RESUMEN
Bacterial zwitterionic capsular polysaccharides (ZPS), such as polysaccharide A (PSA) of the intestinal commensal Bacteroides fragilis, have been shown to modulate T cells, including inducing anti-inflammatory IL-10-secreting T regulatory cells (Tregs). We previously used a genomic screen to identify diverse host-associated bacteria with the predicted genetic capacity to produce ZPSs related to PSA of B. fragilis and hypothesized that genetic disruption (KO) of a key functional gene within these operons would reduce the anti-inflammatory activity of these bacteria. We found that ZPS-KO bacteria in two common gut commensals, Bacteroides uniformis and Bacteroides cellulosilyticus, had a reduced ability to induce Tregs and IL-10 in stimulations of human peripheral blood mononuclear cells (PBMCs). Additionally, we found that macrophage stimulated with either wildtype B. fragilis or B. uniformis produced significantly more IL-10 than KOs, indicating a potentially novel function of ZPS of shifting the cytokine response in macrophages to a more anti-inflammatory state. These findings support the hypothesis that these related ZPS may represent a shared strategy to modulate host immune responses.
Asunto(s)
Interleucina-10 , Leucocitos Mononucleares , Humanos , Interleucina-10/genética , Polisacáridos Bacterianos , Bacteroides fragilis/genética , Antiinflamatorios , BacteriasRESUMEN
Recent studies suggest that HIV infection is an independent risk factor for the development of chronic obstructive pulmonary disease (COPD). We hypothesized that HIV infection and cigarette smoking synergize to alter the function of alveolar macrophages (AMs). To test this hypothesis, global transcriptome analysis was performed on purified AMs from 20 individuals split evenly between HIV-uninfected nonsmokers and smokers and untreated HIV-infected nonsmokers and smokers. Differential expression analysis identified 143 genes significantly altered by the combination of HIV infection and smoking. Of the differentially expressed genes, chitinase 1 (CHIT1) and cytochrome P450 family 1 subfamily B member 1 (CYP1B1), both previously associated with COPD, were among the most upregulated genes (5- and 26-fold, respectively) in the untreated HIV-infected smoker cohort compared with HIV-uninfected nonsmokers. Expression of CHIT1 and CYP1B1 correlated with the expression of genes involved in extracellular matrix organization, oxidative stress, immune response, and cell death. Using time-of-flight mass cytometry to characterize AMs, a significantly decreased expression of CD163, an M2 marker, was seen in HIV-infected subjects, and CD163 inversely correlated with CYP1B1 expression in AMs. CHIT1 protein levels were significantly upregulated in bronchoalveolar lavage fluid from HIV-infected smokers, and increased CHIT1 levels negatively correlated with lung function measurements. Overall, these findings raise the possibility that elevated CHIT1 and CYP1B1 are early indicators of COPD development in HIV-infected smokers that may serve as biomarkers for determining this risk.
Asunto(s)
Infecciones por VIH/metabolismo , Hexosaminidasas/metabolismo , Macrófagos Alveolares/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Regulación hacia Arriba , Adulto , Biomarcadores/análisis , Biomarcadores/metabolismo , Femenino , Infecciones por VIH/inmunología , Hexosaminidasas/genética , Hexosaminidasas/inmunología , Humanos , Macrófagos Alveolares/inmunología , Masculino , Persona de Mediana Edad , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Fumadores , Regulación hacia Arriba/inmunología , Adulto JovenRESUMEN
Genetic risk for autoimmunity in HLA genes is most often attributed to structural specificity resulting in presentation of self-antigens. Autoimmune vitiligo is strongly associated with the MHC class II region. Here, we fine-map vitiligo MHC class II genetic risk to three SNPs only 47 bp apart, located within a predicted super-enhancer in an intergenic region between HLA-DRB1 and HLA-DQA1, localized by a genome-wide association study of 2,853 Caucasian vitiligo patients. The super-enhancer corresponds to an expression quantitative trait locus for expression of HLA-DR and HLA-DQ RNA; we observed elevated surface expression of HLA-DR (P = 0.008) and HLA-DQ (P = 0.02) on monocytes from healthy subjects homozygous for the high-risk SNP haplotype. Unexpectedly, pathogen-stimulated peripheral blood mononuclear cells from subjects homozygous for the high-risk super-enhancer haplotype exhibited greater increase in production of IFN-γ and IL-1ß than cells from subjects homozygous for the low-risk haplotype. Specifically, production of IFN-γ on stimulation of dectin-1, mannose, and Toll-like receptors with Candida albicans and Staphylococcus epidermidis was 2.5- and 2.9-fold higher in high-risk subjects than in low-risk subjects, respectively (P = 0.007 and P = 0.01). Similarly, production of IL-1ß was fivefold higher in high-risk subjects than in low-risk subjects (P = 0.02). Increased production of immunostimulatory cytokines in subjects carrying the high-risk haplotype may act as an "adjuvant" during the presentation of autoantigens, tying together genetic variation in the MHC with the development of autoimmunity. This study demonstrates that for risk of autoimmune vitiligo, expression level of HLA class II molecules is as or more important than antigen specificity.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Citocinas/biosíntesis , Antígenos HLA-DQ/inmunología , Antígenos HLA-DR/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Vitíligo/inmunología , Elementos de Facilitación Genéticos , Haplotipos , Humanos , Polimorfismo de Nucleótido SimpleRESUMEN
Similar to IL-1α and IL-33, IL-1 family member IL-37b translocates to the nucleus and is associated with suppression of innate and adaptive immunity. Here we demonstrate an extracellular function of the IL-37 precursor and a processed form. Recombinant IL-37 precursor reduced LPS-induced IL-6 by 50% (P < 0.001) in highly inflammatory human blood-derived M1 differentiated macrophages derived from selective subjects but not M2 macrophages. In contrast, a neutralizing monoclonal anti-IL-37 increased LPS-induced IL-6, TNFα and IL-1ß (P < 0.01). The suppression by IL-37 was consistently observed at low picomolar but not nanomolar concentrations. Whereas LPS induced a 12-fold increase in TNFα mRNA, IL-37 pretreatment decreased the expression to only 3-fold over background (P < 0.01). Mechanistically, LPS-induced p38 and pERK were reduced by IL-37. Recombinant IL-37 bound to the immobilized ligand binding α-chain of the IL-18 receptor as well as to the decoy receptor IL-1R8. In M1 macrophages, LPS increased the surface expression of IL-1R8. Compared with human blood monocytes, resting M1 cells express more surface IL-1R8 as well as total IL-1R8; there was a 16-fold increase in IL-1R8 mRNA levels when pretreated with IL-37. IL-37 reduced LPS-induced TNFα and IL-6 by 50-55% in mouse bone marrow-derived dendritic cells, but not in dendritic cells derived from IL-1R8-deficient mice. In mice subjected to systemic LPS-induced inflammation, pretreatment with IL-37 reduced circulating and organ cytokine levels. Thus, in addition to a nuclear function, IL-37 acts as an extracellular cytokine by binding to the IL-18 receptor but using the IL-1R8 for its anti-inflammatory properties.
Asunto(s)
Inmunidad Innata , Inflamación/inmunología , Interleucina-1/química , Interleucina-1/metabolismo , Receptores de Interleucina-1/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Endotoxemia/metabolismo , Endotoxemia/patología , Activación Enzimática/efectos de los fármacos , Espacio Extracelular/química , Citometría de Flujo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas Inmovilizadas/metabolismo , Inflamación/patología , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Macrófagos/metabolismo , Ratones , Pruebas de Neutralización , Estructura Terciaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Interleucina-1/química , Proteínas Recombinantes/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Varicella-zoster virus (VZV) vasculopathy produces stroke, giant cell arteritis, and granulomatous aortitis, and it develops after virus reactivates from ganglia and spreads transaxonally to arterial adventitia, resulting in persistent inflammation and pathological vascular remodeling. The mechanism(s) by which inflammatory cells persist in VZV-infected arteries is unknown; however, virus-induced dysregulation of programmed death ligand 1 (PD-L1) may play a role. Specifically, PD-L1 can be expressed on virtually all nucleated cells and suppresses the immune system by interacting with the programmed cell death protein receptor 1, found exclusively on immune cells; thus, downregulation of PD-L1 may promote inflammation, as seen in some autoimmune diseases. Both flow cytometry and immunofluorescence analyses to test whether VZV infection of adventitial cells downregulates PD-L1 showed decreased PD-L1 expression in VZV-infected compared to mock-infected human brain vascular adventitial fibroblasts (HBVAFs), perineural cells (HPNCs), and fetal lung fibroblasts (HFLs) at 72 h postinfection. Quantitative RT-PCR analyses showed no change in PD-L1 transcript levels between mock- and VZV-infected cells, indicating a posttranscriptional mechanism for VZV-mediated downregulation of PD-L1. Flow cytometry analyses showed decreased major histocompatibility complex class I (MHC-I) expression in VZV-infected cells and adjacent uninfected cells compared to mock-infected cells. These data suggest that reduced PD-L1 expression in VZV-infected adventitial cells contribute to persistent vascular inflammation observed in virus-infected arteries from patients with VZV vasculopathy, while downregulation of MHC-I prevents viral clearance. IMPORTANCE: Here, we provide the first demonstration that VZV downregulates PD-L1 expression in infected HBVAFs, HPNCs, and HFLs, which, together with the noted VZV-mediated downregulation of MHC-I, might foster persistent inflammation in vessels, leading to pathological vascular remodeling during VZV vasculopathy and persistent inflammation in infected lungs to promote subsequent infection of T cells and hematogenous virus spread. Identification of a potential mechanism by which persistent inflammation in the absence of effective viral clearance occurs in VZV vasculopathy and VZV infection of the lung is a step toward targeted therapy of VZV-induced disease.
Asunto(s)
Antígeno B7-H1/metabolismo , Herpesvirus Humano 3/patogenicidad , Antígenos de Histocompatibilidad Clase I/metabolismo , Adventicia/irrigación sanguínea , Adventicia/inmunología , Adventicia/virología , Antígeno B7-H1/genética , Encéfalo/irrigación sanguínea , Encéfalo/inmunología , Encéfalo/virología , Células Cultivadas , Regulación hacia Abajo , Fibroblastos/inmunología , Fibroblastos/virología , Infecciones por Herpesviridae/etiología , Infecciones por Herpesviridae/inmunología , Interacciones Huésped-Patógeno/inmunología , Humanos , Pulmón/inmunología , Pulmón/virología , Neuroglía/inmunología , Neuroglía/virologíaRESUMEN
Cytotoxic CD8+ T Lymphocytes (CTL) efficiently control acute virus infections but can become exhausted when a chronic infection develops. Signaling of the inhibitory receptor PD-1 is an important mechanism for the development of virus-specific CD8+ T cell dysfunction. However, it has recently been shown that during the initial phase of infection virus-specific CD8+ T cells express high levels of PD-1, but are fully competent in producing cytokines and killing virus-infected target cells. To better understand the role of the PD-1 signaling pathway in CD8+ T cell cytotoxicity during acute viral infections we analyzed the expression of the ligand on retrovirus-infected cells targeted by CTLs. We observed increased levels of PD-L1 expression after infection of cells with the murine Friend retrovirus (FV) or with HIV. In FV infected mice, virus-specific CTLs efficiently eliminated infected target cells that expressed low levels of PD-L1 or that were deficient for PD-L1 but the population of PD-L1high cells escaped elimination and formed a reservoir for chronic FV replication. Infected cells with high PD-L1 expression mediated a negative feedback on CD8+ T cells and inhibited their expansion and cytotoxic functions. These findings provide evidence for a novel immune escape mechanism during acute retroviral infection based on PD-L1 expression levels on virus infected target cells.
Asunto(s)
Antígeno B7-H1/inmunología , Linfocitos T CD8-positivos/inmunología , Citotoxicidad Inmunológica/inmunología , Evasión Inmune/inmunología , Infecciones por Retroviridae/inmunología , Animales , Linfocitos T CD8-positivos/virología , Citometría de Flujo , Humanos , Ratones , Ratones Endogámicos C57BL , Receptor de Muerte Celular Programada 1/inmunología , Reacción en Cadena en Tiempo Real de la Polimerasa , Retroviridae/inmunologíaRESUMEN
Excessive or persistent programmed death 1 (PD-1) expression on virus- or tumor-specific T cells during chronic viral infection or malignancy has been associated with impaired immune control. To assess the role of the PD-1 pathway in allogeneic stem cell transplantation (SCT), we examined PD-1 expression and maturation phenotype on T cells from 42 patients early (day 55 to 85) after cord blood (CB), matched unrelated donor, and matched related donor transplantation. Expression of PD-1 on CD4+ T cells was significantly elevated in all transplantation types, with the highest level observed in CB subjects. Elevated PD-1 expression on CD4+ T cells early after transplantation was observed in nonsurvivors (median, 40.2%; range, 15.1 to 86.1) compared with survivors (median, 23.6%; range, 8.4 to 55.2; P = .001), indicating its association with increased risk for mortality, especially with CB transplantations, where PD-1 was increased in nonsurvivors (median, 64.6%; range, 36.5 to 86.1) compared with survivors (median, 34.1%; range, 15.9 to 55.2; P = .01). Furthermore, T cell subset analysis revealed that PD-1 expression was further elevated on CD4+ T central memory in nonsurvivors (median, 49.8%; range, 15.1 to 83.4) compared with survivors (median, 24.8%; range, 8.9 to 71.3; P = .002) and on T effector memory cells in nonsurvivors (median, 69.1%; range, 24.7 to 92.6) compared with survivors (median, 43.7%; range, 13.9 to 96.5; P = .0003). Our findings suggest that elevation of PD-1 expression on CD4+ T cells is associated with mortality in CB and possibly all SCT recipients.
Asunto(s)
Linfocitos T CD4-Positivos/química , Trasplante de Células Madre de Sangre del Cordón Umbilical/métodos , Receptor de Muerte Celular Programada 1/análisis , Adulto , Anciano , Trasplante de Células Madre de Sangre del Cordón Umbilical/mortalidad , Humanos , Persona de Mediana Edad , Mortalidad , Pronóstico , Sobrevivientes , Subgrupos de Linfocitos T/química , Trasplante Homólogo , Adulto JovenRESUMEN
RATIONALE: Lymphocytic alveolitis in HIV-1-infected individuals is associated with multiple pulmonary complications and a poor prognosis. Although lymphocytic alveolitis has been associated with viremia and an increased number of CD8(+) T cells in the lung, its exact cause is unknown. OBJECTIVES: To determine if HIV-1-specific T cells are associated with lymphocytic alveolitis in HIV-1-infected individuals. METHODS: Using blood and bronchoalveolar lavage (BAL) cells from normal control subjects and untreated HIV-1-infected individuals, we examined the frequency and functional capacity of HIV-1-specific T cells. MEASUREMENTS AND MAIN RESULTS: We found that HIV-1-specific T cells were significantly elevated in the BAL compared with blood of HIV-1-infected individuals and strongly correlated with T-cell alveolitis. Expression of Ki67, a marker of in vivo proliferation, was significantly reduced on HIV-1-specific T cells in BAL compared with blood, suggesting a diminished proliferative capacity. In addition, HIV-1-specific CD4(+) and CD8(+) T cells in BAL had higher expression of programmed death 1 (PD-1) and lower cytotoxic T-lymphocyte antigen 4 (CTLA-4) expression than those in the blood. A strong correlation between PD-1, but not CTLA-4, and HIV-1-specific T-cell proliferation was seen, and blockade of the PD-1/PD-L1 pathway augmented HIV-1-specific T-cell proliferation, suggesting that the PD-1 pathway was the main cause of reduced proliferation in the lung. CONCLUSIONS: These findings suggest that alveolitis associated with HIV-1 infection is caused by the recruitment of HIV-1-specific CD4(+) and CD8(+) T cells to the lung. These antigen-specific T cells display an impaired proliferative capacity that is caused by increased expression of PD-1.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Infecciones por VIH/inmunología , VIH-1/inmunología , Enfermedades Pulmonares/virología , Biomarcadores/metabolismo , Líquido del Lavado Bronquioalveolar/inmunología , Líquido del Lavado Bronquioalveolar/virología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Estudios de Casos y Controles , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Antígenos VIH/inmunología , Infecciones por VIH/complicaciones , Infecciones por VIH/virología , Humanos , Pulmón/inmunología , Pulmón/virología , Enfermedades Pulmonares/inmunología , Activación de Linfocitos/inmunología , Receptor de Muerte Celular Programada 1/metabolismoRESUMEN
The programmed death-1 (PD-1) pathway limits the function of virus-specific T cells during chronic infection. We previously showed that blockade of the PD-1 pathway increases HIV-1-associated T cell function in vitro. However, the effect of PD-1 blockade on HIV-1 disease progression in vivo has not been examined. As in humans, HIV-1-infected humanized BALB/c-Rag2(-/-)γc(-/-) (Rag-hu) mice express elevated levels of PD-1 on T cells during chronic infection. To examine the effect of PD-1 blockade on disease progression, Rag-hu mice with chronic HIV-1 infection were treated with a blocking mAb directed against programmed cell death-1 ligand-1, the ligand for PD-1. Programmed cell death-1 ligand-1-treated Rag-hu mice exhibited a progressive decrease in the HIV-1 plasma viral load, with a 7-fold decrease by day 7, a 20-fold decrease by day 14, a 178-fold decrease by day 21, and a 269-fold decrease by day 28 postinitiation of treatment. By day 7, the percentage of CD4(+) T cells was statistically higher in the treated compared with the untreated group, and this trend was sustained throughout the 28-d treatment period. Moreover, there was a strong inverse correlation between plasma viral load and the percentage of both CD4(+) (r = -0.66; p < 0.0001) and CD8(+) (r = -0.64; p < 0.0001) T cells in the treated mice but not the untreated mice. This study provides "proof of concept" that humanized mice can be used to examine the effects of immunotherapeutic interventions on HIV-1 infection. Furthermore, to our knowledge, these data demonstrate for the first time that blockade of the PD-1 pathway reduces HIV-1 viral loads.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Regulación hacia Abajo/inmunología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/fisiología , Carga Viral/inmunología , Animales , Antígeno B7-H1/inmunología , Recuento de Linfocito CD4 , Linfocitos T CD4-Positivos/citología , Infecciones por VIH/inmunología , Infecciones por VIH/patología , VIH-1/crecimiento & desarrollo , VIH-1/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Receptor de Muerte Celular Programada 1/biosíntesis , Regulación hacia Arriba/inmunologíaRESUMEN
One of the most formidable impediments to clinical translation of RNA interference (RNAi) is safe and effective delivery of the siRNAs to the desired target tissue at therapeutic doses. We previously described in vivo cell type-specific delivery of anti-HIV small-interfering RNAs (siRNAs) through covalent conjugation to an anti-gp120 aptamer. In order to improve the utility of aptamers as siRNA delivery vehicles, we chemically synthesized the gp120 aptamer with a 3' 7-carbon linker (7C3), which in turn is attached to a 16-nucleotide 2' OMe/2' Fl GC-rich bridge sequence. This bridge facilitates the noncovalent binding and interchange of various siRNAs with the same aptamer. We show here that this aptamer-bridge-construct complexed with three different Dicer substrate siRNAs (DsiRNAs) results in effective delivery of the cocktail of DsiRNAs in vivo, resulting in knockdown of target mRNAs and potent inhibition of HIV-1 replication. Following cessation of the aptamer-siRNA cocktail treatment, HIV levels rebounded facilitating a follow-up treatment with the aptamer cocktail of DsiRNAs. This follow-up injection resulted in complete suppression of HIV-1 viral loads that extended several weeks beyond the final injection. Collectively, these data demonstrate a facile, targeted approach for combinatorial delivery of antiviral and host DsiRNAs for HIV-1 therapy in vivo.
Asunto(s)
Aptámeros de Nucleótidos/genética , VIH-1/genética , ARN Interferente Pequeño/genética , Animales , Secuencia de Bases , Linfocitos T CD4-Positivos/inmunología , Proteína gp120 de Envoltorio del VIH/genética , Depleción Linfocítica , Ratones , Ratones NoqueadosRESUMEN
We evaluated the in vivo efficacy of structurally flexible, cationic PAMAM dendrimers as a small interfering RNA (siRNA) delivery system in a Rag2(-)/-γc-/- (RAG-hu) humanized mouse model for HIV-1 infection. HIV-infected humanized Rag2-/-γc-/- mice (RAG-hu) were injected intravenously (i.v.) with dendrimer-siRNA nanoparticles consisting of a cocktail of dicer substrate siRNAs (dsiRNAs) targeting both viral and cellular transcripts. We report in this study that the dendrimer-dsiRNA treatment suppressed HIV-1 infection by several orders of magnitude and protected against viral induced CD4(+) T-cell depletion. We also demonstrated that follow-up injections of the dendrimer-cocktailed dsiRNAs following viral rebound resulted in complete inhibition of HIV-1 titers. Biodistribution studies demonstrate that the dendrimer-dsiRNAs preferentially accumulate in peripheral blood mononuclear cells (PBMCs) and liver and do not exhibit any discernable toxicity. These data demonstrate for the first time efficacious combinatorial delivery of anti-host and -viral siRNAs for HIV-1 treatment in vivo. The dendrimer delivery approach therefore represents a promising method for systemic delivery of combinations of siRNAs for treatment of HIV-1 infection.
Asunto(s)
Linfocitos T CD4-Positivos/virología , Infecciones por VIH/prevención & control , Infecciones por VIH/virología , VIH-1/fisiología , Nanopartículas/administración & dosificación , ARN Interferente Pequeño/administración & dosificación , Animales , Linfocitos T CD4-Positivos/inmunología , ARN Helicasas DEAD-box/metabolismo , Proteínas de Unión al ADN/fisiología , Dendrímeros , Modelos Animales de Enfermedad , Citometría de Flujo , Infecciones por VIH/genética , Humanos , Subunidad gamma Común de Receptores de Interleucina/fisiología , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/virología , Hígado/citología , Hígado/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , ARN Interferente Pequeño/genética , ARN Viral/genética , Ribonucleasa III/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/virología , Carga Viral , Viremia/genética , Viremia/prevención & control , Viremia/virología , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
The envelope glycoprotein of human immunodeficiency virus (HIV) consists of an exterior glycoprotein (gp120) and a trans-membrane domain (gp41) and has an important role in viral entry into cells. HIV-1 entry has been validated as a clinically relevant anti-viral strategy for drug discovery. In the present work, several 2'-F substituted RNA aptamers that bind to the HIV-1(BaL) gp120 protein with nanomole affinity were isolated from a RNA library by the SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure. From two of these aptamers we created a series of new dual inhibitory function anti-gp120 aptamer-siRNA chimeras. The aptamers and aptamer-siRNA chimeras specifically bind to and are internalized into cells expressing HIV gp160. The Dicer-substrate siRNA delivered by the aptamers is functionally processed by Dicer, resulting in specific inhibition of HIV-1 replication and infectivity in cultured CEM T-cells and primary blood mononuclear cells (PBMCs). Moreover, we have introduced a 'sticky' sequence onto a chemically synthesized aptamer which facilitates attachment of the Dicer substrate siRNAs for potential multiplexing. Our results provide a set of novel inhibitory agents for blocking HIV replication and further validate the use of aptamers for delivery of Dicer substrate siRNAs.
Asunto(s)
Fármacos Anti-VIH/química , Aptámeros de Nucleótidos/química , Proteína gp120 de Envoltorio del VIH/antagonistas & inhibidores , VIH-1/fisiología , ARN Interferente Pequeño/química , Animales , Fármacos Anti-VIH/metabolismo , Aptámeros de Nucleótidos/metabolismo , Secuencia de Bases , Células Cultivadas , Proteína gp120 de Envoltorio del VIH/genética , Proteínas gp160 de Envoltorio del VIH/metabolismo , VIH-1/genética , VIH-1/metabolismo , Humanos , Interferón Tipo I/biosíntesis , Datos de Secuencia Molecular , ARN Interferente Pequeño/metabolismo , Ribonucleasa III/metabolismo , Técnica SELEX de Producción de Aptámeros , Linfocitos T/inmunologíaRESUMEN
Men who have sex with men (MSM), regardless of HIV infection status, have an intestinal microbiome that is compositionally distinct from men who have sex with women (MSW) and women. We recently showed HIV-negative MSM have elevated levels of intestinal CD4+ T cells expressing CCR5, a critical co-receptor for HIV. Whether elevated expression of CCR5 is driven by the altered gut microbiome composition in MSM has not been explored. Here we used in vitro stimulation of gut Lamina Propria Mononuclear Cells (LPMCs) with whole intact microbial cells isolated from stool to demonstrate that fecal bacterial communities (FBCs) from HIV-positive/negative MSM induced higher frequencies of CCR5+ CD4+ T cells compared to FBCs from HIV-negative MSW and women. To identify potential microbial drivers, we related the frequency of CCR5+ CD4+ T cells to the abundance of individual microbial taxa in rectal biopsy of HIV-positive/negative MSM and controls, and Holdemanella biformis was strongly associated with increased frequency of CCR5+ CD4+ T cells. We used in vitro stimulation of gut LPMCs with the type strain of H. biformis, a second strain of H.biformis and an isolate of the closely related Holdemanella porci , cultured from either a HIV-positive or a HIV-negative MSM stool. H. porci elevated the frequency of both CCR5+ CD4+ T cells and the ratio of TNF-α/IL-10 Genomic comparisons of the 3 Holdemanella isolates revealed unique cell wall and capsular components, which may be responsible for their differences in immunogenicity. These findings describe a novel mechanism potentially linking intestinal dysbiosis in MSM to HIV transmission and mucosal pathogenesis.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Firmicutes/inmunología , Microbioma Gastrointestinal/inmunología , Infecciones por VIH/microbiología , Homosexualidad Masculina , Mucosa Intestinal/inmunología , Receptores CCR5/metabolismo , Citocinas/metabolismo , Disbiosis/inmunología , Disbiosis/microbiología , Heces/microbiología , Femenino , Firmicutes/clasificación , Firmicutes/genética , Firmicutes/aislamiento & purificación , Genoma Bacteriano/genética , Infecciones por VIH/inmunología , Infecciones por VIH/transmisión , Humanos , Leucocitos Mononucleares/metabolismo , Masculino , Minorías Sexuales y de GéneroRESUMEN
Following publication of the original article [1], the authors reported an error in Fig. 2. The original Fig. 2 has been incorrectly replaced with the Supplementary Fig. 2. The correct Fig. 2 is presented here.
RESUMEN
Gaining a complete understanding of transmission risk factors will assist in efforts to reduce new HIV infections, especially within the disproportionally affected population of men who have sex with men (MSM). We recently reported that the fecal microbiota of MSM elevates immune activation in gnotobiotic mice and enhances HIV infection in vitro over that of fecal microbiota from men who have sex with women. We also demonstrated elevation of the gut homing marker CD103 (integrin αE) on CD4+ T cells by MSM-microbiota. Here we provide additional evidence that the gut microbiota is a risk factor for HIV transmission in MSM by showing elevated frequencies of the HIV co-receptor CCR5 on CD4+ T cells in human rectosigmoid colon biopsies. We discuss our interest in specific MSM-associated bacteria and propose the influx of CD103+ and CCR5+ CD4+ T cells into the colon as a potential link between the MSM microbiota and HIV transmission.
Asunto(s)
Microbioma Gastrointestinal , Infecciones por VIH/microbiología , Infecciones por VIH/transmisión , Minorías Sexuales y de Género , Linfocitopenia-T Idiopática CD4-Positiva/inmunología , Adolescente , Adulto , Antígenos CD/inmunología , Biopsia , Colon/inmunología , Colon/microbiología , Femenino , Infecciones por VIH/inmunología , Humanos , Cadenas alfa de Integrinas/inmunología , Masculino , Persona de Mediana Edad , Receptores CCR5/inmunología , Factores de Riesgo , Conducta Sexual , Linfocitopenia-T Idiopática CD4-Positiva/microbiología , Adulto JovenRESUMEN
The positive-sense transcripts of Sindbis virus (SINV) resemble cellular mRNAs in that they possess a 5' cap and a 3' poly(A) tail. It is likely, therefore, that SINV RNAs must successfully overcome the cytoplasmic mRNA decay machinery of the cell in order to establish an efficient, productive infection. In this study, we have taken advantage of a temperature-sensitive polymerase to shut off viral transcription, and we demonstrate that SINV RNAs are subject to decay during a viral infection in both C6/36 (Aedes albopictus) and baby hamster kidney cells. Interestingly, in contrast to most cellular mRNAs, the decay of SINV RNAs was not initiated by poly(A) tail shortening in either cell line except when most of the 3' untranslated region (UTR) was deleted from the virus. This block in deadenylation of viral transcripts was recapitulated in vitro using C6/36 mosquito cell cytoplasmic extracts. Two distinct regions of the 319-base SINV 3' UTR, the repeat sequence elements and a U-rich domain, were shown to be responsible for mediating the repression of deadenylation of viral mRNAs. Through competition studies performed in parallel with UV cross-linking and functional assays, mosquito cell factors-including a 38-kDa protein-were implicated in the repression of deadenylation mediated by the SINV 3' UTR. This same 38-kDa protein was also implicated in mediating the repression of deadenylation by the 3' UTR of another alphavirus, Venezuelan equine encephalitis virus. In summary, these data provide clear evidence that SINV transcripts do indeed interface with the cellular mRNA decay machinery during an infection and that the virus has evolved a way to avoid the major deadenylation-dependent pathway of mRNA decay.
Asunto(s)
Regiones no Traducidas 3'/fisiología , ARN Viral/metabolismo , Virus Sindbis/genética , Regiones no Traducidas 3'/genética , Aedes , Animales , Línea Celular , Chlorocebus aethiops , Cricetinae , Interacciones Huésped-Patógeno , Peso Molecular , Proteínas/química , Proteínas/aislamiento & purificación , Estabilidad del ARN , Eliminación de Secuencia , Virus Sindbis/metabolismoRESUMEN
OBJECTIVE: To test whether varicella zoster virus (VZV) infection of human brain vascular cells and of lung fibroblasts directly increases proinflammatory cytokine levels, consistent with VZV as a causative agent in intracerebral VZV vasculopathy and giant-cell arteritis (GCA). METHODS: Conditioned supernatant from mock- and VZV-infected human brain vascular adventitial fibroblasts (HBVAFs), human perineurial cells (HPNCs), human brain vascular smooth muscle cells (HBVSMCs), and human fetal lung fibroblasts (HFLs) were collected at 72 hours postinfection and analyzed for levels of 30 proinflammatory cytokines using the Meso Scale Discovery Multiplex ELISA platform. RESULTS: Compared with mock infection, VZV infection led to significantly increased levels of the following: interleukin-8 (IL-8) in all cell lines examined; IL-6 in HBVAFs, HPNCs, and HFLs, with no change in HBVSMCs; and vascular endothelial growth factor A in HBVAFs, HBVSMCs, and HFLs, with a significant decrease in HPNCs. Other cytokines, including IL-2, IL-4, IL-15, IL-16, TGF-b, Eotaxin-1, Eotaxin-3, IP-10, MCP-1, and granulocyte macrophage colony-stimulating factor, were also significantly altered upon VZV infection in a cell type-specific manner. CONCLUSIONS: VZV infection of vascular cells can directly produce a proinflammatory environment that may potentially lead to prolonged arterial wall inflammation and vasculitis. The VZV-mediated increase in IL-8 and IL-6 is consistent with that seen in the CSF of patients with intracerebral VZV vasculopathy, and the VZV-mediated increase in IL-6 is consistent with the cytokine's elevated levels in temporal arteries and plasma of patients with GCA.
RESUMEN
Zwitterionic capsular polysaccharides (ZPSs) are bacterial products that modulate T cells, including inducing anti-inflammatory IL-10-secreting T regulatory cells (Tregs). However, only a few diverse bacteria are known to modulate the host immune system via ZPS. We present a genomic screen for bacteria encoding ZPS molecules. We identify diverse host-associated bacteria, including commensals and pathogens with known anti-inflammatory properties, with the capacity to produce ZPSs. Human mononuclear cells stimulated with lysates from putative ZPS-producing bacteria induce significantly greater IL-10 production and higher proportions of Tregs than lysates from non-ZPS-encoding relatives or a commensal strain of Bacteroides cellulosilyticus in which a putative ZPS biosynthetic operon was genetically disrupted. Similarly, wild-type B. cellulosilyticus DSM 14838, but not a close relative lacking a putative ZPS, attenuated experimental colitis in mice. Collectively, this screen identifies bacterial strains that may use ZPSs to interact with the host as well as those with potential probiotic properties.
Asunto(s)
Antiinflamatorios/metabolismo , Bacterias/química , Tolerancia Inmunológica , Interleucina-10/metabolismo , Polisacáridos Bacterianos/metabolismo , Linfocitos T Reguladores/inmunología , Animales , Colitis/patología , Modelos Animales de Enfermedad , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , RatonesRESUMEN
The new generation broadly neutralizing antibody VRC01 against HIV-1 shows great potential as a topically administered microbicide to prevent sexual transmission. We evaluated its efficacy in a RAG-hu humanized mouse model of vaginal HIV-1 transmission. Mice were challenged vaginally with R5 tropic HIV-1 BaL an hour after intravaginal application of the VRC01 (1 mg/ml concentration) gel. A combination of four first generation bNAbs, namely b12, 2F5, 4E10 and 2G12, was used as a positive efficacy control whereas a non-specific dengue MAb 4G2 was used as negative control. Our results showed that seven out of nine VRC01 antibody administered mice and all of the mice receiving the four bNAb antibody combination were protected against HIV-1 challenge. These findings demonstrate the efficacy of the new bNAb VRC01 as a topical microbicide to protect against HIV-1 vaginal transmission and highlight the use of the RAG-hu mouse model for testing HIV prevention strategies.