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The CC chemokine ligand 2 (CCL2)/CC chemokine receptor 2 axis plays key roles in the pathogenesis of human immunodeficiency virus type 1 (HIV-1) infection. We previously reported that exposure of monocyte-derived macrophages (MDMs) to CCL2 neutralizing antibody (αCCL2 Ab) restricted HIV-1 replication at post-entry steps of the viral life cycle. This effect was associated with induction of transcripts coding for innate antiviral proteins, amongst which apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3A (APOBEC3A) and radical S-adenosyl methionine domain containing 2 (RSAD2). This study aimed at identifying the signaling pathways involved in induction of these factors by CCL2 blocking in MDMs. Through a combination of pharmacologic inhibition, quantitative RT-PCR, western blotting, and confocal laser-scanning microscopy, we demonstrated that CCL2 neutralization activates the canonical NF-kB and JAK/STAT pathways, as assessed by time-dependent phosphorylation of IkB, STAT1, and STAT3 and p65 nuclear translocation. Furthermore, pharmacologic inhibition of I kappa B kinase and JAKs strongly reduced APOBEC3A and RSAD2 transcript accumulation elicited by αCCL2 Ab treatment. Interestingly, exposure of MDMs to αCCL2 Ab resulted in induction of IL-6 family cytokines, and interfering with glycoprotein 130, the common signal-transducing receptor subunit shared by these cytokines, inhibited APOBEC3A and RSAD2 up-regulation triggered by CCL2 neutralization. These results provide novel insights into the signal transduction pathways underlying the activation of innate responses triggered by CCL2 neutralization in macrophages. Since this response was found to be associated with protective antiviral effects, the new findings may help design innovative therapeutic approaches targeting CCL2 to strengthen host innate immunity.
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London is one of the world's most important coastal cities and is located around the Thames Estuary, United Kingdom (UK). Quantifying changes in sea levels in the Thames Estuary over the 20th century and early part of the 21st century is vital to inform future management of flood risk in London. However, there are currently relatively few long, digital records of sea level available in the Thames. Here we present a new extensive sea level dataset that we have digitised from historical hand-written tabulated ledgers of high and low water, from the Port of London Authority (PLA). We captured 463 years of data, from across 15 tide gauge sites, for the period 1911 to 1995. When these historical datasets are combined with digital records available from the PLA since 1995, the sea level time-series span the 111-year period from 1911 to 2021. This new dataset will be of great importance for ongoing monitoring of mean sea-level rise, and changes in tidal range and extreme sea levels in the Thames Estuary.
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Macrophages are key targets of human immunodeficiency virus type 1 (HIV-1) infection and main producers of the proinflammatory chemokine CC chemokine ligand 2 (CCL2), whose expression is induced by HIV-1 both in vitro and in vivo. We previously found that CCL2 neutralization in monocyte-derived macrophages (MDMs) strongly inhibited HIV-1 replication affecting post-entry steps of the viral life cycle. Here, we used RNA-sequencing to deeply characterize the cellular factors and pathways modulated by CCL2 blocking in MDMs and involved in HIV-1 replication restriction. We report that exposure to CCL2 neutralizing antibody profoundly affected the MDM transcriptome. Functional annotation clustering of up-regulated genes identified two clusters enriched for antiviral defense and immune response pathways, comprising several interferon-stimulated, and restriction factor coding genes. Transcripts in the clusters were enriched for RELA and NFKB1 targets, suggesting the activation of the canonical nuclear factor κB pathway as part of a regulatory network involving miR-155 up-regulation. Furthermore, while HIV-1 infection caused small changes to the MDM transcriptome, with no evidence of host defense gene expression and type I interferon signature, CCL2 blocking enabled the activation of a strong host innate response in infected macrophage cultures, and potently inhibited viral genes expression. Notably, an inverse correlation was found between levels of viral transcripts and of the restriction factors APOBEC3A (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 A), ISG15, and MX1. These findings highlight an association between activation of innate immune pathways and HIV-1 restriction upon CCL2 blocking and identify this chemokine as an endogenous factor contributing to the defective macrophage response to HIV-1. Therapeutic targeting of CCL2 may thus strengthen host innate immunity and restrict HIV-1 replication.
Asunto(s)
Anticuerpos Neutralizantes/farmacología , Quimiocina CCL2/farmacología , Perfilación de la Expresión Génica , Regulación Viral de la Expresión Génica/efectos de los fármacos , VIH-1/genética , Inmunidad Innata , Macrófagos/metabolismo , Anticuerpos Neutralizantes/inmunología , Especificidad de Anticuerpos , Células Cultivadas , Quimiocina CCL2/antagonistas & inhibidores , Quimiocina CCL2/inmunología , Citidina Desaminasa/fisiología , Conjuntos de Datos como Asunto , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/virología , MicroARNs/biosíntesis , MicroARNs/genética , Anotación de Secuencia Molecular , FN-kappa B/metabolismo , Proteínas/fisiología , ARN Viral/biosíntesis , ARN Viral/genética , RNA-Seq , Reacción en Cadena en Tiempo Real de la Polimerasa , Latencia del Virus , Replicación ViralRESUMEN
SAMHD1 is a host restriction factor that functions to restrict both retroviruses and DNA viruses, based on its nuclear deoxynucleotide triphosphate (dNTP) hydrolase activity that limits availability of intracellular dNTP pools. In the present study, we demonstrate that SAMHD1 expression was increased following human cytomegalovirus (HCMV) infection, with only a modest effect on infectious virus production. SAMHD1 was rapidly phosphorylated at residue T592 after infection by cellular cyclin-dependent kinases, especially Cdk2, and by the viral kinase pUL97, resulting in a significant fraction of phosho-SAMHD1 being relocalized to the cytoplasm of infected fibroblasts, in association with viral particles and dense bodies. Thus, our findings indicate that HCMV-dependent SAMHD1 cytoplasmic delocalization and inactivation may represent a potential novel mechanism of HCMV evasion from host antiviral restriction activities.
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Infecciones por Citomegalovirus/virología , Citomegalovirus/patogenicidad , Infecciones por Herpesviridae/metabolismo , Proteína 1 que Contiene Dominios SAM y HD/genética , Antivirales/farmacología , Quinasas Ciclina-Dependientes/metabolismo , Citomegalovirus/genética , Citoplasma/metabolismo , Citoplasma/virología , Humanos , Proteínas de Unión al GTP Monoméricas/metabolismo , Fosforilación , Replicación Viral/efectos de los fármacosAsunto(s)
Infecciones por Coronavirus , Interferón Tipo I , Pandemias , Neumonía Viral , Vitamina D , Betacoronavirus , COVID-19 , Humanos , SARS-CoV-2RESUMEN
The chemokine system mediates acute inflammation by driving leukocyte migration to damaged or infected tissues. However, elevated expression of chemokines and their receptors can contribute to chronic inflammation and malignancy. Thus, great effort has been taken to target these molecules. The first hint of the druggability of the chemokine system was derived from the role of chemokine receptors in HIV infection. CCR5 and CXCR4 function as essential co-receptors for HIV entry, with the former accounting for most new HIV infections worldwide. Not by chance, an anti-CCR5 compound, maraviroc, was the first FDA-approved chemokine receptor-targeting drug. CCR5, by directing leukocytes to sites of inflammation and regulating their activation, also represents an important player in the inflammatory response. This function is shared with CCR2 and its selective ligand CCL2, which constitute the primary chemokine axis driving the recruitment of monocytes/macrophages to inflammatory sites. Both receptors are indeed involved in the pathogenesis of several immune-mediated diseases, and dual CCR5/CCR2 targeting is emerging as a more efficacious strategy than targeting either receptor alone in the treatment of complex human disorders. In this review, we focus on the distinctive and complementary contributions of CCR5 and CCR2/CCL2 in HIV infection, multiple sclerosis, liver fibrosis and associated hepatocellular carcinoma. The emerging therapeutic approaches based on the inhibition of these chemokine axes are highlighted.
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Quimiocina CCL2/genética , Inflamación/genética , Receptores CCR2/genética , Receptores CCR5/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/terapia , Marcación de Gen , VIH/genética , VIH/patogenicidad , Infecciones por VIH/genética , Infecciones por VIH/terapia , Infecciones por VIH/virología , Humanos , Inflamación/terapia , Cirrosis Hepática/genética , Cirrosis Hepática/terapia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/terapia , Esclerosis Múltiple/genética , Esclerosis Múltiple/terapiaRESUMEN
Apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3) family members are cytidine deaminases that play crucial roles in innate responses to retrovirus infection. The mechanisms by which some of these enzymes restrict human immunodeficiency virus type 1 (HIV-1) replication have been extensively investigated in vitro. However, little is known regarding how APOBEC3 proteins affect the pathogenesis of HIV-1 infection in vivo and how antiretroviral therapy influences their expression. In this work, a longitudinal analysis was performed to evaluate APOBEC3G/3A expression in peripheral blood mononuclear cells of antiretroviral-naive HIV-1-infected individuals treated with cenicriviroc (CVC) or efavirenz (EFV) at baseline and 4, 12, 24, and 48 weeks post-treatment follow-up. While APOBEC3G expression was unaffected by therapy, APOBEC3A levels increased in CVC but not EFV arm at week 48 of treatment. APOBEC3G expression correlated directly with CD4+ cell count and CD4+/CD8+ cell ratio, whereas APOBEC3A levels inversely correlated with plasma soluble CD14. These findings suggest that higher APOBEC3G/3A levels may be associated with protective effects against HIV-1 disease progression and chronic inflammation and warrant further studies.
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Desaminasa APOBEC-3G/genética , Citidina Desaminasa/genética , Regulación de la Expresión Génica , Infecciones por VIH/genética , Infecciones por VIH/virología , VIH-1 , Proteínas/genética , Desaminasa APOBEC-3G/metabolismo , Adulto , Alquinos , Fármacos Anti-VIH/uso terapéutico , Terapia Antirretroviral Altamente Activa , Benzoxazinas/uso terapéutico , Recuento de Linfocito CD4 , Ciclopropanos , Citidina Desaminasa/metabolismo , Progresión de la Enfermedad , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , VIH-1/efectos de los fármacos , Humanos , Imidazoles/uso terapéutico , Masculino , Persona de Mediana Edad , Proteínas/metabolismo , Sulfóxidos , Resultado del Tratamiento , Carga Viral , Adulto JovenRESUMEN
The apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3) family of cytosine deaminases plays crucial roles in innate immunity through the ability of restricting viral replication by deamination and mutation of viral genomes. The antiviral function of these proteins was first discovered when research in the field of HIV infection revealed that one member of the family, namely APOBEC3G, restricts HIV infection in T lymphocytes and that the viral infectivity factor protein drives the proteosomal degradation of this enzyme, thus overriding its antiviral function. Recent advances in cancer genomics, together with biochemical characterization of the APOBEC3 enzymes, have now implicated some family members in somatic mutagenesis during carcinogenesis. While several studies investigated the downstream consequences of APOBEC3 expression and activity, either in the context of viral infection or tumorigenesis, little is known on the upstream mechanisms regulating APOBEC3 expression. Such knowledge would be of huge importance in developing innovative approaches to strengthen antiviral innate immunity on one side and to prevent cancer development on the other. This mini review summarizes research advances on the molecular mechanisms regulating the expression of APOBEC3 family members in selected immune cell populations and cancer cells.
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Desaminasa APOBEC-3G/genética , Regulación Neoplásica de la Expresión Génica , Inmunidad Innata/inmunología , Neoplasias/genética , Neoplasias/inmunología , Desaminasa APOBEC-3G/metabolismo , Animales , Humanos , Neoplasias/patologíaRESUMEN
Persistent low grade immune activation and chronic inflammation are nowadays considered main driving forces of the progressive immunologic failure in effective antiretroviral therapy treated HIV-1 infected individuals. Among the factors contributing to this phenomenon, microbial translocation has emerged as a key driver of persistent immune activation. Indeed, the rapid depletion of gastrointestinal CD4⺠T lymphocytes occurring during the early phases of infection leads to a deterioration of the gut epithelium followed by the translocation of microbial products into the systemic circulation and the subsequent activation of innate immunity. In this context, monocytes/macrophages are increasingly recognized as an important source of inflammation, linked to HIV-1 disease progression and to non-AIDS complications, such as cardiovascular disease and neurocognitive decline, which are currently main challenges in treated patients. Lipid signaling plays a central role in modulating monocyte/macrophage activation, immune functions and inflammatory responses. Phospholipase-mediated phospholipid hydrolysis leads to the production of lipid mediators or second messengers that affect signal transduction, thus regulating a variety of physiologic and pathophysiologic processes. In this review, we discuss the contribution of phospholipases to monocyte/macrophage activation in the context of HIV-1 infection, focusing on their involvement in virus-associated chronic inflammation and co-morbidities.
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Infecciones por VIH/inmunología , VIH-1/inmunología , Inflamación/inmunología , Macrófagos/enzimología , Macrófagos/virología , Fosfolipasas/inmunología , Traslocación Bacteriana , Linfocitos T CD4-Positivos/inmunología , Enfermedades Cardiovasculares/complicaciones , Diferenciación Celular , Citocinas/metabolismo , VIH-1/patogenicidad , Humanos , Inmunidad Innata , Monocitos/enzimología , Monocitos/virología , Trastornos Neurocognitivos/complicaciones , Fosfolipasas/metabolismo , Transducción de SeñalRESUMEN
Multiple host factors and their interactions with viral proteins contribute to the complexity of HIV-1 pathogenesis and disease progression. The virus exploits the cell-signaling networks to prepare the ground for viral replication, to affect functions of either infected or uninfected bystander cells, and to evade the immune response. These events are hallmarks of HIV-1 pathogenesis that lead toward AIDS. Phospholipases are essential mediators of intracellular and intercellular signaling. They function as phospholipid-hydrolyzing enzymes, generating many bioactive lipid mediators or second messengers, which control multiple cellular functions, thus regulating a variety of physiologic and pathophysiologic processes. These enzymes also represent important components of the cell-signaling networks exploited by HIV-1 and its proteins to favor viral replication and persistence, as well as immune response dysfunction. Although some individual phospholipases were studied in the context of HIV-1 infection, the mechanisms whereby they regulate diverse infection-associated processes, as well as the interaction among different phospholipases have yet to be fully elucidated. In this review, we discuss the principal aspects of the complex interaction between phospholipases, HIV-1, and the immune system. A thorough understanding of the signaling networks that involve phospholipases in both HIV-1-infected cells and individuals is essential to determine whether therapeutic targeting of these enzymes may represent a novel approach to control viral replication, as well as the associated inflammation and comorbidities.
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Infecciones por VIH/enzimología , VIH-1/inmunología , Sistema Inmunológico/enzimología , Fosfolipasas/metabolismo , Humanos , Modelos Biológicos , Transducción de SeñalRESUMEN
The identification of chemokine receptors as necessary co-receptors for HIV entry into target cells represented a breakthrough in the understanding of the pathogenesis of this viral infection. Since this initial discovery, it was unraveled that chemokines, in addition to their role in blocking viral entry by binding to co-receptors, have other functions in HIV pathogenesis. Indeed, chemokines can either inhibit or enhance HIV replication, and these effects may involve both entry and post-entry events of the viral life cycle. Depending on the balance of their negative versus positive effects on HIV replication and spreading, chemokines contribute to different outcomes of HIV pathogenesis. CCL2 is unique among the chemokines in that mostly enhancing effects on viral replication and pathogenesis have been reported. Either HIV infection itself or exposure to viral products can induce the expression of this chemokine and of its receptor CCR2, and high levels of CCL2/CCR2 are indeed found in HIV-infected subjects. The CCL2/CCR2 axis is tightly linked to the high level of immune activation and inflammation that is the hallmark of HIV infection even in patients undergoing antiretroviral therapy. In addition, more direct effects of CCL2 on HIV replication are becoming apparent. Thus, modulation of CCL2/CCR2-driven effects may have significant impact on HIV disease progression. In this review, we will discuss the complex interaction between CCL2/CCR2 and HIV and the emerging therapeutic approaches based on the inhibition of this axis.
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Antirretrovirales/farmacología , Quimiocina CCL2/inmunología , Infecciones por VIH , VIH-1 , Receptores CCR2/inmunología , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Descubrimiento de Drogas , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , VIH-1/efectos de los fármacos , VIH-1/fisiología , HumanosRESUMEN
Lactoferrin (LF) exhibits a wide range of immunomodulatory activities including modulation of cytokine and chemokine secretion. In this study, we demonstrate that bovine LF (bLF) up-modulates, in a concentration- and time-dependent manner, CCL1 secretion in monocytes (Mo) at the early stage of differentiation toward dendritic cells (DCs), and in fully differentiated immature Mo-derived DCs (MoDCs). In both cell types, up-modulation of CCL1 secretion is an early event following bLF-mediated enhanced accumulation of CCL1 transcripts. Notably, bLF-mediated up-regulation of CCL1 involves the engagement of distinct surface receptors in MoDCs and their Mo precursors. We show that bLF-mediated engagement of CD36 contributes to CCL1 induction in differentiating Mo. Conversely, toll-like receptor (TLR)2 blocking markedly reduces bLF-induced CCL1 production in MoDCs. These findings add further evidence for cell-specific differential responses elicited by bLF through the engagement of distinct TLRs and surface receptors. Furthermore, the different responses observed at early and late stages of Mo differentiation towards DCs may be relevant in mediating bLF effects in specific body districts, where these cell types may be differently represented in physiopathological conditions.
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Quimiocina CCL1/metabolismo , Células Dendríticas/efectos de los fármacos , Lactoferrina/farmacología , Monocitos/efectos de los fármacos , Animales , Bovinos , Células Cultivadas , Quimiocina CCL1/genética , Células Dendríticas/metabolismo , Humanos , Monocitos/citología , Monocitos/metabolismo , ARN Mensajero/metabolismoRESUMEN
BACKGROUND: Macrophages are key targets of HIV-1 infection. We have previously described that the expression of CC chemokine ligand 2 (CCL2) increases during monocyte differentiation to macrophages and it is further up-modulated by HIV-1 exposure. Moreover, CCL2 acts as an autocrine factor that promotes viral replication in infected macrophages. In this study, we dissected the molecular mechanisms by which CCL2 neutralization inhibits HIV-1 replication in monocyte-derived macrophages (MDM), and the potential involvement of the innate restriction factors protein sterile alpha motif (SAM) histidine/aspartic acid (HD) domain containing 1 (SAMHD1) and apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) family members. RESULTS: CCL2 neutralization potently reduced the number of p24 Gag+ cells during the course of either productive or single cycle infection with HIV-1. In contrast, CCL2 blocking did not modify entry of HIV-1 based Virus Like Particles, thus demonstrating that the restriction involves post-entry steps of the viral life cycle. Notably, the accumulation of viral DNA, both total, integrated and 2-LTR circles, was strongly impaired by neutralization of CCL2. Looking for correlates of HIV-1 DNA accumulation inhibition, we found that the antiviral effect of CCL2 neutralization was independent of the modulation of SAMHD1 expression or function. Conversely, a strong and selective induction of APOBEC3A expression, to levels comparable to those of freshly isolated monocytes, was associated with the inhibition of HIV-1 replication mediated by CCL2 blocking. Interestingly, the CCL2 neutralization mediated increase of APOBEC3A expression was type I IFN independent. Moreover, the transcriptome analysis of the effect of CCL2 blocking on global gene expression revealed that the neutralization of this chemokine resulted in the upmodulation of additional genes involved in the defence response to viruses. CONCLUSIONS: Neutralization of endogenous CCL2 determines a profound restriction of HIV-1 replication in primary MDM affecting post-entry steps of the viral life cycle with a mechanism independent of SAMHD1. In addition, CCL2 blocking is associated with induction of APOBEC3A expression, thus unravelling a novel mechanism which might contribute to regulate the expression of innate intracellular viral antagonists in vivo. Thus, our study may potentially lead to the development of new therapeutic strategies for enhancing innate cellular defences against HIV-1 and protecting macrophages from infection.
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Quimiocina CCL2/antagonistas & inhibidores , ADN Viral/metabolismo , VIH-1/fisiología , Macrófagos/virología , Replicación Viral , Células Cultivadas , Quimiocina CCL2/inmunología , Citidina Desaminasa/antagonistas & inhibidores , Citidina Desaminasa/genética , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Proteínas de Unión al GTP Monoméricas/genética , Proteínas/antagonistas & inhibidores , Proteínas/genética , Proteína 1 que Contiene Dominios SAM y HD , Internalización del VirusRESUMEN
HIV-1 envelope glycoprotein gp120 induces, independently of infection, the release of CCL2 from macrophages. In turn, this chemokine acts as an autocrine factor enhancing viral replication. In this study, we show for the first time that phosphoinositide-specific phospholipase C (PI-PLC) is required for the production of CCL2 triggered by gp120 in macrophages. Using a combination of confocal laser-scanner microscopy, pharmacologic inhibition, western blotting and fluorescence-activated cell sorter analysis, we demonstrate that gp120 interaction with CCR5 leads to nuclear localization of the PI-PLC ß1 isozyme mediated by mitogen-activated protein kinase ERK-1/2. Notably, phosphatidylcholine-specific phospholipase C (PC-PLC), previously reported to be required for NF-kB-mediated CCL2 production induced by gp120 in macrophages, drives both ERK1/2 activation and PI-PLC ß1 nuclear localization induced by gp120. PI-PLC ß1 activation through CCR5 is also triggered by the natural chemokine ligand CCL4, but independently of ERK1/2. Finally, PI-PLC inhibition neither blocks gp120-mediated NF-kB activation nor overall accumulation of CCL2 mRNA, whereas it decreases CCL2 transcript level in the cytoplasm. These results identify nuclear PI-PLC ß1 as a new intermediate in the gp120-triggered PC-PLC-driven signal transduction pathway leading to CCL2 secretion in macrophages. The finding that a concerted gp120-mediated signaling involving both PC- and PI-specific PLCs is required for the expression of CCL2 in macrophages suggests that this signal transduction pathway may also be relevant for the modulation of viral replication in these cells. Thus, this study may contribute to identify novel targets for therapeutic intervention in HIV-1 infection.
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Quimiocina CCL2/metabolismo , Citoplasma/metabolismo , Proteína gp120 de Envoltorio del VIH/metabolismo , Macrófagos/virología , Fosfolipasa C beta/metabolismo , Butadienos/farmacología , Núcleo Celular/metabolismo , Separación Celular , Citometría de Flujo , VIH-1 , Humanos , Sistema de Señalización de MAP Quinasas , Macrófagos/metabolismo , Microscopía Confocal , Monocitos/citología , Nitrilos/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Éteres Fosfolípidos/farmacología , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
Guanidinium groups were introduced through a spacer at the lower rim of calix[4]arenes in the cone conformation to give new potential nonviral vectors for gene delivery. Several structural modifications were explored, such as the presence or absence of a macrocyclic scaffold, lipophilicity of the backbone, length of the spacer, and nature of the charged groups, in order to better understand the factors which affect the DNA condensation ability and transfection efficiency of these derivatives. The most interesting compound was a calix[4]arene unsubstituted at the upper rim and having four guanidinium groups linked at the lower rim through a three carbon atom spacer. This compound, when formulated with DOPE, showed low toxicity and transfection efficiency higher than the commercially available lipofectamine LTX in the treatment of human Rhabdomiosarcoma and Vero cells. Most of the investigated compounds showed a tendency to self-aggregate in pure water or in the presence of salts, as evidenced by NMR and AFM studies, and it was found that the ability to condense DNA plasmids in nanometric globules is a necessary but not sufficient condition for transfection. The superiority of macrocyclic vectors over linear Gemini-type analogues and of guanidinium compared to other ammonium head groups in determining the biological activity of the vectors was also ascertained.
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Calixarenos/química , ADN/administración & dosificación , Guanidina/análogos & derivados , Fenoles/química , Plásmidos/administración & dosificación , Transfección , Animales , Línea Celular Tumoral , Chlorocebus aethiops , Humanos , Solubilidad , Células VeroRESUMEN
Chemokines production in monocytes/macrophages is crucial in modulating immune responses generated through Toll-like receptor (TLR)-mediated recognition of microbes. During microbial onset, multiple pathogen-associated structures can be present at infection sites, and simultaneously trigger different TLRs. We report here that TLR3, TLR4 and TLR8 engagement induce CCL1, CCL2 and CCL4 production in freshly isolated monocytes. While differentiating cells maintain the capacity to secrete CCL2 and CCL4, CCL1 is no longer induced at later differentiation stages. Although different pairs of TLR agonists have been described to synergistically induce cytokine production in different cell types, agonist combinations cooperate in reducing CCL1 and CCL2, but not CCL4 secretion in freshly isolated monocytes, and fail to rescue CCL1 production at later differentiation stages. The effects of single, but not combined, TLR engagement on chemokine expression mostly occur at transcriptional level, and are IL-10 independent. Conversely, inhibition of CCL1 secretion upon combined TLR engagement is partially rescued by blocking IL-23. A different chemotactic activity of monocyte-conditioned medium on blood mononuclear cells as well as antigen uptake capacity of TLR agonist activated monocytes parallel the regulated production of chemokines. Overall, these findings indicate that simultaneous engagement of TLRs may lead to different patterns of chemokine expression depending on cellular differentiation state, chemokine, and TLR agonist combination. These different responses may be relevant for the distinct but complementary functions of monocytes and macrophages in the immune response, and may have important implications for the therapeutic manipulation of the innate immune system.
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Antígenos/metabolismo , Quimiocinas CC/biosíntesis , Monocitos/inmunología , Receptor Cross-Talk/efectos de los fármacos , Receptores Toll-Like/metabolismo , Movimiento Celular/inmunología , Células Cultivadas , Citocinas/biosíntesis , Regulación hacia Abajo/genética , Humanos , Imidazoles/farmacología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Fagocitosis/inmunología , Receptores Toll-Like/agonistas , Transcripción GenéticaRESUMEN
OBJECTIVE: In patients with hepatitis C virus (HCV)/HIV co-infection, a faster progression of liver fibrosis to cirrhosis has been reported. In this study, an investigation was carried out to determine whether gp120, an HIV envelope protein, modulates the biology of human hepatic stellate cells (HSCs), key cell types in the pathogenesis of fibrosis. METHODS: Myofibroblastic HSCs were isolated from normal human liver tissue. Gene expression was measured by real-time PCR. Cell migration was assessed in Boyden chambers. Intracellular signalling pathways were evaluated using phosphorylation-specific antibodies or by transfection of a reporter plasmid. RESULTS: Transcripts for the chemokine receptors CCR5 and CXCR4, which bind gp120, were detectable in human HSCs. Upon exposure to M-tropic recombinant gp120, which binds CCR5, a significant increase in HSC chemotaxis was observed (1.6+/-0.3-fold, p=0.03). The effects of gp120 were prevented by protein inactivation. gp120 also resulted in a significant increase in secretion (1.5+/-0.3-fold, p=0.03) and gene expression (1.47+/-0.13-fold, p=0.02) of the proinflammatory chemokine monocyte chemoattractant protein-1, and in increased gene expression of tissue inhibitor of metalloprotease-1 and interleukin-6 (2.03+/-0.57-fold, p=0.02). gp120-induced migration required Akt activation. gp120 also induced activation of nuclear factor-kappaB (NF-kappaB) and p38(MAPK). Preincubation of HSCs with TAK779, a CCR5 receptor antagonist, prevented gp120-mediated chemotaxis and monocyte chemoattractant protein-1 secretion. Expression of CCR5 was detectable in areas of inflammation and fibrogenesis in liver biopsies of patients with HCV/HIV co-infection. CONCLUSIONS: This study shows that HIV gp120 modulates different aspects of HSC biology, including directional cell movement and expression of proinflammatory cytokines. These results identify a direct pathway possibly linking HIV infection with liver fibrogenesis via envelope proteins.
Asunto(s)
Proteína gp120 de Envoltorio del VIH/farmacología , Infecciones por VIH/complicaciones , Células Estrelladas Hepáticas/efectos de los fármacos , Cirrosis Hepática/virología , Células Cultivadas , Quimiocina CCL2/biosíntesis , Quimiotaxis/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Infecciones por VIH/metabolismo , Células Estrelladas Hepáticas/metabolismo , Humanos , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/genética , Receptores CCR5/biosíntesis , Receptores CCR5/genética , Receptores CXCR4/biosíntesis , Receptores CXCR4/genética , Proteínas Recombinantes/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
CCL2 (MCP-1) has been shown to enhance HIV-1 replication. The expression of this chemokine by macrophages is up-modulated as a consequence of viral infection or gp120 exposure. In this study, we show for the first time that the phosphatidylcholine-specific phospholipase C (PC-PLC) is required for the production of CCL2 triggered by gp120 in human monocyte-derived macrophages (MDMs). Using a combination of pharmacologic inhibition, confocal laser-scanner microscopy, and enzymatic activity assay, we demonstrate that R5 gp120 interaction with CCR5 activates PC-PLC, as assessed by a time-dependent modification of its subcellular distribution and a concentration-dependent increase of its enzymatic activity. Furthermore, PC-PLC is required for NF-kB-mediated CCL2 production triggered by R5 gp120. Notably, PC-PLC activation through CCR5 is specifically induced by gp120, since triggering CCR5 through its natural ligand CCL4 (MIP-1beta) does not affect PC-PLC cellular distribution and enzymatic activity, as well as CCL2 secretion, thus suggesting that different signaling pathways can be activated through CCR5 interaction with HIV-1 or chemokine ligands. The identification of PC-PLC as a critical mediator of well-defined gp120-mediated effects in MDMs unravels a novel mechanism involved in bystander activation and may contribute to define potential therapeutic targets to block Env-triggered pathologic responses.
Asunto(s)
Quimiocina CCL2/metabolismo , Proteína gp120 de Envoltorio del VIH/metabolismo , Infecciones por VIH/metabolismo , VIH-1/metabolismo , Macrófagos/metabolismo , FN-kappa B/metabolismo , Receptores CCR5/metabolismo , Fosfolipasas de Tipo C/metabolismo , Efecto Espectador/genética , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL4 , Activación Enzimática/genética , Proteína gp120 de Envoltorio del VIH/genética , Infecciones por VIH/genética , VIH-1/genética , Humanos , Macrófagos/patología , Macrófagos/virología , Microscopía Confocal , FN-kappa B/genética , Receptores CCR5/genética , Transducción de Señal/genética , Factores de Tiempo , Fosfolipasas de Tipo C/genética , Replicación Viral/genéticaRESUMEN
Antigen presenting cell (APC) function is central to the development of an effective anti-viral immune response. Among APC, monocytes, macrophages and dendritic cells (DC) form the principal non-T cell compartment involved in in vivo HIV infection, and these cells play important and well-established roles in multiple aspects of viral pathogenesis. HIV infection may result in APC defects, which could ultimately contribute to the loss of CD4+ T cell responses observed early in HIV infection, when the CD4+ T cell number is still within the normal range. Extensive in vitro studies have demonstrated that the envelope glycoproteins of HIV-1 exert profound influences on various cell populations of the immune system, including hematopoietic progenitors, T and B lymphocytes, monocytes/ macrophages and DC, as well as on neuronal cells. The demonstration of the presence of envelope proteins both free in the circulation and bound to the surface of CD4+ cells suggests that gp120 interactions with non-infected cells can influence cellular functions in vivo, thus contributing to the immunopathogenesis of AIDS. This paper provides an overview of the present knowledge on gp120 binding, signal transduction triggering and interference with macrophage and DC functions and it highlights the importance of this interaction in the pathogenesis of AIDS.