HMGB1 activates replication of latent HIV-1 in a monocytic cell-line, but inhibits HIV-1 replication in primary macrophages

High mobility group box protein 1 (HMGB1) is an abundant component of mammalian cells that can be released into extracellular milieu actively or by cells that undergo necrosis. Exposure of inflammatory and endothelial cells to HMGB1 leads to the release of cytokines, including TNF-alpha and IL-6. To evaluate the impact of exogenous HMGB1 on viral replication in HIV-1 infected cells, we studied models of latent and acute infection. Extracellular HMGB1 dose dependently increased HIV-1 replication in the monocytic cells, U1, which is an established model for studying latent HIV-1 infection. Dexamethasone, a known inhibitor of NF-kappaB signaling in U1 cells, inhibited HMGB1-induced stimulation of the viral production. Addition of HMGB1 to primary monocytic cells with active HIV-1 infection elicited the opposite effect, due to suppression of the viral replication. The mechanism of this unexpected finding was explained by an HMGB1-mediated increased release of chemokines (RANTES, MIP-1alpha, and MIP-1beta) that are known to inhibit HIV-1 replication. The stimulatory effect of the HMGB1 was not present when latently infected T-cells (ACH-2) were used as target cells. Our data suggest that extracellular HMGB1 has a dichotomic effect on the HIV-1 infection in monocytes but not in lymphocytes. Both activation of latent HIV-1 infection and inhibition of active replication can thus be seen in vitro.     

HIV-1 replication in dendritic cells occurs through a tetraspanin-containing compartment enriched in AP-3

Dendritic cells (DC) are crucial components of the early events of HIV infection. Dendritic cells capture and internalize HIV at mucosal surfaces and efficiently transfer the virus to CD4+ T cells in trans through infectious synapses (trans-infection pathway). Alternatively, HIV-1 replicates in DC (R5-HIV-1) (cis-infection pathway). Here, we analyzed HIV trafficking in DC during the trans-infection pathway as well as the cis-infection pathway. Confocal immunofluorescence microscopy demonstrated that after capture by DC, R5-HIV-1 and HIV-1 pseudotyped with vesicular stomatitis virus protein G colocalized in a viral compartment enriched in tetraspanins including CD81, CD82 and CD9, although at different levels, indicating a role of the viral envelope in targeting to the tetraspanin-rich compartment. Replication of R5-HIV-1 in DC (cis-infection pathway) also led to the accumulation, in an envelope-independent manner, of mature viral particles in a tetraspanin-rich compartment. A fraction of the HIV-1-containing compartments appeared directly accessible from the cell surface. In sharp contrast with the trans-infection pathway, the delta-subunit of the adaptor protein 3 (AP-3) complex was enriched on the HIV-1-containing compartment during R5-HIV-1 replication in DC (cis-infection pathway). Downregulation of AP-3 delta-adaptin reduced significantly viral particle release from HIV-1-infected DC. Together, these studies demonstrate a role for AP-3 in HIV replication in a tetraspanin-rich compartment in DC and contribute to the elucidation of the trafficking pathways required for DC-T cell transfer of HIV-1 infection, a critical step during the early events of HIV infection.     

Qa-1(b)-dependent modulation of dendritic cell and NK cell cross-talk in vivo

Dendritic cells (DC) trigger activation and IFN-gamma release by NK cells in lymphoid tissues, a process important for the polarization of Th1 responses. Little is known about the molecular signals that regulate DC-induced NK cell IFN-gamma synthesis. In this study, we analyzed whether the interaction between Qa-1(b) expressed on DC and its CD94/NKG2A receptor on NK cells affects this process. Activation of DC using CpG-oligodeoxynucleotides in Qa-1(b)-deficient mice, or transfer of CpG-oligodeoxynucleotide-activated Qa-1(b)-deficient DC into wild-type mice, resulted in dramatically increased IFN-gamma production by NK cells, as compared with that induced by Qa-1(b)-expressing DC. Masking the CD94/NKG2A inhibitory receptor on NK cells in wild-type mice similarly enhanced the IFN-gamma response of these cells to Qa-1(b)-expressing DC. Furthermore, NK cells from CD94/NKG2A-deficient mice displayed higher IFN-gamma production upon DC stimulation. These results demonstrate that Qa-1(b) is critically involved in regulating IFN-gamma synthesis by NK cells in vivo through its interaction with CD94/NKG2A inhibitory receptors. This receptor-ligand interaction may be essential to prevent unabated cytokine production by NK cells during an inflammatory response.     

HIV-1 replication in Langerhans and interstitial dendritic cells is inhibited by neutralizing and Fc-mediated inhibitory antibodies

Langerhans cells (LCs) and interstitial dendritic cells (IDCs) may be among the first human immunodeficiency virus type 1 (HIV-1) targets after sexual transmission. We generated cells of these types by differentiation of purified CD34(+) cord blood cells. After in vitro infection with R5-tropic strains, we obtained similar percentages of infected cells for both dendritic cell (DC) subsets. Moreover, LC infection was not increased by blockage of langerin by antilangerin. These results indicate that, under our experimental conditions, there was no evidence of any preference of HIV replication in LCs versus IDCs. The inhibitory activity of HIV-1-specific IgAs and IgGs against HIV-1 replication in LCs and IDCs was analyzed. We found that neutralizing antibodies inhibit HIV-1 infection of both DC subsets. Interestingly, HIV-1 was inhibited more efficiently by the IgGs than the corresponding IgA, due to an Fcγ receptor-dependent mechanism. Moreover, nonneutralizing inhibitory IgGs were able to inhibit infection of both LCs and IDCs. These results underline the importance of HIV-1 inhibition by the binding of the Fc part of IgGs to Fcγ receptors and suggest that the induction of neutralizing and nonneutralizing inhibitory IgGs in addition to neutralizing IgAs at mucosal sites may contribute to protection against sexual transmission of HIV-1.     

Immunologic escape as a mechanism of viral persistence in HIV-1 infection

Two specific immune responses to HIV, the cytolytic T cell response to epitopes in the core/envelope proteins and the antibody neutralization response to the V3 epitope in the envelope, are reviewed. Substantial data has accumulated indicating that virus variants can be isolated from infected people that are not recognized by the early immune response. Furthermore, genomic changes in the virus show host dependence and emerge to prominence with a temporal pattern that is consistent with selection for escape from an earlier immune response. Escape from immune recognition may therefore be a major factor in allowing persistent viral replication in HIV infection.     

Productive replication and evolution of HIV-1 in ferret cells

A rodent or other small animal model for HIV-1 has not been forthcoming, with the principal obstacles being species-specific restriction mechanisms and deficits in HIV-1 dependency factors. Some Carnivorans may harbor comparatively fewer impediments. For example, in contrast to mice, the domestic cat genome encodes essential nonreceptor HIV-1 dependency factors. All Feliformia species and at least one Caniformia species also lack a major lentiviral restriction mechanism (TRIM5α/TRIMCyp proteins). Here we investigated cells from two species in another carnivore family, the Mustelidae, for permissiveness to the HIV-1 life cycle. Mustela putorius furo (domesticated ferret) primary cells and cell lines did not restrict HIV-1, feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), or N-tropic murine leukemia virus (MLV) postentry and supported late HIV-1 life cycle steps comparably to human cells. The ferret TRIM5α gene exon 8, which encodes the B30.2 domain, was found to be pseudogenized. Strikingly, ferret (but not mink) cells engineered to express human HIV-1 entry receptors supported productive spreading replication, amplification, and serial passage of wild-type HIV-1. Nevertheless, produced virions had relatively reduced infectivity and the virus accrued G→A hypermutations, consistent with APOBEC3 protein pressure. Ferret cell-passaged HIV-1 also evolved amino acid changes in the capsid cyclophilin A binding loop. We conclude that the genome of this carnivore can provide essential nonreceptor HIV-1 dependency factors and that ferret APOBEC3 proteins with activity against HIV-1 are likely. Even so, unlike in cat cells, HIV-1 can replicate in ferret cells without vif substitution. The virus evolves in this novel nonprimate cell adaptive landscape. We suggest that further characterization of HIV-1 adaptation in ferret cells and delineation of Mustelidae restriction factor repertoires are warranted, with a view to the potential for an HIV-1 animal model.     

Salmonella rapidly kill dendritic cells via a caspase-1-dependent mechanism

Dendritic cells provide a critical link between innate and acquired immunity. In this study, we demonstrate that the bacterial pathogen Salmonella enterica serovar Typhimurium can efficiently kill these professional phagocytes via a mechanism that is dependent on sipB and the Salmonella pathogenicity island 1-encoded type III protein secretion system. Rapid phosphatidylserine redistribution, caspase activation, and loss of plasma membrane integrity were characteristic of dendritic cells infected with wild-type Salmonella, but not sipB mutant bacteria. Caspase-1 was particularly important in this process because Salmonella-induced dendritic cell death was dramatically reduced in the presence of a caspase-1-specific inhibitor. Furthermore, dendritic cells obtained from caspase-1-deficient mice, but not heterozygous littermate control mice, were resistant to Salmonella-induced cytotoxicity. We hypothesize that Salmonella have evolved the ability to selectively kill professional APCs to combat, exploit, or evade immune defense mechanisms.     

The CD85j+ NK cell subset potently controls HIV-1 replication in autologous dendritic cells

Natural killer (NK) cells and dendritic cells (DC) are thought to play critical roles in the first phases of HIV infection. In this study, we examined changes in the NK cell repertoire and functions occurring in response to early interaction with HIV-infected DC, using an autologous in vitro NK/DC coculture system. We show that NK cell interaction with HIV-1-infected autologous monocyte-derived DC (MDDC) modulates NK receptor expression. In particular, expression of the CD85j receptor on NK cells was strongly down-regulated upon coculture with HIV-1-infected MDDC. We demonstrate that CD85j(+) NK cells exert potent control of HIV-1 replication in single-round and productively HIV-1-infected MDDC, whereas CD85j(-) NK cells induce a modest and transient decrease of HIV-1 replication. HIV-1 suppression in MDCC by CD85j(+) NK cells required cell-to-cell contact and did not appear mediated by cytotoxicity or by soluble factors. HIV-1 inhibition was abolished when NK-MDDC interaction through the CD85j receptor was blocked with a recombinant CD85j molecule, whereas inhibition was only slightly counteracted by blocking HLA class I molecules, which are known CD85j ligands. After masking HLA class I molecules with specific antibodies, a fraction of HIV-1 infected MDDC was still strongly stained by a recombinant CD85j protein. These results suggest that CD85j(+) NK cell inhibition of HIV-1 replication in MDDC is mainly mediated by CD85j interaction with an unknown ligand (distinct from HLA class I molecules) preferentially expressed on HIV-1-infected MDDC.     

HMGB1, an alarmin promoting HIV dissemination and latency in dendritic cells

Dendritic cells (DCs) initiate immune responses by transporting antigens and migrating to lymphoid tissues to initiate T-cell responses. DCs are located in the mucosal surfaces that are involved in human immunodeficiency virus (HIV) transmission and they are probably among the earliest targets of HIV-1 infection. DCs have an important role in viral transmission and dissemination, and HIV-1 has evolved different strategies to evade DC antiviral activity. High mobility group box 1 (HMGB1) is a DNA-binding nuclear protein that can act as an alarmin, a danger signal to alert the innate immune system for the initiation of host defense. It is the prototypic damage-associated molecular pattern molecule, and it can be secreted by innate cells, including DCs and natural killer (NK) cells. The fate of DCs is dependent on a cognate interaction with NK cells, which involves HMGB1 expressed at NK–DC synapse. HMGB1 is essential for DC maturation, migration to lymphoid tissues and functional type-1 polarization of naïve T cells. This review highlights the latest advances in our understanding of the impact of HIV on the interactions between HMGB1 and DCs, focusing on the mechanisms of HMGB1-dependent viral dissemination and persistence in DCs, and discussing the consequences on antiviral innate immunity, immune activation and HIV pathogenesis.     

HIV-1 selectively infects a subset of nonmaturing BDCA1-positive dendritic cells in human blood

The infection of cultured monocyte-derived dendritic cells (DCs) with HIV-1 involves CD4 and CCR5 receptors, while transmission to T cells is enhanced at least in part by the lectin DC-SIGN/CD209. In the present study, we studied BDCA-1+ myeloid DCs isolated directly from human blood. These cells express CD4 and low levels of CCR5 and CXCR4 coreceptors, but not DC-SIGN. The myeloid DCs replicate two R5 viruses, BaL and YU2, and transfer infection to activated T cells. The virus productively infects a small fraction of the blood DCs that fail to mature in culture, as indicated by the maturation markers CD83 and DC-LAMP/CD208, and the expression of high CD86 and MHC class II, in contrast to many noninfected DCs. A greater proportion of BDCA-1+ DCs are infected when the virus is pseudotyped with the vesicular stomatitis envelope VSV-G (5-15%), as compared with the R5 virus (0.3-3.5%), indicating that HIV-1 coreceptors may limit the susceptibility of DCs to become infected, or the endocytic route of viral entry used by HIV/vesicular stomatitis virus enhances infectivity. When infected and noninfected cells are purified by cell sorting, the former uniformly express HIV p24 gag and are virtually inactive as stimulators of the allogeneic MLR, in contrast to potent stimulation by noninfected DCs from the same cultures. These results point to two roles for a small fraction of blood DCs in HIV-1 pathogenesis: to support productive infection and to evade the direct induction of T cell-mediated immunity.     

Molecules from apoptotic pathways modulate HIV-1 replication in Jurkat cells

The replication of viruses involves control of some aspects of host cell homeostasis by modification of target cell metabolism and regulation of the apoptotic machinery. It is not well known whether molecules involved in apoptotic pathways affect human immunodeficiency virus type 1 (HIV-1) replication and regulate viral yields. Using the susceptible Jurkat cell line, we studied the relationship of apoptosis-associated molecules with HIV-1 virus production using a sensitive real-time RT-PCR assay. Here, we found that expression of proapoptotic proteins, including Fas ligand (FasL), FADD, or p53 significantly increased HIV-1 virus production. In contrast, the expression of antiapoptotic molecules, such as FLIP, Bcl-X_L, and XIAP, decreased HIV-1 virus production. Knockdown of Bax with siRNA and FADD with expression of its antisense mRNA also inhibited viral replication and the caspase-3 inhibitor, Z-DEVD, and decreased virus production. These data indicate that HIV-1 infection regulates the apoptosis process to facilitate viral replication and inhibition of apoptosis may inhibit HIV-1 replication and cytopathogenesis. We also discuss the effects of MAPK signaling pathways and apoptosis on HIV-1 replication.     

Opposite effects of IL-10 on the ability of dendritic cells and macrophages to replicate primary CXCR4-dependent HIV-1 strains

We investigated the effect of IL-10 on replication of primary CXCR4-dependent (X4) HIV-1 strains by monocyte-derived dendritic cells (DCs) and macrophages (M Phis). M Phis efficiently replicated CXCR4-dependent HIV-1 (X4 HIV-1) strains NDK and VN44, whereas low levels of p24 were detected in supernatants of infected DCs. IL-10 significantly increased X4 HIV-1 replication by DCs but blocked viral production by M Phis as determined by p24 levels and semiquantitative nested PCR. IL-10 up-regulated CXCR4 mRNA and protein expression on DCs and M Phis, suggesting that IL-10 enhances virus entry in DCs but blocks an entry and/or postentry step in M Phis. The effect of IL-10 on the ability of DCs and M Phis to transmit virus to autologous CD4(+) T lymphocytes was investigated in coculture experiments. DCs exhibited a greater ability than did M Phis to transmit a vigorous infection to CD4(+) T cells despite their very low replication capacity. IL-10 had no effect on HIV-1 replication in DC:T cell cocultures but markedly decreased viral production in M Phi:T cell cocultures. These results demonstrate that IL-10 has opposite effects on the replication of primary X4 HIV-1 strains by DCs and M Phis. IL-10 increases X4-HIV-1 replication in DCs but does not alter their capacity to transmit virus to CD4(+) T lymphocytes. These findings suggest that increased levels of IL-10 observed in HIV-1-infected patients with disease progression may favor the replication of X4 HIV-1 strains in vivo.     

HSV-2 infection of dendritic cells amplifies a highly susceptible HIV-1 cell target

Herpes simplex virus type 2 (HSV-2) increases the risk of HIV-1 infection and, although several reports describe the interaction between these two viruses, the exact mechanism for this increased susceptibility remains unclear. Dendritic cells (DCs) at the site of entry of HSV-2 and HIV-1 contribute to viral spread in the mucosa. Specialized DCs present in the gut-associated lymphoid tissues produce retinoic acid (RA), an important immunomodulator, able to influence HIV-1 replication and a key mediator of integrin α₄β₇ on lymphocytes. α₄β₇ can be engaged by HIV-1 on the cell-surface and CD4⁺ T cells expressing high levels of this integrin (α₄β₇ ^high) are particularly susceptible to HIV-1 infection. Herein we provide in-vivo data in macaques showing an increased percentage of α₄β₇ ^high CD4⁺ T cells in rectal mucosa, iliac lymph nodes and blood within 6 days of rectal exposure to live (n = 11), but not UV-treated (n = 8), HSV-2. We found that CD11c⁺ DCs are a major target of HSV-2 infection in in-vitro exposed PBMCs. We determined that immature monocyte-derived DCs (moDCs) express aldehyde dehydrogenase ALDH1A1, an enzyme essential for RA production, which increases upon HSV-2 infection. Moreover, HSV-2-infected moDCs significantly increase α₄β₇ expression on CD4⁺ T lymphocytes and HIV-1 infection in DC-T cell mixtures in a RA-dependent manner. Thus, we propose that HSV-2 modulates its microenviroment, influencing DC function, increasing RA production capability and amplifying a α₄β₇ ^highCD4⁺ T cells. These factors may play a role in increasing the susceptibility to HIV-1.     

HIV-1 reactivation in HIV-latently infected dendritic cells by oral microorganisms and LPS

Dendritic cells are critical components of the host defense system that play pivotal role in linking innate immunity to adaptive immune responses. In the role of interfacing with pathogens through the action of surface pattern-recognition receptors, dendritic cells are a potential target for retroviral infection and latency. Dendritic cells are a long-lived reservoir of latent virus in HIV (human immunodeficiency virus)-infected patients. It is hypothesized that HIV-latently infected dendritic cells would be stimulated by oral bacteria leading to reactivation of HIV. In our HIV-latently infected dendritic cell models, of both promoter activation and HIV production, significant differences were observed among the bacterial species in their ability to stimulate HIV reactivation. The experimental data support the hypothesis that oral bacteria related to periodontal infections could trigger latently infected dendritic cells in gingival tissues and contribute to HIV recrudescence and undermining anti-retroviral therapy.