[Show abstract][Hide abstract] ABSTRACT: The I-kappa-B kinase epsilon (IKK-ε) has an essential role as a regulator of innate immunity, functioning downstream of pattern recognition receptors to modulate NF-κB and interferon (IFN) signaling. In the present study, we investigated IKKε activation following T cell receptor (TCR)/CD28 stimulation of primary CD4(+) T cells and its role in the stimulation of a type I IFN response. IKK-ε was activated following TCR/CD28 stimulation of primary CD4(+) T cells; however, in T cells treated with poly I:C, TCR/CD28 co-stimulation blocked induction of IFN-β transcription. We demonstrated that IKK-ε phosphorylated transcription factor IRF1 at residues aa215/219/221 in primary CD4(+) T cells and blocked its transcriptional activity. At the mechanistic level, IRF-1 phosphorylation impaired the physical interaction between IRF-1 and the NF-κB RelA subunit, and interfered with PCAF-mediated acetylation of NF-κB RelA. These results demonstrate that TCR/CD28 stimulation of primary T cells stimulates IKKε activation, which in turns contributes to suppression of IFNβ production.
Molecular and cellular biology 01/2014; · 6.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The major obstacle towards HIV-1 eradication is the life-long persistence of the virus in reservoirs of latently infected cells. In these cells the proviral DNA is integrated in the host's genome but it does not actively replicate, becoming invisible to the host immune system and unaffected by existing antiviral drugs. Rebound of viremia and recovery of systemic infection that follows interruption of therapy, necessitates life-long treatments with problems of compliance, toxicity, and untenable costs, especially in developing countries where the infection hits worst. Extensive research efforts have led to the proposal and preliminary testing of several anti-latency compounds, however, overall, eradication strategies have had, so far, limited clinical success while posing several risks for patients. This review will briefly summarize the more recent advances in the elucidation of mechanisms that regulates the establishment/maintenance of latency and therapeutic strategies currently under evaluation in order to eradicate HIV persistence.
[Show abstract][Hide abstract] ABSTRACT: Intensive combined antiretroviral therapy successfully suppresses HIV-1 replication and AIDS disease progression making infection manageable, but it is unable to eradicate the virus that persists in long-lived, drug-insensitive and immune system-insensitive reservoirs thus asking for life-long treatments with problems of compliance, resistance, toxicity and cost. These limitations and recent insights into latency mechanisms have fueled a renewed effort in finding a cure for HIV-1 infection. Proposed eradication strategies involve reactivation of the latent reservoir upon induction of viral transcription followed by the elimination of reactivated virus-producing cells by viral cytopathic effect or host immune response. Several molecules identified by mechanism-directed approaches or in large-scale screenings have been proposed as latency reversing agents. Some of them have already entered clinical testing in humans but with mixed or unsatisfactory results.
[Show abstract][Hide abstract] ABSTRACT: The transcription factor interferon regulatory factor-8 (IRF-8) is crucial for myeloid cell development and immune response and also acts as a tumor suppressor gene. Here, we analyzed the role of IRF-8 in the cross talk between melanoma cells and tumor-infiltrating leukocytes. B16-F10 melanoma cells transplanted into IRF-8-deficient (IRF-8(-/-)) mice grow more rapidly, leading to higher numbers of lung metastasis, with respect to control animals. These events correlated with reduced dendritic cell and T cell infiltration, accumulation of myeloid-derived suppressor cells and a chemokine/chemokine receptor expression profile within the tumor microenvironment supporting tumor growth, angiogenesis, and metastasis. Noticeably, primary tumors developing in IRF-8(-/-) mice displayed a clear-cut inhibition of IRF-8 expression in melanoma cells. Injection of the demethylating agent 5-aza-2'-deoxycytidine into melanoma-bearing IRF-8(-/-) animals induced intratumoral IRF-8 expression and resulted in the re-establishment of a chemokine/ chemokine receptor pattern favoring leukocyte infiltration and melanoma growth arrest. Importantly, intrinsic IRF-8 expression was progressively down-modulated during melanoma growth in mice and in human metastatic melanoma cells with respect to primary tumors. Lastly, IRF-8 expression in melanoma cells was directly modulated by soluble factors, among which interleukin-27 (IL-27), released by immune cells from tumor-bearing mice. Collectively, these results underscore a key role of IRF-8 in the cross talk between melanoma and immune cells, thus revealing its critical function within the tumor microenvironment in regulating melanoma progression and invasiveness.
Neoplasia (New York, N.Y.) 12/2012; 14(12):1223-35. · 5.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Antiretroviral therapy (ART) has proved highly effective in suppressing HIV-1 replication and disease progression. Nevertheless, ART has failed to eliminate the virus from infected individuals. The main obstacle to HIV-1 eradication is the persistence of cellular viral reservoirs. Therefore, the "shock-and-kill" strategy was proposed consisting of inducing HIV-1 escape from latency, in the presence of ART. This is followed by the elimination of reactivated, virus-producing cells. Immune modulators, including protein kinase C (PKC) activators, anti-leukemic drugs and histone deacetylase inhibitors (HDACis) have all demonstrated efficacy in the reactivation of latent virus replication. This review will focus on the potential use of these small molecules in the "shock and kill" strategy, the molecular basis for their action and the potential advantages of their immune-modulating activities.
[Show abstract][Hide abstract] ABSTRACT: Thirty years after the first isolation of the etiological agent of AIDS, the virus HIV-1 is still a major threat worldwide with millions of individuals currently infected. Although current combination therapies allow viral replication to be controlled, HIV-1 is not eradicated and persists in drug- and immune system-insensitive reservoirs and a cure is still lacking. Pathogens such as HIV-1 that cause chronic infections are able to adapt to the host in a manner that ensures long term residence and survival, via the evolution of numerous mechanisms that evade various aspects of the innate and adaptive immune response. One such mechanism is targeted to members of the interferon (IFN) regulatory factor (IRF) family of proteins. These transcription factors regulate a variety of biological processes including interferon induction, immune cell activation and downstream pattern recognition receptors (PRRs). HIV-1 renders IRFs harmless and hijacks them to its own advantage in order to facilitate its replication and evasion of immune responses. Type I interferon (IFN), the canonical antiviral innate response, can be induced in both acute and chronic HIV-1 infection in vivo, but in the majority of individuals this initial response is not protective and can contribute to disease progression. Type I IFN expression is largely inhibited in T cells and macrophages in order to successfully establish productive infection, whereas sustained IFN production by plasmacytoid dendritic cells is considered an important source of chronic immune activation, a hallmark to AIDS progression.
[Show abstract][Hide abstract] ABSTRACT: Crucial steps in high-risk human papillomavirus (HR-HPV)-related carcinogenesis are the integration of HR-HPV into the host genome and loss of viral episomes. The mechanisms that promote cervical neoplastic progression are, however, not clearly understood. During HR-HPV infection, the HPV E5 protein is expressed in precancerous stages but not after viral integration. Given that it has been reported that loss of HPV16 episomes and cervical tumor progression are associated with increased expression of antiviral genes that are inducible by type I interferon (IFN), we asked whether E5, expressed in early phases of cervical carcinogenesis, affects IFN-β signaling. We show that the HPV type 16 (HPV16) E5 protein expression per se stimulates IFN-β expression. This stimulation is specifically mediated by the induction of interferon regulatory factor 1 (IRF-1) which, in turn, induces transcriptional activation of IRF-1-targeted interferon-stimulated genes (ISGs) as double-stranded RNA-dependent protein kinase R (PKR) and caspase 8. Our data show a new and unexpected role for HR-HPV E5 protein and indicate that HPV16 E5 may contribute to the mechanisms responsible for cervical carcinogenesis in part via stimulation of IFN-β and an IFN signature, with IRF-1 playing a pivotal role. HPV16 E5 and IRF-1 may thus serve as potential therapeutic targets in HPV-associated premalignant lesions.
Journal of Virology 03/2011; 85(10):5070-80. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Despite extensive studies that unraveled ligands and signal transduction pathways triggered by TLRs, little is known about the regulation of TLR gene expression. TLR3 plays a crucial role in the recognition of viral pathogens and induction of immune responses by myeloid DCs. IFN regulatory factor (IRF)-8, a member of the IRF family, is a transcriptional regulator that plays essential roles in the development and function of myeloid lineage, affecting different subsets of myeloid DCs. In this study, we show that IRF-8 negatively controls TLR3 gene expression by suppressing IRF-1- and/or polyinosinic-polycytidylic acid-stimulated TLR3 expression in primary human monocyte-derived DCs (MDDCs). MDDCs expressed TLR3 increasingly during their differentiation from monocytes to DCs with a peak at day 5, when TLR3 expression was further enhanced upon stimulation with polyinosinic-polycytidylic acid and then was promptly downregulated. We found that both IRF-1 and IRF-8 bind the human TLR3 promoter during MDDC differentiation in vitro and in vivo but with different kinetic and functional effects. We demonstrate that IRF-8-induced repression of TLR3 is specifically mediated by ligand-activated Src homology 2 domain-containing protein tyrosine phosphatase association. Indeed, Src homology 2 domain-containing protein tyrosine phosphatase-dephosphorylated IRF-8 bound to the human TLR3 promoter competing with IRF-1 and quashing its activity by recruitment of histone deacetylase 3. Our findings identify IRF-8 as a key player in the control of intracellular viral dsRNA-induced responses and highlight a new mechanism for negative regulation of TLR3 expression that can be exploited to block excessive TLR activation.
The Journal of Immunology 02/2011; 186(4):1951-62. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Glucocorticoid receptor (GR) agonists increase erythropoiesis in vivo and in vitro. To clarify the effect of the dominant negative GRβ isoform (unable to bind STAT-5) on erythropoiesis, erythroblast (EB) expansion cultures of mononuclear cells from 18 healthy (nondiseased) donors (NDs) and 16 patients with polycythemia vera (PV) were studied. GRβ was expressed in all PV EBs but only in EBs from 1 ND. The A3669G polymorphism, which stabilizes GRβ mRNA, had greater frequency in PV (55%; n = 22; P = .0028) and myelofibrosis (35%; n = 20) patients than in NDs (9%; n = 22) or patients with essential thrombocythemia (6%; n = 15). Dexamethasone stimulation of ND cultures increased the number of immature EBs characterized by low GATA1 and β-globin expression, but PV cultures generated great numbers of immature EBs with low levels of GATA1 and β-globin irrespective of dexamethasone stimulation. In ND EBs, STAT-5 was not phosphorylated after dexamethasone and erythropoietin treatment and did not form transcriptionally active complexes with GRα, whereas in PV EBs, STAT-5 was constitutively phosphorylated, but the formation of GR/STAT-5 complexes was prevented by expression of GRβ. These data indicate that GRβ expression and the presence of A3669G likely contribute to development of erythrocytosis in PV and provide a potential target for identification of novel therapeutic agents.
[Show abstract][Hide abstract] ABSTRACT: Infections with hepatitis C virus (HCV) progress to chronic phase in 80% of patients. To date, the effect produced by HCV on the expression of microRNAs (miRs) involved in the interferon-β (IFN-β) antiviral pathway has not been explored in details. Thus, we compared the expression profile of 24 selected miRs in IFN-β-treated Huh-7 cells and in three different clones of Huh-7 cells carrying a self-replicating HCV RNA which express all viral proteins (HCV replicon system).
The expression profile of 24 selected miRs in IFN-β-treated Huh-7 cells and in HCV replicon 21-5 clone with respect to Huh-7 parental cells was analysed by real-time PCR. To exclude clone specific variations, the level of 16 out of 24 miRs, found to be modulated in 21-5 clone, was evaluated in two other HCV replicon clones, 22-6 and 21-7. Prediction of target genes of 3 miRs, confirmed in all HCV clones, was performed by means of miRGator program. The gene dataset obtained from microarray analysis of HCV clones was farther used to validate target prediction.
The expression profile revealed that 16 out of 24 miRs were modulated in HCV replicon clone 21-5. Analysis in HCV replicon clones 22-6 and 21-7 indicated that 3 out of 16 miRs, (miR-128a, miR-196a and miR-142-3p) were modulated in a concerted fashion in all three HCV clones. Microarray analysis revealed that 37 out of 1981 genes, predicted targets of the 3 miRs, showed an inverse expression relationship with the corresponding miR in HCV clones, as expected for true targets. Classification of the 37 genes by Panther System indicated that the dataset contains genes involved in biological processes that sustain HCV replication and/or in pathways potentially implicated in the control of antiviral response by HCV infection.
The present findings reveal that 3 IFN-β-regulated miRs and 37 genes, which are likely their functional targets, were commonly modulated by HCV in three replicon clones. The future use of miR inhibitors or mimics and/or siRNAs might be useful for the development of diagnostic and therapeutic strategies aimed at the recovering of protective innate responses in HCV infections.
[Show abstract][Hide abstract] ABSTRACT: In T lymphocytes, the internalization of the R2 chain of the IFN-γ receptor (IFN-γR2) prevents the switching-on of pro-apoptotic and anti-proliferative genes induced by the IFN-γ/STAT1 pathway. In fibroblasts, a critical role of controlling the IFN-γR2 internalization is played by the LI(255-256) intracellular motif. Here we show that, in human malignant T cells, the expression of a mutated IFN-γR2 chain in which the LI(255-256) internalization motif is replaced by two alanines (LI(255-256)AA) induces cell surface accumulation of the receptor and reinstates the cell sensitivity to IFN-γ. In comparison with T cells that expressed wild-type IFN-γR2, cells that expressed the mutated receptor displayed, in response to IFN-γ a sustained activation of STAT1. The activation of this signaling pathway leads to higher induction of MHC class I and FasL expression and triggered apoptosis. Malignant ST4 cells transduced with either wild-type or mutated receptor were able to grow in SCID mice, but only the proliferation of T cells expressing the mutated receptor was inhibited by IFN-γ. Finally, lentiviral-mediated transduction of the mutated receptor in T lymphoblasts from healthy donors reinstated their IFN-γ-dependent apoptosis. As a whole, these data indicate that perturbation of IFN-γR2 internalization by mutating the LI(255-256) motif induces a timely coordinated activation of IFN-γ/STAT1 signaling pathways that leads to the apoptosis of T cells.
[Show abstract][Hide abstract] ABSTRACT: Genetic vaccines are safe cost-effective approaches to immunization but DNA immunization is an inefficient process. There is, therefore, a pressing need for adjuvants capable of enhancing the immunogenicity and effectiveness of these vaccines. This is particularly important for diseases for which successful vaccines are still lacking, such as cancer and infectious diseases including HIV-1/AIDS. Here we report an approach to enhance the immunogenicity of DNA vaccines involving the use of transcription factors of the Interferon regulatory factor (IRF) family, specifically IRF-1, IRF-3, and IRF-7 using the tat gene as model antigen. Balb/c mice were immunized by three intramuscular inoculations, using a DNA prime-protein boost protocol, with a DNA encoding tat of HIV-1 and the indicated IRFs and immune responses were compared to those induced by vaccination with tat DNA alone. In vivo administration of plasmid DNA encoding IRF-1, or a mutated version of IRF-1 deleted of the DNA-binding domain, enhanced Tat-specific immune responses and shifted them towards a predominant T helper 1-type immune response with increased IFN-gamma production and cytotoxic T lymphocytes responses. Conversely, the use of IRF-3 or IRF-7 did not affect the tat-induced responses. These findings define IRF-1 and its mutated form as efficacious T helper 1-inducing adjuvants in the context of tat-based vaccination and also providing a new promising candidate for genetic vaccine development.
Journal of Cellular Physiology 09/2010; 224(3):702-9. · 4.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Members of the interferon regulatory factor (IRF) family are transcription factors implicated in the regulation of a variety of biological processes. Originally identified as intracellular mediators of the induction and biological activities of interferons, their central role in host resistance to pathogens has recently been confirmed by the recognition of their involvement in the regulation of gene expression in responses triggered by Toll-like receptors and other pattern recognition receptors (PRRs). Their function in regulating the development as well as the activity of hematopoietic cells puts them at the interface between innate and adaptive immune responses. IRFs also regulate cell growth and apoptosis in several cell types, thereby affecting susceptibility to and the progression of cancer. In this review the role of some members of the family more deeply involved in the differentiation of hematopoietic cells and in immune regulation is addressed, with a specific focus on T cells and dendritic cells.
Journal of interferon & cytokine research: the official journal of the International Society for Interferon and Cytokine Research 11/2009; 29(12):765-80. · 1.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Several innate cellular antiviral factors exist in mammalian cells that prevent the replication of retroviruses. Among them, the tripartite motif protein (TRIM)5alpha has been shown to block human immunodeficiency virus type 1 (HIV-1) infection in several types of Old World monkey cells. Here we report a novel HIV-1 chronically infected monkey B cell line, F6/HIV-1, characterized by very low levels of TRIM5alpha expression that allows HIV-1 to overcome the restriction. Virus produced by F6/HIV-1 cells fails to infect monkey cells but retains the ability to infect human peripheral blood mononuclear cells (PBMCs) and T cell lines, although with a reduced infectivity compared to the input virus. Ultrastructural analyses revealed the presence of budding virions at the F6/HIV-1 cells plasma membrane characterized by a typical conical core shell. To our knowledge F6/HIV-1 is the first monkey cell line chronically infected by HIV-1 and able to release infectious particles thus representing a useful tool to gain further insights into the molecular mechanisms of HIV-1 pathogenesis.
Journal of Cellular Physiology 09/2009; 221(3):760-5. · 4.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to identify whether the rapid membrane-associated pathway of the glucocorticoid receptor (GR) is active in erythroid cells and plays any role in determining the reversible inhibition on erythroid maturation exerted by GR.
First we determined the biological effects (inhibition of apoptosis and induction of beta-globin expression) induced in primary erythroblasts by erythropoietin (EPO) and the GR agonist dexamethasone (DXM), alone and in combination. Next, by biochemical analysis, we determined the association between GR and EPO receptor in proerythroblasts generated in vitro from 10 normal adult donors. These studies also analyzed the levels of signal transducers and activators of transcription-5 (STAT-5) phosphorylation induced when the cells were stimulated with DXM alone or in combination with EPO.
DXM antagonized the beta-globin messenger RNA increases, but not the inhibition of apoptosis induced by EPO in primary cells. DXM also antagonized the ability of EPO to induce STAT-5 phosphorylation in these cells. In fact, EPO and DXM alone, but not in combination, induced phosphorylation and nuclear translocation of STAT-5. The inhibition likely occurred through an interaction between the two receptors because GR became associated with the EPO receptor and STAT-5 in cells stimulated with EPO and DXM.
These data suggest that glucocorticoids inhibit erythroid maturation not only through a transcriptional mechanism, but also through a rapid membrane-associated pathway that interferes with EPO receptor signaling.
[Show abstract][Hide abstract] ABSTRACT: Regulatory T (Treg) cells are critical in inducing and maintaining tolerance. Despite progress in understanding the basis of immune tolerance, mechanisms and molecules involved in the generation of Treg cells remain poorly understood. IFN regulatory factor (IRF)-1 is a pleiotropic transcription factor implicated in the regulation of various immune processes. In this study, we report that IRF-1 negatively regulates CD4(+)CD25(+) Treg cell development and function by specifically repressing Foxp3 expression. IRF-1-deficient (IRF-1(-/-)) mice showed a selective and marked increase of highly activated and differentiated CD4(+)CD25(+)Foxp3(+) Treg cells in thymus and in all peripheral lymphoid organs. Furthermore, IRF-1(-/-) CD4(+)CD25(-) T cells showed extremely high bent to differentiate into CD4(+)CD25(+)Foxp3(+) Treg cells, whereas restoring IRF-1 expression in IRF-1(-/-) CD4(+)CD25(-) T cells impaired their differentiation into CD25(+)Foxp3(+) cells. Functionally, both isolated and TGF-beta-induced CD4(+)CD25(+) Treg cells from IRF-1(-/-) mice exhibited more increased suppressive activity than wild-type Treg cells. Such phenotype and functional characteristics were explained at a mechanistic level by the finding that IRF-1 binds a highly conserved IRF consensus element sequence (IRF-E) in the foxp3 gene promoter in vivo and negatively regulates its transcriptional activity. We conclude that IRF-1 is a key negative regulator of CD4(+)CD25(+) Treg cells through direct repression of Foxp3 expression.
The Journal of Immunology 08/2008; 181(3):1673-82. · 5.52 Impact Factor