TNF-alpha inhibits HIV-1 replication in peripheral blood monocytes and alveolar macrophages by inducing the production of RANTES and decreasing C-C chemokine receptor 5 (CCR5) expression.

The pathogenesis of HIV-1 infection is influenced by the immunoregulatory responses of the host. Macrophages present in the lymphoid tissue are susceptible to infection with HIV-1, but are relatively resistant to its cytopathic effects and serve as a reservoir for the virus during the course of disease. Previous investigators have demonstrated that increased serum levels of TNF-alpha contribute to the clinical symptoms of AIDS and that TNF-alpha stimulates the production of HIV-1 in chronically infected lymphocytic and monocytic cell lines by increasing HIV-1 gene expression. Although previous studies have suggested that TNF-alpha may increase HIV-1 infection of primary human mononuclear cells, some recent studies have indicated that TNF-alpha suppresses HIV-1 infection of macrophages. We now demonstrate that TNF-alpha suppresses HIV-1 replication in freshly infected peripheral blood monocytes (PBM) and alveolar macrophages (AM) in a dose-dependent manner. As TNF-alpha has been shown to increase the production of C-C chemokine receptor (CCR5)-binding chemokines under certain circumstances, we hypothesized that TNF-alpha inhibits HIV-1 replication by increasing the expression of these HIV-suppressive factors. We now show that TNF-alpha treatment of PBM and AM increases the production of the C-C chemokine, RANTES. Immunodepletion of RANTES alone or in combination with macrophage inflammatory protein-1alpha and -1beta block the ability of TNF-alpha to suppress viral replication in PBM and AM. In addition, we found that TNF-alpha treatment reduces CCR5 expression on PBM and AM. These findings suggest that TNF-alpha plays a significant role in inhibiting monocytotropic strains of HIV-1 by two distinct, but complementary, mechanisms.     

Nef enhances human immunodeficiency virus replication and responsiveness to interleukin-2 in human lymphoid tissue ex vivo

The nef gene is important for the pathogenicity associated with simian immunodeficiency virus infection in rhesus monkeys and with human immunodeficiency virus type 1 (HIV-1) infection in humans. The mechanisms by which nef contributes to pathogenesis in vivo remain unclear. We investigated the contribution of nef to HIV-1 replication in human lymphoid tissue ex vivo by studying infection with parental HIV-1 strain NL4-3 and with a nef mutant (DeltanefNL4-3). In human tonsillar histocultures, NL4-3 replicated to higher levels than DeltanefNL4-3 did. Increased virus production with NL4-3 infection was associated with increased numbers of productively infected cells and greater loss of CD4(+) T cells over time. While the numbers of productively infected T cells were increased in the presence of nef, the levels of viral expression and production per infected T cell were similar whether the nef gene was present or not. Exogenous interleukin-2 (IL-2) increased HIV-1 production in NL4-3-infected tissue in a dose-dependent manner. In contrast, DeltanefNL4-3 production was enhanced only marginally by IL-2. Thus, Nef can facilitate HIV-1 replication in human lymphoid tissue ex vivo by increasing the numbers of productively infected cells and by increasing the responsiveness to IL-2 stimulation.     

Tumor necrosis factor-alpha and nitric oxide in vertically HIV-1-infected children: implications for pathogenesis

We performed a cross-sectional study to investigate the plasma TNF-alpha and nitric oxide (NO) production in 44 vertically HIV-1-infected children, and the relationship with immunological status and viral replication. As a control group, 36 healthy, uninfected children were studied. Plasma TNF-alpha and NO levels were determined by ELISA. Viral load was quantified using standard assays. Cell proliferation, apoptosis and viral replication were evaluated in vitro by incorporation of (3H)-thymidine, flow cytometry and p24 antigen, respectively. Higher plasma TNF-alpha and NO levels were observed in HIV-1-infected children compared with healthy controls. We found a very strong correlation between plasma TNF-alpha and NO levels in HIV-1-infected children (r = 0.98; p < 0.001). Moreover, HIV-1-infected children with higher viral load (> 4.7 log10) showed higher TNF-alpha and NO levels than those with viral load below this threshold. Interestingly, we detected inducible nitric oxide synthase (iNOS) mRNA in T-lymphocytes from HIV-1-infected children. To address their possible patho-physiological significance, we tested the in vitro effects of NO and TNF-alpha in HIV-1 replication. Addition of TNF-alpha and NO donors to mitogen-activated, HIV-1-infected PBMC cultures produced a significant increase in viral replication. Moreover, HIV-1 replication in mitogen-stimulated, PBMC cultures was partially inhibited by iNOS specific inhibitors, and a neutralising, anti-TNF-alpha monoclonal antibody. Our results indicate that TNF-alpha and NO correlated with high viral load in HIV-1-infected children and favoured HIV-1 in vitro replication. These data suggest a detrimental role of NO in HIV-1 infection, and that NOS inhibitors may have some therapeutic benefit in HIV-1-infection.     

Cell-specific differences in activation of NF-kappa B regulatory elements of human immunodeficiency virus and beta interferon promoters by tumor necrosis factor.

Three aspects of the involvement of tumor necrosis factor in human immunodeficiency virus (HIV) pathogenesis were examined. Tumor necrosis factor alpha (TNF-alpha) mRNA production was analyzed by polymerase chain reaction amplification in monocytic U937 cells and in a chronically HIV infected U937 cell line (U9-IIIB). TNF-alpha RNA was undetectable in U937 cells, whereas a low constitutive level was detected in U9-IIIB cells. Paramyxovirus infection induced a 5- to 10-fold increase in the steady-state level of TNF-alpha RNA in U9-IIIB cells compared with U937 cells, suggesting that HIV-infected monocytic cells produced higher levels of TNF-alpha than did normal cells after a secondary virus infection. The effects of TNF-alpha on gene expression were examined by transient expression assays using reporter chloramphenicol acetyltransferase plasmids linked to regulatory elements from the HIV long terminal repeat (LTR) and the beta interferon promoter. In U937 and Jurkat T lymphoid cells, the inducibility of the different hybrid promoters by TNF-alpha or phorbol ester varied in a cell type- and promoter context-specific manner; the levels of gene activity of NF-kappa B-containing plasmids correlated directly with induction of NF-kappa B DNA-binding activity. Although the intact beta interferon promoter was only weakly stimulated by phorbol ester or TNF-alpha, multimers of the PRDII NF-kappa B-binding domain were inducible by both agents. TNF-alpha was able to increase expression of the HIV LTR in T cells, but in monocytic cells, TNF-alpha did not induce the HIV LTR above a constitutive level of activity. This level of NF-kappa B-independent activity appears to be sufficient for virus multiplication, since TNF-alpha treatment had no effect on the kinetics of de novo HIV type 1 (HIV-1) infection and viral RNA production in U937 cells. However, in Jurkat cells, TNF-alpha dramatically enhanced the spread of HIV-1 through the cell population and increased viral RNA synthesis, indicating that in T cells HIV-1 multiplication was stimulated by TNF-alpha treatment.     

Differences among HIV-1 variants in their ability to elicit secretion of TNF-alpha

HIV-1 infection of human PBMC has been shown to elicit secretion of several different cytokines. TNF-alpha secretion induced by this virus has been of particular interest because it has been associated with the development of HIV-1 dementia and because TNF-alpha increases viral replication by enhancing NF-kappaB interaction with the viral promoter, the HIV-1 long terminal repeat. Thus, an autocrine pathway is potentially created in which HIV-1 stimulates its own replication. Conflicting reports exist, however, on the ability of HIV-1 to induce TNF-alpha secretion in vitro or in vivo. Using experimental protocols that controlled for potential bacterial endotoxin-induced TNF-alpha secretion, the current study demonstrates significant differences in TNF-alpha-eliciting properties among primary and laboratory obtained HIV-1. The relative TNF-alpha-inducing ability of different variants is conserved when tested using PBMC from different individuals. Elicitation of TNF-alpha secretion was not blocked by exposure of cells to zidovudine, indicating that viral integration was not required to induce secretion. Rather, the interaction between the virus and cell surface is critical for TNF-alpha induction, as Abs against CD4 or CCR5 blocked the induction of TNF-alpha synthesis by PBMC when added before virus exposure. Furthermore, the ability to induce TNF-alpha secretion mapped to a region of the HIV-1 env gene that includes the third hypervariable domain. Differences in the ability of different HIV-1 variants to elicit TNF-alpha may account for individual differences in HIV-1 disease course.     

Viral DNA and mRNA expression correlate with the stage of human immunodeficiency virus (HIV) type 1 infection in humans: evidence for viral replication in all stages of HIV disease.

Studies of cultivatable human immunodeficiency virus type 1 (HIV-1) from plasma samples from infected patients have shown a correspondence between increasing viral burden and disease progression, but these measurements are selective and thus nonrepresentative of the in vivo viral load. Quantitation of proviral DNA sequences by the polymerase chain reaction in purified CD4+ T cells has shown a similar relationship but does not provide a measure of viral gene expression. We have studied viral DNA, genomic RNA, and spliced mRNA expression of HIV-1 in infected patients with a quantitative polymerase chain reaction assay. Viral RNA expression is detected in all stages of infection. These data show that the natural history of HIV infection is associated with a shift in the balance of viral expression favoring the production of genomic RNA without a preceding period of true viral latency.     

Oscillation of the human immunodeficiency virus surface receptor is regulated by the state of viral activation in a CD4+ cell model of chronic infection.

We have developed a unique physiologic model of chronic human immunodeficiency virus type 1 (HIV-1) infection, OM-10.1, clonally derived from infected HL-60 promyelocytes and harboring a single integrated provirus. Unlike other models of chronic infection, OM-10.1 cultures remain CD4+ under normal culture conditions, during which less than 10% of the cells constitutively express HIV-1 proteins. However, when treated with tumor necrosis factor alpha (TNF-alpha), OM-10.1 cultures dramatically increased (greater than 35-fold) HIV-1 expression and rapidly down-modulated surface CD4, as greater than 95% of the cells became HIV-1+. The complete loss of surface CD4 following viral activation was neither associated with apparent cytopathicity nor due to a decline of available CD4 mRNA. There was, however, a temporal association between CD4 down-modulation and the accumulation of intracellular HIV-1 gp 160/120; in addition, intracellular CD4-gp 160 complexes were identifiable in OM-10.1 cell lysates at time points following TNF-alpha induction after surface CD4 was no longer detectable. Surface CD4 expression by OM-10.1 cells returned once viral activation ceased and could be repeatedly oscillated upon HIV-1 reactivation. Furthermore, inhibition of protein kinase activity following maximal TNF-alpha stimulation of OM-10.1 cells quickly returned activated HIV-1 to a state of latency, as evidenced by an accelerated return of surface CD4. These results with the new OM-10.1 cell line demonstrate that CD4 surface expression can be maintained during chronic infection and is critically dependent on the state of viral activation, that CD4-gp 160 intracellular complexing is involved in CD4 down-modulation, and that protein kinase pathways not only function in the primary induction of latent HIV-1 but also are required for maintaining the state of viral activation.     

beta-Chemokine production in CD40L-stimulated monocyte-derived macrophages requires activation of MAPK signaling pathways

CD40 ligand is a cell surface molecule on CD4(+) T cells that interacts with its receptor, CD40, on antigen presenting cells to mediate humoral and cellular immune responses. Our previous studies demonstrated that a trimeric soluble form of CD40L (CD40LT) activates macrophages to produce beta-chemokines and decrease CCR5 and CD4 cell surface expression, thus inducing resistance to HIV-1 infection. However, the mechanism(s) by which CD40LT mediates these effects in primary macrophages remains unclear. In this report, we demonstrate that CD40LT induces synthesis of beta-chemokines through the activation of MAPK signaling pathways. Treatment of macrophages with CD40LT results in a rapid activation of p38 and ERK1/2 mitogen-activated protein kinases. Inhibitors of these MAPKs blocked beta-chemokine production, while protein kinase A and C inhibitors had little or no effect. We also provide evidence that CD40LT stimulates beta-chemokine production directly, as well as indirectly via a TNF-alpha-dependent mechanism. At the early time points, CD40LT directly stimulated beta-chemokine production, whereas at later time points the effect was mediated to some extent by TNF-alpha. In conclusion, our results suggest that CD40-CD40L interactions are important for the activation of monocyte-derived macrophage antiviral response affecting both viral replication and the recruitment of immune cells.     

Alterations in mast cell function and survival following in vitro infection with human immunodeficiency viruses-1 through CXCR4

HIV-1 infection leads to a disease that attacks the central regulatory mechanisms of the immune response. As mucosal tissue is one of the primary sites infected with HIV in vivo, we examined the effects of HIV exposure on human mast cells, important components of mucosal defense. Using the human mast cell line, HMC-1, which expresses CXCR4 but not CCR5 on the cell surface, we found that several HIV-1 X4 tropic lab (IIIB, RF) and primary isolates but not R5 (BAL, ADA) isolates productively infected these cells. Furthermore, stem cell factor-dependent mast cells derived from primary fetal liver or cord blood cultures were also productively infected with both X4 and R5 HIV-1 strains. Infection was blocked at the level of viral entry using monoclonal antibodies to CXCR4 and CD4. Treatment of HMC-1 with TNF-alpha and TGF-beta stimulated cell surface expression of CCR5 and up-regulated expression of both CCR5 and CXCR4 on primary mast cells, leading to increased susceptibility to both X4 and R5 viral isolates. HIV-1 infection also resulted in histamine release from these mast cells, most due in part to HIV-mediated cell death. These results demonstrate that X4 viruses can use CD4 and the CXCR4 receptor to infect mast cells, suggesting that mast cell-T cell interactions may contribute to HIV mediated immune dysfunction in the mucosa.     

In vivo evidence for instability of episomal human immunodeficiency virus type 1 cDNA

Current regimens for the management of human immunodeficiency virus type 1 (HIV-1) infection suppress plasma viremia to below detectable levels for prolonged intervals. Nevertheless, there is a rapid resumption in plasma viremia if therapy is interrupted. Attempts to characterize the extent of viral replication under conditions of potent suppression and undetectable plasma viremia have been hampered by a lack of convenient assays that can distinguish latent from ongoing viral replication. Using episomal viral cDNA as a surrogate for ongoing replication, we previously presented evidence that viral replication persists in the majority of infected individuals with a sustained aviremic status. The labile nature of viral episomes and hence their validity as surrogate markers of ongoing replication in individuals with long-term-suppressed HIV-1 infection have been analyzed in short-term in vitro experiments with conflicting results. Since these in vitro experiments do not shed light on the long-term in vivo dynamics of episomal cDNA or recapitulate the natural targets of infection in vivo, we have analyzed the dynamics of episomal cDNA turnover in vivo by following the emergence of an M184V polymorphism in plasma viral RNA, in episomal cDNA, and in proviral DNA in patients on suboptimal therapies. We demonstrate that during acquisition of drug resistance, wild-type episomal cDNAs are replaced by M184V-harboring episomes. Importantly, a complete replacement of wild-type episomes with M184V-containing episomes occurred while proviruses remained wild type. This indicates that episomal cDNAs are turned over by degradation rather than through death or tissue redistribution of the infected cell itself. Therefore, evolution of episomal viral cDNAs is a valid surrogate of ongoing viral replication in HIV-1-infected individuals.     

Direct evidence of lower viral replication rates in vivo in human immunodeficiency virus type 2 (HIV-2) infection than in HIV-1 infection

Studies have shown that human immunodeficiency virus type 2 (HIV-2) is less pathogenic than HIV-1, with a lower rate of disease progression. Similarly, plasma viral loads are lower in HIV-2 infection, suggesting that HIV-2 replication is restricted in vivo in comparison to that of HIV-1. However, to date, in vivo studies characterizing replication intermediates in the viral life cycle of HIV-2 have been limited. In order to test the hypothesis that HIV-2 has a lower replication rate in vivo than HIV-1 does, we quantified total viral DNA, integrated proviral DNA, cell-associated viral mRNA, and plasma viral loads in peripheral blood samples from groups of therapy-na?ve HIV-1-infected (n = 21) and HIV-2-infected (n = 18) individuals from Dakar, Senegal, with CD4(+) T-cell counts of >200/microl. Consistent with our previous findings, total viral DNA loads were similar between HIV-1 and HIV-2 and plasma viral loads were higher among HIV-1-infected individuals. Proportions of DNA in the integrated form were also similar between these viruses. In contrast, levels of viral mRNA were lower in HIV-2 infection. Our study indicates that HIV-2 is able to establish a stable, integrated proviral infection in vivo, but that accumulation of viral mRNA is attenuated in HIV-2 infection relative to that in HIV-1 infection. The differences in viral mRNA are consistent with the differences in plasma viral loads between HIV-1 and HIV-2 and suggest that lower plasma viral loads, and possibly the attenuated pathogenesis of HIV-2, can be explained by lower rates of viral replication in vivo.     

The adenovirus E3 14.7-kilodalton protein which inhibits cytolysis by tumor necrosis factor increases the virulence of vaccinia virus in a murine pneumonia model.

The 14.7-kilodalton protein (14.7K protein) encoded by the adenovirus (Ad) E3 region inhibits tumor necrosis factor alpha (TNF-alpha)-mediated lysis of cells in tissue culture experiments, but the relevance of this effect in vivo is incompletely understood. To examine the effect of the ability of the Ad 14.7K protein to block TNF lysis upon viral pathogenesis in a murine model, we cloned the 14.7K protein-encoding gene into vaccinia virus (VV), permitting its study in isolation from other Ad E3 immunomodulatory proteins. The gene for murine TNF-alpha was inserted into the same VV containing the 14.7K gene to ensure that each cell infected with the VV recombinant would express both the agonist (TNF) and its antagonist (14.7K). VV was utilized as the vector because it accommodates large and multiple inserts of foreign DNA with faithful, high-level expression of the protein products. In addition, infection of mice with VV induces disease with quantifiable morbidity, mortality, and virus replication. The results of intranasal infections of BALB/c mice with these VV recombinants indicate that the Ad 14.7K protein increases the virulence of VV carrying the TNF-alpha gene by reversing the attenuating effect of TNF-alpha on VV pathogenicity. This was demonstrated by increased mortality, pulmonary pathology, and viral titers in lung tissue following infection with VV coexpressing the 14.7K protein and TNF-alpha, compared with the control virus expressing TNF-alpha alone. These results suggest that the 14.7K protein, which is nonessential for Ad replication in tissue culture, is an immunoregulatory protein which functions in vivo to help counteract the antiviral effects of TNF-alpha.