Nuclear Factor-Kappa B Family Member RelB Inhibits Human Immunodeficiency Virus-1 Tat-Induced Tumor Necrosis Factor-Alpha Production

Human Immunodeficiency Virus-1 (HIV-1)-associated neurocognitive disorder (HAND) is likely neuroinflammatory in origin, believed to be triggered by inflammatory and oxidative stress responses to cytokines and HIV protein gene products such as the HIV transactivator of transcription (Tat). Here we demonstrate increased messenger RNA for nuclear factor-kappa B (NF-κB) family member, transcription factor RelB, in the brain of doxycycline-induced Tat transgenic mice, and increased RelB synthesis in Tat-exposed microglial cells. Since genetic ablation of RelB in mice leads to multi-organ inflammation, we hypothesized that Tat-induced, newly synthesized RelB inhibits cytokine production by microglial cells, possibly through the formation of transcriptionally inactive RelB/RelA complexes. Indeed, tumor necrosis factor-alpha (TNFα) production in monocytes isolated from RelB deficient mice was significantly higher than in monocytes isolated from RelB expressing controls. Moreover, RelB overexpression in microglial cells inhibited Tat-induced TNFα synthesis in a manner that involved transcriptional repression of the TNFα promoter, and increased phosphorylation of RelA at serine 276, a prerequisite for increased RelB/RelA protein interactions. The Rel-homology-domain within RelB was necessary for this interaction. Overexpression of RelA itself, in turn, significantly increased TNFα promoter activity, an effect that was completely blocked by RelB overexpression. We conclude that RelB regulates TNFα cytokine synthesis by competitive interference binding with RelA, which leads to downregulation of TNFα production. Moreover, because Tat activates both RelB and TNFα in microglia, and because Tat induces inflammatory TNFα synthesis via NF-κB, we posit that RelB serves as a cryoprotective, anti-inflammatory, counter-regulatory mechanism for pathogenic NF-κB activation. These findings identify a novel regulatory pathway for controlling HIV-induced microglial activation and cytokine production that may have important therapeutic implications for the management of HAND

Hiv-1 Tat Potentiates Tnf-Induced Nf-Kappa-B Activation and Cytotoxicity

This study demonstrates that human immunodeficiency virus type 1 (HTV-1) Tat protein amplifies the activity of tumor necrosis factor (TNF), a cytokine that stimulates HTV-1 replication through activation of NF-kappa B. In HeLa cells stably transfected with the HIV-1 tat gene (HeLa-tat cells), expression of the Tat protein enhanced both TNF-induced activation of NF-kappa B and TNF-mediated cytotoxicity. A similar potentiation of TNF effects was observed in Jurkat T cells and HeLa cells treated with soluble Tat protein, TNF-mediated activation of NF-kappa B and cytotoxicity involves the intracellular formation of reactive oxygen intermediates. Therefore, Tat-mediated effects on the cellular redox state were analyzed. In both T cells and HeLa cells HIV-1 Tat suppressed the expression of Mn-dependent superoxide dismutase (Mn-SOD), a mitochondrial enzyme that is part of the cellular defense system against oxidative stress. Thus, Mn-SOD RNA protein levels and activity were markedly reduced in the presence of Tat. Decreased Mn-SOD expression was associated with decreased levels of glutathione and a lower ratio of reduced:oxidized glutathione. A truncated Tat protein (Tat(1-72)), known to transactivate the HIV-1 long terminal repeat (LTR), no longer affected Mn-SOD expression, the cellular redox state or TNF-mediated cytotoxicity. Thus, our experiments demonstrate that the C-terminal region of HIV-1 Tat is required to suppress Mn-SOD expression and to induce pro-oxidative conditions reflected by a drop in reduced glutathione (GSH) and the GSH:oxidized GSH (GSSG) ratio. They further imply a distinct mechanism of Mn-SOD suppression as compared,vith HIV-1 LTR transactivation by Tat. Taken together, our data suggest that Tat expressed in HIV-1-infected cells and Tat taken up by non-infected cells modulates TNF activity by altering the cellular redox state. These findings may be relevant for HIV-1 replication and for T cell depletion in acquired immune deficiency syndrome

Gene silencing approaches in mast cells and primary human basophils.

The ability to silence gene expression is an invaluable tool for elucidating the importance of intracellular signaling proteins which contribute to the effector functions of mast cells and basophils. However, primary mast cells and their terminally differentiated blood counterpart, namely basophils, pose a difficult challenge for gene silencing approaches given not only their state of maturation and difficulty to transfect, but also because their functions are readily altered by cell-handling conditions. Here, we describe a method using lipofection which has been successfully employed to silence gene expression using siRNA in human LAD2 mast cells as well as primary human basophils

Apoptosis signal-regulating kinase 1 (ASK1) and HIF-1 alpha protein are essential factors for nitric oxide-dependent accumulation of p53 in THP-1 human myeloid macrophages

Nitric oxide (NO) is a reactive secondary mediator, which has been found to participate in cell cycle regulation and apoptosis in myeloid macrophages, the key effectors of inflammatory and innate immune responses. However, the molecular mechanisms of nitric oxide-induced death of myeloid macrophages are not well understood. In this study we have found that NO derived from S-nitrosoglutathione (GSNO) activates ASK1 in THP-1 human myeloid macrophages in a concentration and time-dependent manner. It also induces accumulation of HIF-1 alpha protein in a concentration-dependent manner, which peaks at 4 h of exposure to 1 mM GSNO. GSNO does not affect the level of HIF-1 alpha mRNA as detected by the RT-PCR. In addition, GSNO was found to induce accumulation of p53 in normal but not HIF-1 alpha knockdown THP-1 cells, where expression of this protein was silenced by specific siRNA. It has also been found that GSNO-mediated accumulation of p53 depends on activation of ASK1 since no GSNO-induced p53 stabilisation was observed in THP-1 cells transfected with dominant-negative form of this kinase. However, in both HIF-1 alpha knockdown THP-1 cells and those transfected with the dominant-negative form of ASK1, GSNO was able to induce cell death as detected by the MTS cell viability assay leading to an increase in release of LDH

Involvement of xanthine oxidase and hypoxia-inducible factor 1 in Toll-like receptor 7/8-mediated activation of caspase 1 and interleukin-1?

Inflammatory reactions to ssRNA viruses are induced by the endosomal Toll-like receptors (TLRs) 7 and 8. TLR7/8-mediated inflammatory reaction results in activation of the Nalp3 inflammasome via an unknown mechanism. Here we report for the first time that TLR7/8 mediate activation of xanthine oxidase (XOD) in an HIF-1?-dependent manner. XOD produces uric acid and reactive oxygen species, which could activate Nalp3 and therefore induce activation of caspase 1, known to convert inactive pro-IL-1? into active IL-1?. Specific inhibition of the XOD activity attenuates TLR7/8-mediated activation of caspase 1 and IL-1? release. These results were obtained using human THP-1 myeloid macrophages. The findings were verified by conducting in vivo experiments on mice