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(The FASEB Journal. 2006;20:2198-2208.)
© 2006 FASEB

The immunoregulatory effects of HIV-1 Nef on dendritic cells and the pathogenesis of AIDS

Maria Giovanna Quaranta, Benedetta Mattioli, Luciana Giordani and Marina Viora1

Department of Drug Research and Evaluation, Istituto Superiore di Sanità, Rome, Italy

1Correspondence: Department of Drug Research and Evaluation, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. E-mail: viora{at}iss.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 ROLE OF DC IN...
 HIV-1 NEF PROTEIN
 EFFECT OF NEF ON...
 Phenotype
 Morphology
 Cytokine and chemokine...
 Stimulatory capacity
 Signal transduction
 CONCLUSIONS
 REFERENCES
 
Dendritic cells (DC) play a crucial role in the generation and regulation of immunity, and their interaction with HIV is relevant in the pathogenesis of AIDS favoring both the initial establishment and spread of the infection and the development of antiviral immunity. HIV-1 Nef is an essential factor for efficient viral replication and pathogenesis, and several studies have been addressed to assess the possible influence of endogenous or exogenous Nef on DC biology. Our findings and other reported data described in this review demonstrate that Nef subverts DC biology interfering with phenotypical, morphological, and functional DC developmental programs, thus representing a viral tool underlying AIDS pathogenesis. This review provides an overview on the mechanism by which Nef, hijacking DC functional activity, may favor both the replication of HIV-1 and the escape from immune surveillance. Overall, the findings described here may contribute to the understanding of Nef function, mechanism of action, and cellular partners. Further elucidation of genes induced through Nef signaling in DC could reveal pathways used by DC to drive HIV spread and will be critical to identify therapeutic strategies to bias the DC system toward activation of antiviral immunity instead of facilitating virus dissemination.—Quaranta, M. G., Mattioli, B., Giordani, L., Viora, M. The immunoregulatory effects of HIV-1 Nef on dendritic cells and the pathogenesis of AIDS.


Key Words: antigen presenting cells • viral spreading • immune evasion


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
ROLE OF DC IN...
 HIV-1 NEF PROTEIN
 EFFECT OF NEF ON...
 Phenotype
 Morphology
 Cytokine and chemokine...
 Stimulatory capacity
 Signal transduction
 CONCLUSIONS
 REFERENCES
 
DURING HIV INFECTION, the perturbation of the adaptive and innate immune responses contributes to the progressive immunosuppression leading to an increased susceptibility to opportunistic infections and neoplastic diseases. Several impairments observed in HIV-infected patients include a gradual loss of CD4+ T cells, CD8+ T cell dysfunction and a decreased number and function of natural killer (NK) cells. Such defects may be a consequence of the functional impairment and variation in the number of dendritic cells (DC) associated with HIV infection. DC represent a viral reservoir acting as primary target and HIV carriers for infection of permissive CD4+ T cells. HIV-1 takes advantage of DC biology to facilitate the onset of infection and its dissemination to surrounding permissive cells. HIV-1 codes for proteins, including the structural and accessory proteins, that interacting with DC may contribute to AIDS pathogenesis (1) .

This review summarizes recent progress in the field of DC-HIV interactions, focusing on the immunomodulatory effect of the accessory Nef protein and its role in the pathogenesis of HIV-1 infection.


   ROLE OF DC IN HIV-1 INFECTION
TOP
 ABSTRACT
 INTRODUCTION
 ROLE OF DC IN...
HIV-1 NEF PROTEIN
 EFFECT OF NEF ON...
 Phenotype
 Morphology
 Cytokine and chemokine...
 Stimulatory capacity
 Signal transduction
 CONCLUSIONS
 REFERENCES
 
Upon viral infection, the immune system delivers a coordinate response, including recruitment of innate immune effectors, such as neutrophils and NK cells, the activation of antigen-specific memory B and T lymphocytes into effector cells, and the priming of naive lymphocytes. This complex program is orchestrated by DC (2 3 4) .

DC are instrumental in the development of pathogen-specific immune responses and are well equipped for activation of both the innate and adaptive immune response. Their priming ability is acquired upon maturation and is characterized by the activation of different transcriptional factors, leading to the modulation of genes involved in cytoskeleton rearrangement, cell-to-cell communication, antigen processing, control of migration, and regulation of inflammatory responses (5 6 7 8) . Regulated migration of DC is central to the induction of physiological immune responses, and this process necessitates plasticity of the cytoskeleton. DC, unlike other antigen presenting cells (APC), actively polarize their actin cytoskeleton during interaction with T cells, leading to the formation of the immunological synapse (9 , 10) .

It has been demonstrated that some DC-tropic viruses, such as influenza virus, leave DC function intact (11) , whereas other DC-tropic viruses, such as HIV and cytomegalovirus (CMV), have evolved strategies to impair DC functions, thereby enhancing the chance of virus to persist and escape immune surveillance (12 13 14) .

DC interaction with HIV is relevant in the pathogenesis of AIDS, favoring both the initial establishment and spread of the infection and the development of antiviral immunity (15 , 16) . Immature DC (iDC) able to capture antigens are present in the skin and mucosa and therefore could be among the first cells encountering HIV (17) . Mucosal and blood DC represent the first HIV-1 targets following sexual transmission (18) and transmission via blood (19) , respectively. Both myeloid DC (myDC) and plasmacytoid DC (pDC) express the required receptors for HIV-1 entry, i.e., CD4, CXCR4, and CCR5, suggesting that they could be infected, although with a lower efficiency than CD4+ T cells or macrophages (20 21 22) . The evidence currently available indicates that very few DC appear to be infected in vivo. Studies of splenic tissue have shown an infection rate of 1/3000 DC (23) , and similar studies of lymph nodes have also shown low rates of infection of DC (24) .

In addition to CD4, CCR5, and CXCR4, iDC express DC-SIGN (CD209), a C-type lectin that binds gp120 and might play a role in virus capture and transmission occurring in the virologic synapse (18 , 25) . DC-SIGN is expressed in DC derived from blood monocytes or found in lymphoid tissues and beneath genital surfaces (26) . Binding of gp120 by DC-SIGN does not lead to viral fusion; however, the virus is internalized through DC-SIGN into a low-pH compartment in which incoming virus escapes lysosomal degradation and remains infectious for several days before transmission to T cells (27) . Contrary to previous observations, it has been recently demonstrated that DC-SIGN-mediated HIV internalization is dispensable for both trans-enhancement of T cell infection and retention of viral infectivity. Moreover, long-term transfer of HIV to T cells requires viral fusion and occurs exclusively through DC infection and transmission of viral progeny. Both DC-SIGN-mediated cis infection of DC and trans-enhancement of T cell infection may occur in vivo, either in mucosal surfaces or in lymphnodes (28) . Skin DC lack DC-SIGN but can bind HIV by other C-type lectins such as the mannose receptor (CD206) and langerin (29 , 30) . Although mature DC (mDC) do not replicate the virus, they are able to transmit virus to T cells in draining lymphoid tissues (31 , 32) . Therefore, the developmental stage of DC can influence the interaction of these cells with HIV-1. In addition, the mechanisms whereby DC promote HIV-1 replication may vary in different sites. At body surface, iDC may directly replicate virus. In peripheral lymphnodes during acute infection, virus may be transmitted via DC to T cells in the T cell areas, whereas in chronic infection virus may be transmitted primarily via DC interacting with T cells in germinal centers. At mucosal surfaces, activated DC can support virus replication and transmit virus to adjacent T cells (31) .

Several functional impairments and variations in the number of DC have been reported during HIV infection (33) . The number of both myDC and pDC is significantly decreased in the blood of HIV+ progressors, remaining unaltered in HIV+ long-term nonprogressors. These findings support the existence of a direct interaction between the two DC subsets and HIV-1. DC could be lost as a consequence either of direct lytic infection or as targets for specific CTL or through a block in DC development from peripheral CD34+ stem cells (33) . Accumulating evidence supports the notion that both myDC and pDC show impaired functional capacity in HIV+ patients (34) . Both ex vivo cultured myDC and pDC from HIV-infected individuals exhibit very weak allogeneic or autologous immunostimulatory function. Moreover, it has been reported that infected freshly isolated myDC fail to mature in culture (35) . This may be relevant, considering that iDC can induce tolerance rather than immunity (36 , 37) .

Since both DC subsets participate in the initiation of innate and adaptive immune responses, infection, depletion, and dysfunction of DC may contribute to the immunosuppression seen in HIV-1 disease. Therefore, DC play a dual role in HIV infection: they trigger both innate and adaptive immune responses to control the infection and represent a viral reservoir acting as target and HIV carriers for infection of permissive CD4+ T cells.


   HIV-1 NEF PROTEIN
TOP
 ABSTRACT
 INTRODUCTION
 ROLE OF DC IN...
 HIV-1 NEF PROTEIN
EFFECT OF NEF ON...
 Phenotype
 Morphology
 Cytokine and chemokine...
 Stimulatory capacity
 Signal transduction
 CONCLUSIONS
 REFERENCES
 
Besides three prototypical retroviral proteins (Gag, Pol, and Env) and two regulatory proteins (Tat, Rev) that are essential for viral replication, HIV (and simian immunodeficiency virus) also encodes four so-called accessory proteins Nef, Vif, Vpr, and Vpu. Studies in vivo or in primary cell types susceptible to HIV infection, have demonstrated that the accessory gene products can dramatically alter the course and severity of viral infection, replication and disease progression. Moreover, soluble HIV proteins exert bystander effects on neighboring immune cells in the absence of productive infection (38) .

HIV-1 Nef is a 27 kDa protein abundantly produced shortly after virus infection and associates with the cell membranes through N-terminal myristoylation (39 , 40) . In vivo, Nef is an essential factor for efficient viral replication and pathogenesis; in vitro, Nef also facilitates virus replication and enhances virions infectivity.

Nef exerts pleiotropic effects, involving membrane-bound or cytoplasmic stages and, depending on its intracellular localization, interferes with cellular signal transduction pathways (41 42 43 44 45) and modulates the cell surface expression of multiple membrane-associated proteins. By down-regulating major histocompatibility complex class I (MHC-I) surface molecules, Nef may reduce the recognition of HIV-infected cells by cytotoxic T lymphocytes (CTL) and may provide a selective advantage for viral persistence and replication in vivo (46 , 47) . Nef selectively down-regulates human leukocyte antigen (HLA)-A and HLA-B known to present antigens to CTL but not HLA-C and HLA-E known to protect cells from lysis by NK cells (48) . By down-regulating the CD4 molecule (49) , Nef might enhance HIV replication by preventing superinfection (50) and might interfere with TCR signaling to the advantage of the virus (51) . The down-regulation of CD4 and MHC-I occurs via two distinct mechanisms. Nef-induced CD4 down-modulation involves the internalization of surface CD4 followed by degradation via the endosomal/lysosomal pathways, and it is blocked by molecular inhibitors of clathrin coated pit-mediated endocytosis. Differently, Nef-induced MHC-I down-modulation is clathrin independent resulting in sequestration in the trans-Golgi network (52) . In addition to MHC-I and CD4 molecules, the list of membrane proteins in which intracellular trafficking is affected by Nef now includes MHC class II molecules, the costimulatory CD28 molecule, the lectin DC-SIGN, and CCR5 (53 54 55 56) . Moreover, a novel activity of Nef is the post-translational down-regulation of the MR (57) . Finally, Nef up-regulates Fas ligand (FasL) expression on the surface of the infected cells inducing apoptotic killing of attacking CTL (58) . These alterations likely promote immune evasion of infected cells and enhance the spread of viruses within the host during the natural course of HIV infection.


   EFFECT OF NEF ON DC BIOLOGY
TOP
 ABSTRACT
 INTRODUCTION
 ROLE OF DC IN...
 HIV-1 NEF PROTEIN
 EFFECT OF NEF ON...
Phenotype
 Morphology
 Cytokine and chemokine...
 Stimulatory capacity
 Signal transduction
 CONCLUSIONS
 REFERENCES
 
HIV-1 takes advantage of DC biology to facilitate the onset of infection and its dissemination to surrounding permissive cells. HIV/SIV nef-defective strains are attenuated in their ability to cause persistent infection and disease (59) . Initial studies have demonstrated deficient replication of {Delta}nef in DC-T cell cultures (60 , 61) . The replication of {Delta}nef depends on the maturation status of the DC: in iDC-T cell mixtures the level of replication of {Delta}nef is significantly lower than the wild-type (WT). In contrast, in cultures of mDC and T cells, WT and {Delta}nef replication rates are similar. Since the activation of the culture with a superantigen enables {Delta}nef replication, this suggests that Nef induces the necessary signals in iDC. It has been reported that Nef expression in CD34+ cord blood cells does not affect DC generation and maturation in suspension culture and in the thymus (62) . Thus, the block in DC development from CD34+ stem cells in HIV infection is unlikely to result from Nef expression in DC precursors. Several studies have suggested the possible influence of endogenous or exogenous Nef on DC function (63 64 65 66 67 68 69) . However, the results have been conflicting, and no clear consensus has emerged. The influence of endogenous Nef on DC function is an important question, since it is now generally accepted that HIV-1 can infect DC (at least in vitro) and infected DC express Nef.

The observation that exogenous Nef is efficiently internalized by iDC (68) led us to evaluate its effects on these cells. In addition to containing large amounts of Nef in the cytoplasm and membrane, HIV-1-infected cells release Nef into the extracellular environment (70) . Although it is not clear how a membrane-associated protein such as Nef might be secreted from an HIV-1-infected cell, soluble Nef protein is detected in sera from HIV+ patients. Fujii et al. (70) reported that a high percentage of sera from HIV-1 infected individuals contain soluble Nef (from 1 to 10 ng/ml), while sera of patients in which Nef is not detectable contain high titers of anti-Nef antibodies. Nef concentration may be even higher in lymphnodes where high levels of HIV replication is associated with close interaction between DC and T lymphocytes. Qiao et al. (71) found abundant Nef in the germinal centers of infected lymphoid follicles, and they found that IgD+ B cells contain Nef, but lacked p17, p24 and viral RNA. In addition, they found that Nef localizes together with CD21+ follicular DC and CD21– cells, including CD3+ T cells, CD11c+ DC, and CD68+ macrophages. Moreover, they reported that Nef penetrates in B cells in vitro and we previously demonstrated that Nef enters CD14+ monocytes and U937 promonocytic cell line (72) . Thus, uninfected DC and B cells might accumulate Nef not as a result of endogenous synthesis but instead as a result of internalization from the extracellular environment. HIV-1-infected cells would release Nef through a nonclassical secretory pathway or after lysis (70) . Gould et al. (73) proposed the "Trojan horse" hypothesis, which suggests that the exosomal secretion of viral proteins not packaged in viral particles includes Nef.

Until now no mechanism whereby infected cells release Nef in the extracellular environment has been provided and no such targeting motifs have been identifies on the protein. It has been reported that Nef interacts with the clathrin-associated activating protein (AP)-1 and AP-3 adaptor complexes stabilizing their association with endosomal membranes. This stabilization may facilitate coat formation and stimulate the trafficking of multiple cellular proteins (74) . Moreover, Nef interact with the small GTPase ARF6 that regulates a recycling branch of short dynamic tubular intermediates involved in traffic along the endocytic and secretory routes. Moreover, Nef induces an increase of the number of multivescicular bodies (75) . Therefore, Nef affecting these signaling pathways might increase the release of Nef-containing vesicles. Moreover, Nef is possibly released by virions (76 , 77) . Uninfected cells could internalize Nef or multivescicular bodies containing Nef via endocytosis, pinocytosis or other unknown mechanisms. Highly immunogenic soluble Nef raises both humoral and cell-mediated immune responses.


   Phenotype
TOP
 ABSTRACT
 INTRODUCTION
 ROLE OF DC IN...
 HIV-1 NEF PROTEIN
 EFFECT OF NEF ON...
 Phenotype
Morphology
 Cytokine and chemokine...
 Stimulatory capacity
 Signal transduction
 CONCLUSIONS
 REFERENCES
 
DC express several molecules that are associated to different stages of activation, some of which may be important in HIV/SIV replication and/or DC-T cell communication (15 , 16 , 31) . Therefore, the assessment of Nef impact on these markers could be of great interest. The regulation of the expression of specific surface receptors in charge of the host immune response may represent a viral strategy to survive within host cells. Among these, antigen presenting molecules such as MHC-I and MHC-II are modulated on iDC surface. Contrary to reports suggesting that adenoviral vector expressing Nef in DC does not affect expression of MHC-I (61 , 64 , 65) , other reports demonstrate that both exogenous (68) and endogenous (64 , 78) Nef down-regulates HLA-A,B,C molecules, critical for the initiation of specific CTL responses, impairing antigen presentation to HIV-specific CD8+ T lymphocytes. The different approaches used possibly explain the experimental differences observed. Besides Nef, recombinant gp120 is able to down-regulate MHC-I in iDC (79) . Exposure of iDC to exogenous Nef leads to an up-regulation of MHC-II molecules, specialized in the presentation of antigenic peptides, favoring CD4+ T cell activation (68) , thus increasing the "pool" of lymphocytes permissive to infection. Differently, endogenous Nef does not modulate MHC-II surface expression (63) . Notably, it has been recently demonstrated that Nef down-regulates MHC-II surface expression in Hela cells (54) . This activity is conserved among different group of primate lentivirus (80) . Moreover, Nef impairs MHC-II antigen presentation up-regulating surface expression of the invariant chain associated with immature MHC-II both in HeLa cells, PBMC, and 221-B7 cells (54 , 80) .

Besides MHC class I and class II, endogenously expressed HIV-1 nef selectively down-regulates CD1a, a molecules in charge of glycolipid/lipid antigens presentation (64) . Endogenously or exogenously provided Nef down-regulates and up-regulates, respectively, surface expression of CD80 and CD86, major costimulatory molecules on APC that are crucial for T cell priming (81 , 82 , 68) . These results underlie the pleiotropy of the effect of Nef on DC: on the one hand, exogenous Nef triggers APC-mediated bystander T cell activation ensuring viral spread, and on the other hand endogenous Nef induces a loss of costimulation favoring immune evasion.

We demonstrated that exogenous Nef down-regulates MR expression on iDC surface (68) . Similarly, Vigerust et al. (57) demonstrated that infection of macrophages with adenovirus expressing HIV-1 Nef results in decreased surface expression of the MR. The down-regulation of the MR expression on iDC surface correlates with the observed down-regulation of Ag capture function (68) . This effect may contribute to immune evasion of HIV, preventing the uptake of new incoming viral antigens. In addition, the Nef-induced down-regulation of antibody (Ab) response (83) and suppression of CD40-dependent immunoglobulin (Ig) class switching (71) may contribute to the humoral immune dysfunction observed in AIDS patients. Of interest is the fact that another HIV-1 protein such as Tat is able to decrease MR expression (84) , indicating that the removal of this receptor from DC and macrophages may confer a variety of advantages to the virus. HIV-1 exploits its binding to MR to enter macrophages and DC via an endocytic route rather than the fusion mechanism described for CD4-dependent entry. Moreover, as previously shown for CD4, the presence of the MR on the surface of macrophages and DC could result in its incorporation into budding virions, which might inhibit infection of neighboring cells, similar to the mechanism proposed for CD4. Down-regulation of the MR from the cell surface would remove this inhibition, allowing successful spread of infection.

Exogenous Nef induces an up-regulation of CXCR4 expression on iDC. It has been demonstrated that the CXCR4 level gradually increases in maturing DC and may drive them first toward the lymphatics and then into T cell areas within the lymphnodes. Therefore, Nef increasing CXCR4 expression might favor migration of infected DC from periphery to lymphnode resulting in HIV transfer to T cells. Moreover, Nef may allow DC infection with T-tropic strains and, because the tissue distribution of CXCR4 is much broader than CCR5, this may allow the virus access to a wider range of potential target cells, or alternatively may permit fusion with more permissive target cells, thereby facilitating HIV-1 replication and spread. This hypothesis is consistent with the findings by Alessandrini et al. (85) that demonstrated that exogenous Nef induces resistance to M-tropic HIV replication in human monocyte-derived macrophages, thereby facilitating the switching from M- to T-tropic HIV strains, frequently observed during AIDS progression. Moreover, since Nef and gp120 have been shown to induce an apoptotic signal and Tat induces a proliferative signal through CXCR4 (86 , 87) in CD4+ T cells, it cannot be ruled out that Nef exerts similar effect on DC contributing to AIDS pathogenesis.

Recently, Michel et al. (55) demonstrated that CCR5 expression is down-regulated in Nef-expressing human TZM cell line. HIV may benefit by the Nef-induced CCR5 down-modulation: first, interference with superinfection may protect the infected cell from the accumulation of unintegrated viral genomes that are cytotoxic and may thus impede virus propagation (55) . In rendering cells refractory to superinfection, HIV also facilitates the spread to yet-uninfected cells by interfering with the loss of newly synthesized virus particles to cells with an already established infection. Second, HIV may prevent premature interactions with the viral Env, and this may ensure proper morphogenesis and thus higher infectivity of viral progeny. Similarly, this down-modulation may counteract HIV Env- or ß-chemokine-induced signaling events via CCR5, which can induce apoptosis (88 , 89) . Third, as an additional viral-evasion function, CCR5 down-modulation may inhibit the chemotaxis of infected DC toward ß-chemokine-secreting CTL (90) .

To boost virion infectivity, Nef increases surface expression of DC-SIGN, resulting in increased clustering of DC with T cells facilitating HIV transmission (56) .


   Morphology
TOP
 ABSTRACT
 INTRODUCTION
 ROLE OF DC IN...
 HIV-1 NEF PROTEIN
 EFFECT OF NEF ON...
 Phenotype
 Morphology
Cytokine and chemokine...
 Stimulatory capacity
 Signal transduction
 CONCLUSIONS
 REFERENCES
 
It has been demonstrated that DC, unlike other APC, actively polarize their actin cytoskeleton during interaction with T cells (91) . DC cytoskeletal rearrangement is critical for both the clustering and the activation of resting T cells playing a key role in the establishment of the immunological synapse. In addition, regulated migration of DC is central to the induction of physiological immune responses and this process necessitates plasticity of the cytoskeleton. We found that exogenous Nef induces actin rearrangement increasing the capacity of DC to form clusters with T cells improving the immunological synapse formation (67) . The increase of DC-T cells clustering may also involve up-regulation of DC-SIGN expression induced by Nef as reported by Sol-Foulon et al. (56) . It has been reported that in human B and T lymphocytes recombinant Nef non-covalently associates with actin in a myristoylation-dependent process (92) whereas in human astrocytes nonmyristoylated Nef colocalizes with astrocyte-specific cytoskeleton protein GFAP (93) . Therefore, we hypothesize that Nef could be retained in DC cytoplasm, probably interfering with a cytoskeletal protein.


   Cytokine and chemokine production
TOP
 ABSTRACT
 INTRODUCTION
 ROLE OF DC IN...
 HIV-1 NEF PROTEIN
 EFFECT OF NEF ON...
 Phenotype
 Morphology
 Cytokine and chemokine...
Stimulatory capacity
 Signal transduction
 CONCLUSIONS
 REFERENCES
 
DC-T cell interaction could also be influenced by cytokine and chemokine secretion profiles required for recruitment and successful activation of T cells. Cytokine and chemokine production by DC normally correlates with complete DC maturation when stimulated with lipopolysaccharide (LPS), CD40 ligand, dsRNA, or mixtures of cytokines. Of note, Nef-pulsed DC produce a wide range of cytokines and chemokines typical of mDC. We found that exogenous Nef triggers secretion of interleukin (IL)-1ß, IL-12, IL-15, TNF-{alpha}, and IL-8 by DC (68) . The up-regulation of cytokine production induced by Nef might promote bystander activation of T cells that cluster around DC and could be responsible for the enhancement of HIV-1 replication in CD4+ T lymphocytes. Moreover, exogenous Nef significantly increases ß-chemokine production, such as MIP-1{alpha} and MIP-1ß, by DC (68) and this may favor both T cell recruitment and variants that can replicate in the presence of high concentrations of these chemokines. Similarly, Nef induces MIP-1{alpha} and MIP-1ß release by infected macrophages promoting lymphocyte chemotaxis and activation (94) .

Likewise exogenous Nef, endogenous Nef was shown to trigger similar patterns of cytokine and chemokine release by iDC (63) . However, the levels of cytokine and chemokine production appear to be greater in response to endogenous DC-derived Nef. How exogenous or endogenous Nef interferes in DC signaling pathways leading to cytokine transcription still remains to be determined, and NF-kB and STAT3 activation could represent a possible mechanism. The activation of transcription factors by Nef will be described in more detail further on.


   Stimulatory capacity
TOP
 ABSTRACT
 INTRODUCTION
 ROLE OF DC IN...
 HIV-1 NEF PROTEIN
 EFFECT OF NEF ON...
 Phenotype
 Morphology
 Cytokine and chemokine...
 Stimulatory capacity
Signal transduction
 CONCLUSIONS
 REFERENCES
 
The Nef-induced down-modulation of surface MHC-I leads to poor presentation of HIV peptides to TCR on CTL. The functional consequence is the impairment of the induction of virus-specific cytotoxic response. More-over, the variability of the Nef protein derived from primary isolates may affect the processing of this viral antigen and impair its presentation to CTL (95) .

Our studies show that Nef-induced down-regulation of HLA-ABC molecules coincides with the reduced capacity of DC to prime alloreactive CD8+ T cell responses down-regulating their proliferation and functional competence. Therefore, CD8+ T cells primed by Nef-treated DC may become anergic. This closely fits with CD8+ T cells anergy in the setting of HIV infection (96 , 97) . It has been demonstrated that in HIV-infected individuals CD8+ T cells may even become prone to undergo Fas-induced apoptosis (98) . HIV-infected macrophages can induce apoptosis of HIV-specific CD8+ T cells, and this may help to explain the accumulation of incompletely mature HIV-specific CTL in infected individuals (99) . In addition, we found that Nef induces apoptotic killing of CD8+ T cells by exploiting DC death receptors, such as TNF-{alpha} and FasL and by activating caspase 8 (69) . On the other hand, Nef simultaneously protects the infected host cells from the same proapoptotic signals interfering with ASK-1 function (100) .

Contrary to a previous report (54) suggesting that Nef impairs MHC-II antigen presentation and surface expression in HeLa cells, exogenous Nef up-regulates the expression of MHC class II molecules on DC surface, increasing allogeneic CD4+ T cells proliferative responses (68) . The up-regulation of CD4+ T cell stimulatory capacity of DC may also be mediated by the increased expression of costimulatory/signaling molecules as well as by the up-regulation of cytokines and chemokines production by iDC (68) . Accordingly, adeno-nef-infected DC induce T cell activation (63) . Thus, Nef strategically promotes bystander CD4+ T cell activation increasing the "pool" of lymphocytes permissive to infection. In this context, our previous findings concerning the effect of exogenous Nef in the activation of unstimulated/suboptimally stimulated T cells (72) indicates that Nef exerts its activating effect principally on cells not sufficiently stimulated/activated, rendering them properly activated for the replication of the virus. On the other hand, Nef does not exert any effect on optimally stimulated PBMC, lamina propria mononuclear cells, that represent a physiologically preactivated cell population, and mDC (72 , 68) .

Besides the ability to stimulate T cells, DC interact with NK cells in a bidirectional crosstalk mostly occurring via cell-to-cell contact (101 , 102) . The Nef-induced up-regulation of IL-12 and IL-15 production by DC could amplify NK cell activation. Moreover, Nef may protect DC from NK cell-mediated lysis selectively down-regulating HLA-A,B but not HLA-C and HLA-E (48) . Experiments are in progress in our laboratory to clarify the interference of Nef in DC/NK crosstalk to verify the role of Nef in the dysregulation of innate immune response observed in HIV infected patients.


   Signal transduction
TOP
 ABSTRACT
 INTRODUCTION
 ROLE OF DC IN...
 HIV-1 NEF PROTEIN
 EFFECT OF NEF ON...
 Phenotype
 Morphology
 Cytokine and chemokine...
 Stimulatory capacity
 Signal transduction
CONCLUSIONS
 REFERENCES
 
It is well established that Nef perturbs cell signaling pathways by interacting with several tyrosine kinases or serine/threonine kinases known to be critical players in multiple signaling cascades (51) . Extensive investigation of the cellular proteins that interact with Nef has been undertaken in T cells and monocytes/macrophages. Although few reports investigate the signaling pathways triggered by Nef in DC, similar molecular partners of Nef have been reported in T cells and DC. Among these, Vav, a protein sitting on a common pathway for cytoskeletal and transcription regulation (103) , has been demonstrated to interact with Nef in both human T cell lines and DC (104 , 67) . Intriguingly, consistent with the observed effects of exogenous Nef on Vav in DC, in NIH3T3 cells the C-terminal SH3 domain of Vav has been shown to interact with Nef, resulting in Vav activation with concomitant cytoskeletal rearrangements and activation of the c-Jun NH2-terminal kinase (JNK) pathway (104) . Moreover, exogenous Nef is shown to enhance GTPase activity of the Rho family p21-GTPase Rac1, by increasing tyrosine phosphorylation of Vav (67) . Activation of Rac1 results in activation of PAK2, one member of the family of p21-activated kinases; in this regard, PAK2 is one of the key cellular targets of Nef. Binding of the GTP-bound GTPases Cdc42 and Rac1 leads to PAK2 autophosphorylation; however, many studies have shown a direct interaction of Nef with PAK2 also resulting in autophosphorylation of PAK2 (105 , 106) . Recent data have shown that Nef associates with a subpopulation of active PAK2 within lipid rafts (107 , 108) . Another cellular pathway usurped by Nef is ARF6 endocytic pathway that controls MHC-I down-regulation. Nef, associates to PACS-1 and moves to trans-Golgi network, leading to the PI3K-dependent ARF6 activation and cell surface MHC-I internalization to the trans-Golgi network (109) .

Several reports (44 , 67 , 110) show that Nef induces STAT3 and NF-kB activation in macrophages and DC. The Nef-induced activation of STAT3 and NF-kB could play an important role in triggering DC to facilitate viral spread. Specifically, signaling through NF-kB is responsible for DC maturation, and both NF-kB and STAT3 regulate the expression of antiapoptotic genes. By preventing apoptosis of the virus-carrying cell, Nef could keep the cell alive for more virus production and better virus spread. However, to date there is no evidence in literature concerning the regulation of antiapoptotic gene induced by Nef in DC. In this context, as described above, Nef induces surface expression of FasL that itself does not induce DC suicide (69) , making it probable that Nef triggers antiapoptotic signals in DC. Recently, Olivetta et al. (111) reported that Nef protects human-monocyte-derived macrophages from HIV-1-induced apoptosis inactivating the proapoptotic Bad protein. Taken together the above results clearly delineate that the functional defects in APC from HIV-infected patients are due to a direct effect of Nef exposure. In addition, an altered APC function may occur as a consequence of primary CD4+ T cell reduced expression of CD154 (CD40 ligand). Notably, it has been demonstrated that in nef-transgenic mice CD4+ T cells exhibit a blunted CD154 expression perturbing CD40 signaling in bystander DC (112 , 113) . Thus, Nef manipulation of signaling receptor and molecules represents a finely tuned mechanism for the virus to achieve its spreading.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 ROLE OF DC IN...
 HIV-1 NEF PROTEIN
 EFFECT OF NEF ON...
 Phenotype
 Morphology
 Cytokine and chemokine...
 Stimulatory capacity
 Signal transduction
 CONCLUSIONS
REFERENCES
 
The selective effects of the accessory Nef protein in subverting DC biology may constitute a viral tool underlying AIDS pathogenesis. Our findings and other reported data, outline a novel mechanism by which Nef, hijacking DC functional activity, may favor both the replication of HIV-1, via bystander activation of CD4+ T cells promoting virus spread and the escape of HIV-1 from immune surveillance by blocking CD8+ T cells functional competence. Exogenous Nef partially recapitulates the function of endogeous Nef (Table 1 ). Thus, Nef assists HIV-1 in the control of its host, promoting unresponsiveness and cell death of neighboring uninfected cells, yet protecting DC from the same fate. Moreover, Nef may contribute to the immune evasion hindering NK cell-mediated cytolytic and noncytolytic antiviral functions occurring on contact with DC and protecting DC from NK cell-mediated lysis (our unpublished observations). These mechanisms might contribute to the impairment of natural immunity during HIV-1 infection. The inhibition of the specific Ab response and the down-regulation of DC antigen capture function may represent additional strategies to escape immune surveillance. Hence, Nef may play a role in the impairment of both humoral and cellular (innate and adaptive) host immune responses. Nef interfering with phenotypical, morphological, and functional DC developmental program directly contributes to AIDS pathogenesis. Therefore, the development of opportunistic infections and/or progression of retrovirus-induced immunodeficiency may not require intact replicating virus.


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  		Table 1. Effect of HIV-1 Nef on DC biology

Further elucidation of genes induced through Nef signaling in DC could reveal pathways used by DC to drive HIV spread and will be critical to identify therapeutic strategies to bias the DC system toward activation of antiviral immunity instead of facilitating virus dissemination. A schematic representation illustrating the effects of interactions between DC and Nef leading dysregulation of innate and adaptive immune responses and their role in the pathogenesis of AIDS is shown in Fig. 1 .


Figure 1
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  		Figure 1. Schematic representation illustrating the effects of interactions between DC and Nef leading dysregulation of innate and adaptive immune responses. The model mainly illustrates results obtained by our group using exogenous Nef (65 66 67) . Virus transmission from DC to permissive CD4+ T cells (54) and their productive infection induced by endogenously expressed Nef (58 , 59 , 61) are also illustrated.

Correlating the numerous reported Nef activities described in vitro to in vivo pathogenicity has been difficult. In patients infected with HIV-1, there is a correlation between infection with nef defective viruses and decreased rate of disease progression. Since, it has been demonstrated that DC functionality is impaired in HIV+ progressor, while unaltered in HIV+ long-term nonprogressor, it cannot be rule out that this may be a Nef specific effect. Phenotype and function of DC from patients infected with viruses lacking Nef should be the focus of future in vivo analysis of Nef function.

Overall, the findings described in this review may contribute to the understanding of the function, the mechanism of action, and the cellular partners of Nef and to aid the discovery of suitable anti-Nef drugs. Such inhibitors, combined with agents targeting reverse transcripase, proteases, or other factors that are crucial for viral replication, could represent the basis for the pharmaco-vaccination of HIV-infected individuals.


   ACKNOWLEDGMENTS
 
This work was supported by a grant from the Italian Ministry of Health (Progetto di ricerca sull’AIDS). We thank Dr. Manuela Colucci for assistance with computer graphics.

Received for publication April 7, 2006. Accepted for publication June 6, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 ROLE OF DC IN...
 HIV-1 NEF PROTEIN
 EFFECT OF NEF ON...
 Phenotype
 Morphology
 Cytokine and chemokine...
 Stimulatory capacity
 Signal transduction
 CONCLUSIONS
 REFERENCES
 

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