E5564 inhibits immunosuppressive cytokine IL-10 induction promoted by HIV-1 Tat protein

Background

In HIV-1 infected patients, production of interleukin-10 (IL-10), a highly immunosuppressive cytokine, is associated with progression of infection toward AIDS. HIV-1 Tat protein, by interacting with TLR4-MD2 at the membrane level, induces IL-10 production by primary human monocytes and macrophages. In the present study we evaluated the effect of the TLR4 antagonist Eritoran tetrasodium (E5564) on HIV-1 Tat-induced IL-10 production.

Findings

Here, we confirm that the recombinant HIV-1 Tat protein and the GST-Tat 1–45 fusion protein efficiently stimulate IL-10 production by primary monocytes and macrophages and that this stimulation is inhibited by blocking anti-TLR4 mAbs. We show that a similar inhibition is observed by preincubating the cells with the TLR4 antagonist E5564.

Conclusion

This study provides compelling data showing for the first time that the TLR4 antagonist E5564 inhibits the immunosuppressive cytokine IL-10 production by primary human monocytes and macrophages incubated in the presence of HIV-1 Tat protein.

     

Apoptosis enhancement by the HIV-1 Nef protein

The HIV-1 nef gene, essential for AIDS pathogenesis, encodes a 27-kDa protein (Nef) whose biochemical and biological functions are unclear. It has been suggested that Nef expression contributes to the T cell depletion observed during the disease by promoting their apoptosis. We report that in CD4(+) human lymphoblastoid cell lines transfected with the nef cDNA obtained from three different HIV-1 strains, expression of the Nef protein enhances and accelerates the response to four unrelated apoptotic agents (staurosporine, anisomycin, camptothecin, and etoposide) but not to an anti-Fas agonist Ab. Nef reduces the expression of the anti-apoptotic proteins Bcl-2 and Bcl-X(L) and induces a striking enhancement of apoptotic hallmarks, including mitochondrial depolarization, exposure of phosphatidylserine on the cell surface, activation of caspase-3, and cleavage of the caspase target poly(ADP-ribose) polymerase. Interestingly, the peptide Z-Val-Ala-DL-Asp-fluoromethylketone (a broad-spectrum caspase inhibitor) reduces, but does not abolish, phosphatidylserine exposure, suggesting that Nef also activates a caspase-independent apoptotic pathway. Surprisingly, Nef expression increases DNA degradation but without causing oligonucleosomal fragmentation. An increased apoptotic response and down-modulation of Bcl-2/Bcl-X(L) following Nef expression are observed also in NIH-3T3 fibroblasts. These data show that Nef enhances programmed cell death in different cell types by affecting multiple critical components of the apoptotic machinery independently from the Fas pathway.     

Regulation of interleukin-18 by THP-1 monocytoid cells stimulated with HIV-1 and Nef viral protein

Interleukin (IL)-18 is a proinflammatory cytokine that plays an important role in both innate and adaptive immune responses against several infectious pathogens. Relatively little is known about its production in HIV-1 infection, and there are controversial data on the influence of IL-18 on HIV-1 replication in vitro. In this study, we investigated the effect of HIV-1 infection, and challenge with recombinant HIV-1 proteins, on IL-18 production by THP-1 cells. This is a monocytoid cell line spontaneously producing IL-18, and consequently is particularly suitable for the study of HIV-1 effects on this type of cytokine regulation. The results reported here demonstrate a significant reduction in IL-18 secretion during HIV-infection. In fact, low levels of IL-18 were released until 120 h from viral challenge (15 +/- 11 pg/mL at 24 h and 17 +/- 13 at 96 h and < 12.5 at 120 h), whereas IL-18 production by uninfected control cells was 193 +/- 104 pg/mL and 214 +/- 114 pg/mL at 24 h and 120 h respectively. At 168 h of incubation, IL-18 production by infected and uninfected cells was found to be 164 +/- 88 pg/mL and 325 +/- 101 pg/mL respectively (p = 0.001). Of the following viral proteins: gp 120, p24 and Nef, only the last one induced decreased IL-18 secretion in the supernatants of THP-1 cells. This effect is more evident with the concentrations of 5 -1.25 microg/mL of Nef protein (p < 0.0001). In conclusion, our data show that HIV-1 and its regulatory protein, Nef, are able to down-regulate the release of IL-18, in vitro. These results confirm that a variety of modulating effects on the immune response, induced by HIV-infection, may facilitate progression of HIV-1 infection.     

A specific protein disorder catalyzer of HIV-1 Nef

The HIV-1 auxiliary protein Nef is required for the onset and progression of AIDS in HIV-1-infected persons. Here, we have deciphered the mode of action of a second-generation inhibitor of Nef, DLC27-14, presenting a competitive IC(50) of ～16 μM measured by MALDI-TOF experiments. Thermal protein denaturation experiments revealed a negative effect on stability of Nef in the presence of a saturating concentration of the inhibitor. The destabilizing action of DLC27-14 was confirmed by a HIV protease-based experiment, in which the protease sensitivity of DLC27-14-bound Nef was three times as high as that of apo Nef. The only compatible docking modes of action for DLC27-14 suggest that DLC27-14 promotes an opening of two α-helices that would destabilize the Nef core domain. DLC27-14 thus acts as a specific protein disorder catalyzer that destabilizes the folded conformation of the protein. Our results open novel avenues toward the development of next-generation Nef inhibitors.     

Role of HIV-1 Nef protein for virus replication in vitro

The Nef protein of primate lentiviruses (simian and human immunodeficiency viruses; SIV/HIVs) appears to be multi-functional and plays a pivotal role in viral persistence and pathogenesis in vivo. Of its numerous functions reported to date, the ability to enhance virion infectivity in indicator cell lines and to augment viral replication in peripheral blood mononuclear cells (PBMCs) and lymphocytes (PBLs) is very well conserved among various SIV/HIVs. This review summarizes and organizes current knowledge of HIV-1 Nef with respect to this particularly virological activity for understanding the basis of its in vivo function.     

Blockade of BFA-mediated apoptosis in macrophages by the HIV-1 Nef protein

HIV-1 Nef protein has key roles at almost all stages of the viral life cycle. We assessed the role of Nef and of the translation elongation factor eEF1A in primary human macrophages. Nuclear retention experiments and inhibition of the exportin-t (Exp-t) pathway suggested that cytoplasmic relocalization of eEF1A, mediated by Exp-t occurs in Nef-treated monocyte-derived macrophages (MDMs). We observed the presence of tRNA in the Nef/eEF1A complexes. Nucleocytoplasmic relocalization of the Nef/eEF1A complexes prevented stress-induced apoptosis of MDMs treated with brefeldin A. Blockade of stress-induced apoptosis of MDMs treated with HIV-1 Nef resulted from enhanced nucleocytoplasmic transport of eEF1A with decreased release of mitochondrial cytochrome c, and from increased tRNA binding to cytochrome c, ultimately leading to an inhibition of caspase activation. Our results indicate that HIV-1 Nef, through the nucleocytoplasmic relocalization of eEF1A and tRNAs, enhances resistance to stress-induced apoptosis in primary human macrophages.     

Effects of the exogenous Nef protein on HIV‐1 target cells

Nef is a multifunctional gene of HIV which can increase virus replication either directly or by modulating the target cell's metabolism. Nevertheless the role of the exogenous Nef protein is not yet well understood. To investigate it, we studied the effects of the recombinant Nef protein on the expression of some antigens of lymphoid T‐cells permissive to HIV‐1 replication, and on their proliferation and on apoptosis induction. For this purpose, we utilised MT‐4 and H9 T‐cell lines. We evaluated FN (fibronectin), CD4 and CD71 expression in uninfected and acutely or chronically infected cells, both untreated and treated with Nef. Our studies showed a significant up‐regulation of FN especially in uninfected cells, with a dose of 2·5 μg ml⁻¹; in contrast, a significant down‐modulation of CD4 and CD71 both in uninfected and in acutely or chronically infected cells, was detected. The proliferation of H9 uninfected cells was significantly reduced 24 h after treatment with Nef protein in a dose‐dependent manner (ranging from 0·02 to 2·5 μg ml⁻¹); likewise a significant inhibition of proliferation of acutely and chronically infected cells was evident with 2·5 μg ml⁻¹. Moreover, we demonstrated a dose‐dependent activity of Nef on inducing apoptosis in H9 uninfected cells and no effects of this protein on modulation of INF α and γ production in peripheral blood mononucleated cells of health donors. Nef appeared to be able to increase the effect of apoptotic stimuli. In conclusion, our data suggest that in our experimental system, the exogenous Nef protein can inhibit cellular synthesis facilitating the metabolic pathway involved in virus replication. In addition it modulates the susceptibility to the HIV‐1 infection and finally, that apoptosis induction or enhancement can facilitate the release of neoformed virions. Copyright © 1999 John Wiley & Sons, Ltd.     

The HIV-1 Nef protein and phagocyte NADPH oxidase activation

Nef, a multifunctional HIV protein, activates the Vav/Rac/p21-activated kinase (PAK) signaling pathway. Given the potential role of this pathway in the activation of the phagocyte NADPH oxidase, we have investigated the effect of the HIV-1 Nef protein on the phagocyte respiratory burst. Microglia (cell line and primary culture) were transduced with lentiviral expression vectors. Expression of Nef did not activate the NADPH oxidase by itself but led to a massive enhancement of the responses to a variety of stimuli (Ca(2+) ionophore, formyl peptide, endotoxin). These effects were not caused by up-regulation of phagocyte NADPH oxidase subunits. Nef mutants lacking motifs involved in the interaction with Vav and PAK failed to reproduce the effects of wild type Nef, suggesting a role for the Vav/Rac/PAK signaling pathway. The following results suggest a key role for Rac in the priming effect of Nef. (i) Inactivation of Rac by Clostridium difficile toxin B abolished the Nef effect. (ii) The fraction of activated Rac1 was increased in Nef-transduced cells, and (iii) the dominant positive Rac1(V12) mutant mimicked the effect of Nef. These results are to our knowledge the first analysis of the effect of Rac activation on the NADPH oxidase in intact phagocytes. Rac activation is not sufficient to stimulate the phagocyte NADPH oxidase; however, it markedly enhances the NADPH oxidase response to other stimuli.     

IL-4 and IL-10 are both required for the induction of oral tolerance

Protection from the development of experimental autoimmune uveitis (EAU) can be induced by feeding mice interphotoreceptor retinoid binding protein before uveitogenic challenge with the same protein. Two different regimens are equally effective in inducing protective tolerance, although they seem to do so through different mechanisms: one involving regulatory cytokines (IL-4, IL-10, and TGF-beta), and the other with minimal involvement of cytokines. Here we studied the importance of IL-4 and IL-10 for the development of oral tolerance using mice genetically engineered to lack either one or both of these cytokines. In these animals we were able to protect against EAU only through the regimen inducing cytokine-independent tolerance. When these animals were fed a regimen that in the wild-type animal is thought to predominantly induce regulatory cells and is associated with cytokine secretion, they were not protected from EAU. Interestingly, both regimens were associated with reduced IL-2 production and proliferation in response to interphotoreceptor retinoid binding protein. These findings indicate that both IL-4 and IL-10 are required for induction of protective oral tolerance dependent on regulatory cytokines, and that one cytokine cannot substitute for the other in this process. These data also underscore the fact that oral tolerance, manifested as suppression of proliferation and IL-2 production, is not synonymous with protection from disease.     

Characterization of intramolecular interactions of HIV-1 accessory protein Nef by differential scanning calorimetry

The interactions between the N-terminal arm and the structural core of Nef, an HIV-1 accessory protein, have been studied by high sensitivity differential scanning calorimetry. Critical interactions have been identified by measuring the structural energetics of both truncation and point mutants of the protein. These studies demonstrate that the N-terminal arm of Nef strongly interacts with the core and that it contributes close to 4.3 kcal/mol to the stability of the entire protein. The interactions are not evenly distributed through the N-terminal arm. Two strongly interacting regions have been identified: the region between residues 1 and 40 that contributes 1.6 kcal/mol to the stability of Nef; and, the region between residues 50 and 61 that contributes 2.7 kcal/mol. Other regions (e.g. residues 41 to 49) appear to contribute little to the interaction. The identification of these, until now, elusive interactions provides a physical foundation for allosteric effects between N-terminal arm and protein core elicited by different binding partners.     

HIV-1 Nef disrupts the podocyte actin cytoskeleton by interacting with diaphanous interacting protein

The ability of the human immunodeficiency virus, type 1 (HIV-1) protein Nef to induce cytoskeleton changes in infected host cells is a key event in viral replication. In renal podocytes, we found that Nef induced loss of stress fibers and increased lamellipodia, pathological changes leading to proteinuria in HIV-associated nephropathy. These morphological changes were mediated by Nef-induced Rac1 activation and RhoA inhibition. We identified a new interaction between Nef and diaphanous interacting protein (DIP), a recently described regulator of Rho and Rac signaling. We found that the Src homology 3 binding domain of DIP and the Nef PXXP motif were required for this interaction. Nef also interacts with Vav2 in podocytes. DIP and Vav2 both interact directly with Nef in a competitive manner. DIP interacts with p190RhoGAP, and intact DIP was required for Nef-induced phosphorylation of p190RhoGAP. DIP also interacts with Vav2, and although DIP enhanced baseline phosphorylation of Vav2, it was not required for Nef-induced Vav2 activation. In Nef-infected podocytes, Src kinase induces phosphorylation of DIP, p190RhoGAP, and Vav2, leading to RhoA inhibition and Rac1 activation. Inhibition of the Nef-induced signaling pathway by using a dominant negative of either Src or DIP or siRNA for DIP or p190RhoAGAP restored RhoA activity and stress fiber formation in Nef-infected podocytes, whereas siRNA for Vav2 reduced Rac1 activity and formation of lamellipodia. We conclude that in HIV-infected podocytes, Nef, through the recruitment of DIP and p190RhoAGAP to Nef-Src complex, activates p190RhoAGAP and down-regulates RhoA activity.     

Down-Regulation of CTLA-4 by HIV-1 Nef Protein

HIV-1 Nef protein down-regulates several cell surface receptors through its interference with the cell sorting and trafficking machinery. Here we demonstrate for the first time the ability of Nef to down-regulate cell surface expression of the negative immune modulator CTLA-4. Down-regulation of CTLA-4 required the Nef motifs DD175, EE155 and LL165, all known to be involved in vesicle trafficking. Disruption of the lysosomal functions by pH-neutralizing agents prevented CTLA-4 down-regulation by Nef, demonstrating the implication of the endosomal/lysosomal compartments in this process. Confocal microscopy experiments visualized the co-localization between Nef and CTLA-4 in the early and recycling endosomes but not at the cell surface. Overall, our results provide a novel mechanism by which HIV-1 Nef interferes with the surface expression of the negative regulator of T cell activation CTLA-4. Down-regulation of CTLA-4 may contribute to the mechanisms by which HIV-1 sustains T cell activation, a critical step in viral replication and dissemination.     

HIV-1 Tat protein induces IL-10 production in monocytes by classical and alternative NF-kappaB pathways

The human immunodeficiency virus (HIV) transactivating Tat protein is not only critical for viral replication but also affects the host immune system by inducing the production of cytokines such as IL-10. This anti-inflammatory cytokine is upregulated during the course of HIV infection, representing an important pathway by which HIV may induce immunodeficiency. Here, we show that, by acting at the membrane, Tat induces IL-10 expression in primary monocytes and promonocytic U937 cells by NF-kappaB-dependent pathways. The trans-dominant negative mutants of NF-kappaB-inducing kinase (NIK), IKKalpha and IKKbeta expressed in our transactivation model, in accordance with the nuclear binding of p65 and p52 NF-kappaB subunits to the IL-10 promoter, suggest the involvement of both classical and alternative NF-kappaB pathways. In inactivated cells, IKKalpha is localized predominantly in the cytoplasm. Interestingly, Tat stimulates IKKalpha translocation from the cytoplasm to the nucleus in monocytes. Chromatin immunoprecipitation (ChIP) assay experiments, after Tat treatment, revealed IKKalpha and CBP/p300 recruitment to the IL-10 promoter and histone H3 phosphorylation (Ser 10) and acetylation (Lys 14) in this region, presumably leading to chromatin remodeling. We demonstrate that, upstream of NF-kappaB, PKC, ERK1/2 and p38 MAP kinases are involved in Tat-induced IKKalpha nuclear translocation and histone H3 modifications on the IL-10 promoter in accordance with the role of these three kinases in IL-10 production. As a whole, the study demonstrates that Tat activates at least three signaling pathways concurrently, including the classical, alternative and IKKalpha pathways, to promote production of IL-10.     

HIV-1 Nef stabilizes the association of adaptor protein complexes with membranes

The maximal virulence of HIV-1 requires Nef, a virally encoded peripheral membrane protein. Nef binds to the adaptor protein (AP) complexes of coated vesicles, inducing an expansion of the endosomal compartment and altering the surface expression of cellular proteins including CD4 and class I major histocompatibility complex. Here, we show that Nef stabilizes the association of AP-1 and AP-3 with membranes. These complexes remained with Nef on juxtanuclear membranes despite the treatment of cells with brefeldin A, which induced the release of ADP-ribosylation factor 1 (ARF1) from these membranes to the cytosol. Nef also induced a persistent association of AP-1 and AP-3 with membranes despite the expression of dominant-negative ARF1 or the overexpression of an ARF1-GTPase activating protein. Mutational analysis indicated that the direct binding of Nef to the AP complexes is essential for this stabilization. The leucine residues of the EXXXLL motif found in Nef were required for binding to AP-1 and AP-3 in vitro and for the stabilization of these complexes on membranes in vivo, whereas the glutamic acid residue of this motif was required specifically for the binding and stabilization of AP-3. These data indicate that Nef mediates the persistent attachment of AP-1 and AP-3 to membranes by an ARF1-independent mechanism. The stabilization of these complexes on membranes may underlie the pleiotropic effects of Nef on protein trafficking within the endosomal system.     

HIV-1 Nef protects human-monocyte-derived macrophages from HIV-1-induced apoptosis

HIV-1 Nef is the regulatory protein expressed earliest and most abundantly in the infection cycle. Its expression has been correlated with a plethora of effects detectable either in producer, target, and bystander cells, as well as in the viral particles. Even if the relationship between Nef expression and apoptosis has been already matter of investigation in infected lymphocytes, whose resistance to HIV infection is however limited to few days, this remains to be investigated in cells that in vivo well resist the HIV cytopathic effect. In such an instance, we were interested in establishing whether Nef influences the apoptotic processes in primary human-monocyte-derived macrophages (MDM). High efficiency HIV-1 infection of MDM allowed us to establish that virus-expressed Nef strongly counteracts the HIV-1-induced apoptosis. The Nef mutant analysis suggested that this effect relies on the interaction with different protein partners and cell compartments. We also observed that the Nef protection to the HIV-1-induced apoptosis correlated with the hyper-phosphorylation and consequent inactivation of the pro-apoptotic Bad protein. On the basis of these results, we propose the Nef anti-apoptotic effect as a relevant part of the mechanism of the in vivo establishment of the HIV macrophage reservoirs.     

Dysregulation of autophagy by HIV-1 Nef in human astrocytes

Viruses often exploit autophagy, a common cellular process of degradation of damaged proteins, organelles, and pathogens, to avoid destruction. HIV-1 dysregulates this process in several cell types by means of Nef protein. Nef is a small HIV-1 protein which is expressed abundantly in astrocytes of HIV-1-infected brains and has been suggested to have a role in the pathogenesis of HIV-Associated Neurocognitive Disorders (HAND). In order to explore its effect in the CNS with respect to autophagy, HIV-1 Nef was expressed in primary human fetal astrocytes (PHFA) using an adenovirus vector (Ad-Nef). We observed that Nef expression triggered the accumulation of autophagy markers, ATG8/LC3 and p62 (SQSMT1). Similar results were obtained with Bafilomycin A1, an autophagy inhibitor which blocks the fusion of autophagosome to lysosome. Furthermore co-expression of tandem LC3 vector (mRFP-EGFP-LC3) and Ad-Nef in these cells produced mainly yellow puncta (mRFP+, EGFP+) strongly suggesting that autophagosome fusion to lysosome is blocked in PHFA cells in the presence of Nef. Together these data indicate that HIV-1 Nef mimics Bafilomycin A1 and blocks the last step of autophagy thereby helping HIV-1 virus to avoid autophagic degradation in human astrocytes.