistance in HESN subjects include heightened dendritic cell responses and increased secretion of anti‐viral factors such as β‐chemokines, small anti‐viral factors and defensins. This review will highlight the most current evidence in HESN subjects supporting the role of epithelial microenvironment and the innate immune system in sustaining resistance against HIV‐1 infection. We will argue that as a front‐line defence the innate immune response determines the threshold of infectivity that HIV‐1 must overcome to establish a productive infection.