[Show abstract][Hide abstract] ABSTRACT: The potential role of the human immunodeficiency virus-1 (HIV-1) accessory protein Nef in the pathogenesis of neuroAIDS is still poorly understood. Nef is a molecular adapter that influences several cellular signal transduction events and membrane trafficking. In human macrophages, Nef expression induces the production of extracellular factors (e.g. pro-inflammatory chemokines and cytokines) and the recruitment of T cells, thus favoring their infection and its own transfer to uninfected cells via exosomes, cellular protrusions or cell-to-cell contacts. Murine cells are normally not permissive for HIV-1 but, in transgenic mice, Nef is a major disease determinant. Both in human and murine macrophages, myristoylated Nef (myr+Nef) treatment has been shown to activate NF-κB, MAP kinases and interferon responsive factor 3 (IRF-3), thereby inducing tyrosine phosphorylation of signal transducers and activator of transcription (STAT)-1, STAT-2 and STAT-3 through the production of proinflammatory factors.
We report that treatment of BV-2 murine microglial cells with myr+Nef leads to STAT-1, -2 and -3 tyrosine phosphorylation and upregulates the expression of inducible nitric oxide synthase (iNOS) with production of nitric oxide. We provide evidence that extracellular Nef regulates iNOS expression through NF-κB activation and, at least in part, interferon-β (IFNβ) release that acts in concert with Nef. All of these effects require both myristoylation and a highly conserved acidic cluster in the viral protein. Finally, we report that Nef induces the release of neurotoxic factors in the supernatants of microglial cells.
These results suggest a potential role of extracellular Nef in promoting neuronal injury in the murine model. They also indicate a possible interplay between Nef and host factors in the pathogenesis of neuroAIDS through the production of reactive nitrogen species in microglial cells.
PLoS ONE 06/2015; 10(6):e0130189. DOI:10.1371/journal.pone.0130189 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Atrial natriuretic peptide (ANP), a cardiovascular hormone, elicits different biological actions in the immune system. Aim of the present study was to investigate in THP-1 monocytes the ANP effect on hydrogen peroxide (H2O2)-induced Reactive Oxygen Species (ROS), cell proliferation and migration. A significant increase of H2O2-dependent ROS production was induced by physiological concentration of ANP (10(-10)M). The ANP action was partially affected by cell pretreatment with PD98059, an inhibitor of mitogen activated- protein kinases (MAPK) as well as by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K) and totally suppressed by diphenylene iodonium (DPI), an inhibitor of the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. The hormone effect was mimicked by cANF and an ANP/NPR-C signalling pathway was studied using pertussis toxin (PTX). A significant increase of H2O2-induced cell migration was observed after ANP (10(-10)M) treatment, conversely a decrease of THP-1 proliferation, due to cell death, was found. Both ANP actions were partially prevented by DPI. Moreover, H2O2-induced release of IL-9, TNF-α, MIP-1α and MIP-1β was not counteracted by DPI, whereas no effect was observed in any experimental condition for both IL-6 and IL-1β. Our results support the view that ANP can play a key role during the inflammatory process.
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) are small (typically 22 nucleotides) non-coding, endogenous, single-stranded RNAs. MiRNA genes are evolutionarily conserved and are located within the introns or exons of protein-coding genes, as well as in intergenic areas. Before the discovery of miRNAs, it had been known that a large part of the genome is not translated into proteins. This so called "junk" DNA was thought to be evolution debris with no function. Recently, the explosive research in this area has established miRNAs as powerful regulators of gene expression. While only about 1,424 human miRNA sequences have been identified so far, genomic computational analysis indicates that as many as 50,000 miRNAs may exist in the human genome, and each may have multiple targets based on similar sequences in the 3'-UTR of mRNA. MiRNAs have been implicated in different areas such as the immune response, neural development, DNA repair, apoptosis, oxidative stress response and others and it is impressive the list of diseases which have recently been found to be associated with abnormal miRNA expression. Here, we focus our attention on the importance of cancer regulator miRNAs. They are divided into oncomiRs and anti-oncomiRs that negatively regulate tumor suppressor genes and oncogenes, respectively. Importantly, the association of miRNAs with cancer has prompted additional functional classification of these short RNAs and their potential relevance in cancer diagnosis, prognosis and treatment.
Current Medicinal Chemistry 12/2012; 19(4):461-74. DOI:10.2174/092986712798918798 · 3.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Interferon (IFN)-β inhibits cell proliferation and affects cell cycle in keratinocytes transformed by both mucosal high risk Human Papilloma Virus (HPV) and cutaneous HPV E6 and E7 proteins. In particular, upon longer IFN-β treatments, cutaneous HPV38 expressing cells undergo senescence. IFN-β appears to induce senescence by upregulating the expression of the tumor suppressor PML, a well known IFN-induced gene. Indeed, experiments in gene silencing via specific siRNAs have shown that PML is essential in the execution of the senescence programme and that both p53 and p21 pathways are involved. IFN-β treatment leads to a modulation of p53 phosphorylation and acetylation status and a reduction in the expression of the p53 dominant negative ΔNp73. These effects allow the recovery of p53 transactivating activity of target genes involved in the control of cell proliferation. Taken together, these studies suggest that signaling through the IFN pathway might play an important role in cellular senescence. This additional understanding of IFN antitumor action and mechanisms influencing tumor responsiveness or resistance appears useful in aiding further promising development of biomolecular strategies in the IFN therapy of cancer.
PLoS ONE 05/2012; 7(5):e36909. DOI:10.1371/journal.pone.0036909 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Despite the fact that murine cells are not permissive for human immunodeficiency virus type 1 (HIV-1) infection, several investigators have constructed transgenic (Tg) mice to model HIV-1-induced diseases to overcome this restriction. The generation of Tg mice expressing selected HIV-1 genes revealed that Nef harbors a major disease determinant. HIV-1 Nef protein is a molecular adapter able to interact with several cellular partners, interfering with cellular functions. The phenotype of Nef Tg mice was extensively characterized regarding in vivo development of AIDS-like disease and the effects of Nef expression in T lymphocytes, but the functions eventually corrupted by Nef in monocytes and macrophages were less studied. Nef treatment of human monocyte-derived macrophages induces the internalization of the protein and modulates the production and secretion of different chemokines and cytokines by activating specific intracellular signaling pathways (i.e., NF-κB, MAPK, and IRF3). Therefore we set up an in vitro murine macrophage-based model using stabilized cell lines and primary peritoneal macrophages, and treated them with recombinant myristoylated Nef(SF2) (recNef). Like human cells, murine macrophages responded to Nef treatment, activating IKK-α and IKK-β, JNK, and p38 MAP kinases. Activation of the NF-κB pathway is mandatory for the synthesis and release of a pool of cytokines and chemokines, including IFN-β, that induce tyrosine phosphorylation of the signal transducer and activator of transcription (STAT)-1, STAT-2, and STAT-3, in an autocrine and paracrine manner, confirming that murine macrophages respond to Nef similarly to human ones. These data extend the results previously obtained in human primary macrophages, allowing the use of murine cells in culture to study signaling events modulated by Nef in myeloid-derived cells. In particular, it may be feasible to use macrophages derived from mice knocked out in specific signaling intermediates to obtain greater insight into the mechanism of Nef-induced effects.
[Show abstract][Hide abstract] ABSTRACT: It is now widely accepted that thyroid hormones, l-thyroxine (T(4)) and 3,3',5-triiodo-l-thyronine (T(3)), act as modulators of the immune response. Immune functions such as chemotaxis, phagocytosis, generation of reactive oxygen species, and cytokine synthesis and release, are altered in hypo- and hyper-thyroid conditions, even though for many immune cells no clear correlation has been found between altered levels of T(3) or T(4) and effects on the immune responses. Integrins are extracellular matrix proteins that are important modulators of many cellular responses, and the integrin αvβ3 has been identified as a cell surface receptor for thyroid hormones. Rapid signaling via this plasma membrane binding site appears to be responsible for many nongenomic effects of thyroid hormones, independent of the classic nuclear receptors. Through the integrin αvβ3 receptor the hormone can activate both the ERK1/2 and phosphatidylinositol 3-kinase pathways, with downstream effects including intracellular protein trafficking, angiogenesis and tumor cell proliferation. It has recently become clear that an important downstream target of the thyroid hormone nongenomic pathway may be the mammalian target of rapamycin, mTOR. New results demonstrate the capability of T(3) or T(4) to induce in the short time range important responses related to the immune function, such as reactive oxygen species production and cell migration in THP-1 monocytes. Thus thyroid hormones seem to be able to modulate the immune system by a combination of rapid nongenomic responses interacting with the classical nuclear response.
[Show abstract][Hide abstract] ABSTRACT: HIV-1 Nef is a virulence factor that plays multiple roles during HIV replication. Recently, it has been described that Nef intersects the CD40 signalling in macrophages, leading to modification in the pattern of secreted factors that appear able to recruit, activate and render T lymphocytes susceptible to HIV infection. The engagement of CD40 by CD40L induces the activation of different signalling cascades that require the recruitment of specific tumor necrosis factor receptor-associated factors (i.e. TRAFs). We hypothesized that TRAFs might be involved in the rapid activation of NF-κB, MAPKs and IRF-3 that were previously described in Nef-treated macrophages to induce the synthesis and secretion of proinflammatory cytokines, chemokines and IFNβ to activate STAT1, -2 and -3.
Searching for possible TRAF binding sites on Nef, we found a TRAF2 consensus binding site in the AQEEEE sequence encompassing the conserved four-glutamate acidic cluster. Here we show that all the signalling effects we observed in Nef treated macrophages depend on the integrity of the acidic cluster. In addition, Nef was able to interact in vitro with TRAF2, but not TRAF6, and this interaction involved the acidic cluster. Finally silencing experiments in THP-1 monocytic cells indicate that both TRAF2 and, surprisingly, TRAF6 are required for the Nef-induced tyrosine phosphorylation of STAT1 and STAT2.
Results reported here revealed TRAF2 as a new possible cellular interactor of Nef and highlighted that in monocytes/macrophages this viral protein is able to manipulate both the TRAF/NF-κB and TRAF/IRF-3 signalling axes, thereby inducing the synthesis of proinflammatory cytokines and chemokines as well as IFNβ.
PLoS ONE 08/2011; 6(8):e22982. DOI:10.1371/journal.pone.0022982 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In newborn the innate immune system provides essential protection during primary infections before the generation of an appropriate adaptive immune response that is initially not fully operative. Innate immune response is evoked and perpetuated by molecules derived from microorganisms or by the damage/death of host cells. These are collectively known as damage-associated molecular-pattern (DAMP) molecules. High-mobility group box 1 protein (HMGB1) or amphoterin, which previously was considered to be only a nuclear factor, has been recently identified as a DAMP molecule. When it is actively secreted by inflammatory cells or passively released from necrotic cells, HMGB1 mediates the response to infection, injury and inflammation, inducing dendritic cells maturation and T helper-1-cell responses. To characterize the role of HMGB1 in the innate and immature defense mechanisms in newborns, human cord blood (CB) mononuclear cells, in comparison to adult peripheral blood (PB) mononuclear cells, have been analyzed for its expression. By flow cytometry and western blot analysis, we observed that in CB and PB cells: i) HMGB1 is expressed on cell surface membranes of myeloid dendritic cell precursors, mostly, and lymphocytes (gamma/delta and CD4(+) T cells) to a lesser extent; ii) different pro-inflammatory stimuli or molecules that mimic infection increased cell surface expression of HMGB1 as well as its secretion into extracellular environment; iii) the treatment with synthetic molecules such as aminobisphosphonates (ABs), identified to be γδ T cell antigens, triggered up-regulation of HMGB1 expression on mononuclear cells, as well γδ T lymphocytes, inducing its secretion. The modulation of its secretion and the HMGB1-mediated migration of monocytes indicated HMGB1 as regulator of immune response in an immature system, like CB, through engagement of γδ T lymphocytes and myeloid dendritic cell precursors, essential components of innate immunity. In addition, the increased HMGB1 expression/secretion triggered by ABs, previously characterized for their immuno-modulating and immune-adjuvant capabilities, indicated that immunomodulation might represent a new therapeutical approach for neonatal and adult pathologies.
PLoS ONE 08/2011; 6(8):e23766. DOI:10.1371/journal.pone.0023766 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Anticancer drug-induced tumor suppression may involve mechanisms of protection against neoplastic transformation that are normally latent in mammalian cells and consist in a genetic program implemented during anti-tumoral defense. This defense program results in the self elimination of cells harboring potentially dangerous mutations by triggering cell death through apoptosis and/or autophagy or in the execution of a program that leads to a permanent growth arrest known as senescence. These responses are considered crucial tumor suppressive mechanisms and their study appears to be essential to develop therapeutical procedures based on the enhancement of the different responses. This review summarizes fundamental knowledge on the underlying mechanisms able to limit excessive or aberrant cellular proliferation and on the prognostic value of both apoptosis and senescence detection. In addition, interesting evidence showing that different drugs induce senescence or cell death depending on the genetic features of the tumor cells as well as on the integrity of the relative pathways is reported.
Current Medicinal Chemistry 02/2009; 16(3):287-300. DOI:10.2174/092986709787002691 · 3.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The viral protein Nef is a virulence factor that plays multiple roles during the early and late phases of human immunodeficiency virus (HIV) replication. Nef regulates the cell surface expression of critical proteins (including down-regulation of CD4 and major histocompatibility complex class I), T-cell receptor signaling, and apoptosis, inducing proapoptotic effects in uninfected bystander cells and antiapoptotic effects in infected cells. It has been proposed that Nef intersects the CD40 ligand signaling pathway in macrophages, leading to modification in the pattern of secreted factors that appear able to recruit and activate T lymphocytes, rendering them susceptible to HIV infection. There is also increasing evidence that in vitro cell treatment with Nef induces signaling effects. Exogenous Nef treatment is able to induce apoptosis in uninfected T cells, maturation in dendritic cells, and suppression of CD40-dependent immunoglobulin class switching in B cells. Previously, we reported that Nef treatment of primary human monocyte-derived macrophages (MDMs) induces a cycloheximide-independent activation of NF-kappaB and the synthesis and secretion of a set of chemokines/cytokines that activate STAT1 and STAT3. Here, we show that Nef treatment is capable of hijacking cellular signaling pathways, inducing a very rapid regulatory response in MDMs that is characterized by the rapid and transient phosphorylation of the alpha and beta subunits of the IkappaB kinase complex and of JNK, ERK1/2, and p38 mitogen-activated protein kinase family members. In addition, we have observed the activation of interferon regulatory factor 3, leading to the synthesis of beta interferon mRNA and protein, which in turn induces STAT2 phosphorylation. All of these effects require Nef myristoylation.
Journal of Virology 04/2007; 81(6):2777-91. DOI:10.1128/JVI.01640-06 · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Interferon (IFN) was the first cytokine produced by recombinant DNA technology used in wide-spread clinical treatment of infectious diseases as well as malignancies. The IFN clinical potential was clearly realized from the outset. However, IFN represents one of the most controversial drugs of our time, as remarkable cycles of promise and disappointment have affected its development and use. Considerable evidence regarding anti-tumor activities of IFNs has been reported. In this paper we focus on molecular bases of the IFN system that may relate to its antitumor activities. Many of the numerous genes transcriptionally activated by IFNs have been shown to encode proteins that activate immune recognition of tumor cells, directly or indirectly exert tumor suppressor activity and/or control tumor cell cycle and programmed cell death. In addition, a physiological relevant function for endogenous type I IFN in cancer immunoediting process and a new way to IFN clinical use based on gene therapy or vaccine-like approaches have recently been suggested. The identification of selected tissue-specific and/or tumor-specific target pathways as well as of different type I IFN tumor escape and resistance mechanisms may provide novel approaches in the search for new IFN-based therapeutic strategies to circumvent cancer disease or improve clinical outcome. Promising IFN treatment has been recently defined by using novel pharmaceutical preparations with a more favourable pharmacokinetic response, also in combination with other bioreagents or other modalities of therapy. Translational research, linking both basic and clinical research, will lead to a new rationale for the use of IFN in cancer therapy.
Current Medicinal Chemistry 02/2007; 14(6):667-79. DOI:10.2174/092986707780059616 · 3.85 Impact Factor