RESUMEN
Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor whose expression regulates immune cell differentiation. Single-cell transcriptomic profiling was used to ascertain the heterogeneity of AHR expression in human B cell subpopulations. We identified a unique population of B cells marked by expression of AHR, CD9, and myeloid genes such as CD14 and CXCL8. Results were confirmed directly in human PBMCs and purified B cells at the protein level. TLR9 signaling induced CD14, CD9, and IL-8 protein expression in CD19+ B cells. CD14-expressing CD9+ B cells also highly expressed AHR and atypical B cell markers such as CD11c and TBET. In patients with active lupus disease, CD14+ and CD9+ B cells are dysregulated, with loss of CD9+ B cells strongly predicting disease severity and demonstrating the relevance of CD9+ B cells in systemic lupus erythematosus and autoimmune disease.
RESUMEN
The oral toxicity of recombinant human lactoferrin (rhLF, Helaina rhLF, Effera™) produced in Komagataella phaffii was investigated in adult Sprague Dawley rats by once daily oral gavage for 14 consecutive days. The study used groups of 3-6 rats/sex/dose. The vehicle control group received sodium citrate buffer, and the test groups received daily doses of 200, 1000, and 2000 mg of rhLF in sodium citrate buffer per kg body weight. Bovine LF at 2000 mg/kg body weight per day was used as a comparative control. Clinical observations, body weight, hematology, clinical chemistry, iron parameters, immunophenotyping, and gross examination at necropsy were used as criteria for detecting the effects of treatment in all groups and to help select dose levels for future toxicology studies. Quantitative LF levels were also analyzed as an indication of bioavailability. Overall, administration of Helaina rhLF by once daily oral gavage for 14 days was well tolerated in rats at levels up to 2000 mg/kg/day, or 57 × Helaina's intended commercial use in adults, and indicating that a high dose of 2000 mg/kg/day is appropriate for future definitive toxicology studies.
Asunto(s)
Relación Dosis-Respuesta a Droga , Lactoferrina , Ratas Sprague-Dawley , Proteínas Recombinantes , Animales , Lactoferrina/toxicidad , Proteínas Recombinantes/toxicidad , Masculino , Femenino , Humanos , Ratas , Nivel sin Efectos Adversos Observados , Administración Oral , Peso Corporal/efectos de los fármacos , SaccharomycetalesRESUMEN
Current advances in combined antiretroviral therapy have rendered HIV infection a chronic, manageable disease; however, the problem of persistent immune activation still remains despite treatment. The immune cell receptor SLAMF7 has been shown to be upregulated in diseases characterized by chronic immune activation. In this study, we studied the function of the SLAMF7 receptor in immune cells of HIV patients and the impacts of SLAMF7 signaling on peripheral immune activation. We observed increased frequencies of SLAMF7+ PBMCs in HIV+ individuals in a clinical phenotype-dependent manner, with discordant and long-term nonprogressor patients showing elevated SLAMF7 levels, and elite controllers showing levels comparable to healthy controls. We also noted that SLAMF7 was sensitive to IFN-⺠stimulation, a factor elevated during HIV infection. Further studies revealed SLAMF7 to be a potent inhibitor of the monocyte-derived proinflammatory chemokine CXCL10 (IP-10) and other CXCR3 ligands, except in a subset of HIV+ patients termed SLAMF7 silent (SF7S). Studies utilizing small molecule inhibitors revealed that the mechanism of CXCL10 inhibition is independent of known SLAMF7 binding partners. Furthermore, we determined that SLAMF7 activation on monocytes is able to decrease their susceptibility to HIV-1 infection in vitro via downregulation of CCR5 and upregulation of the CCL3L1 chemokine. Finally, we discovered that neutrophils do not express SLAMF7, are CXCL10+ at baseline, are able to secrete CXCL10 in response to IFN-⺠and LPS, and are nonresponsive to SLAMF7 signaling. These findings implicate the SLAMF7 receptor as an important regulator of IFN-âº-driven innate immune responses during HIV infection.
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Infecciones por VIH/inmunología , VIH-1/fisiología , Interferón-alfa/metabolismo , Neutrófilos/inmunología , Familia de Moléculas Señalizadoras de la Activación Linfocitaria/metabolismo , Células Cultivadas , Quimiocina CCL3/metabolismo , Quimiocina CXCL10/metabolismo , Progresión de la Enfermedad , Susceptibilidad a Enfermedades , Humanos , Fenotipo , Receptores CCR5/metabolismo , Transducción de Señal , Regulación hacia ArribaRESUMEN
Cannabis is widely used in the United States, with an estimated prevalence of 9.5%. Certain cannabinoids in Cannabis sativa, Δ9-tetrahydrocannabinol (THC) in particular, possess immune-modulating and anti-inflammatory activity. Depending on the context, the anti-inflammatory activity of cannabinoids may be beneficial (e.g., in treating inflammatory diseases) or detrimental to normal immune defense against pathogens. The potential beneficial effect of cannabinoids on chronic neuroinflammation has gained recent attention. Monocyte migration to the brain has been implicated as a key event in chronic neuroinflammation and in the etiology of central nervous system diseases including viral infection (e.g., human immunodeficiency virus-associated neurocognitive disorder). In the brain, monocytes can contribute to neuroinflammation through interactions with astrocytes, including inducing astrocyte secretion of cytokines and chemokines. In a human coculture system, monocyte-derived interleukin (IL)-1ß due to Toll-like receptor 7 (TLR7) activation has been identified to promote astrocyte production of monocyte chemoattractant protein (MCP)-1 and IL-6. THC treatment of the TLR7-stimulated coculture suppressed monocyte secretion of IL-1ß, resulting in decreased astrocyte production of MCP-1 and IL-6. Furthermore, THC displayed direct inhibition of monocytes, as TLR7-stimulated monocyte monocultures treated with THC also showed suppressed IL-1ß production. The cannabinoid receptor 2 (CB2) agonist, JWH-015, impaired monocyte IL-1ß production similar to that of THC, suggesting that THC acts, in part, through CB2. THC also suppressed key elements of the IL-1ß production pathway, including IL1B mRNA levels and caspase-1 activity. Collectively, this study demonstrates that the anti-inflammatory properties of THC suppress TLR7-induced monocyte secretion of IL-1ß through CB2, which results in decreased astrocyte secretion of MCP-1 and IL-6. SIGNIFICANCE STATEMENT: Because cannabis use is highly prevalent in the United States and has putative anti-inflammatory properties, it is important to investigate the effect of cannabinoids on immune cell function. Furthermore, cannabinoids have garnered particular interest due to their potential beneficial effects on attenuating viral-induced chronic neuroinflammation. This study utilized a primary human coculture system to demonstrate that the major psychotropic cannabinoid in cannabis, Δ9-tetrahydrocannabinol, and a cannabinoid receptor-2 selective agonist suppress specific monocyte-mediated astrocyte inflammatory responses.
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Astrocitos/efectos de los fármacos , Agonistas de Receptores de Cannabinoides/farmacología , Quimiocina CCL2/metabolismo , Dronabinol/farmacología , Interleucina-6/metabolismo , Monocitos/efectos de los fármacos , Receptor Toll-Like 7/metabolismo , Astrocitos/metabolismo , Células Cultivadas , Quimiocina CCL2/genética , Técnicas de Cocultivo , Humanos , Indoles/farmacología , Interleucina-6/genética , Monocitos/metabolismoRESUMEN
Plasmacytoid dendritic cells (pDC) compose 0.2-0.5% of circulating leukocytes but play a significant role in mounting host immune responses. Elevated and chronic activation of pDC are implicated in autoimmune disease like systemic lupus erythematosus and rheumatoid arthritis. Δ9-tetrahydrocannabinol (THC) is a well characterized cannabinoid with potent anti-inflammatory activity, but acceptance of THC as a treatment for autoimmune disorders has been hindered due to psychotropic activity. The psychotropic effects of THC are mediated through cannabinoid receptor 1 (CB1) expressed in the central nervous system while the immunomodulatory effects of THC result from THC binding to CB1 and CB2 on immune cells. Synthetic CB2-selective agonists have been developed to explore immune modulation by cannabinoids in the absence of psychotropic effects. The goal of these studies was to determine if the CB2-selective agonists, JWH-015 and JWH-133, have comparable efficacy to THC in modulating IFNα and TNFα responses by primary human pDC. Treatment with JWH-133 and JWH-015 inhibited CpG-induced IFNα and TNFα responses by pDC. Further, the phosphorylation of IRF7, TBK1, NFκB, and IKKγ, key events in pDC activation, were suppressed by THC, JWH-133, and JWH-015. Likewise, the phosphorylation of AKT at the S473 and T308 residues were differentially modulated by treatment with THC and both JWH compounds. Collectively, these results demonstrate the potential for CB2 targeted therapeutics for treatment of inflammatory conditions involving aberrant pDC activity.
Asunto(s)
Antiinflamatorios/farmacología , Agonistas de Receptores de Cannabinoides/farmacología , Cannabinoides/farmacología , Células Dendríticas/efectos de los fármacos , Indoles/farmacología , Interferón gamma/metabolismo , Oligodesoxirribonucleótidos/farmacología , Receptor Cannabinoide CB2/agonistas , Factor de Necrosis Tumoral alfa/metabolismo , Células Cultivadas , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Humanos , Fosforilación , Receptor Cannabinoide CB2/metabolismo , Transducción de SeñalRESUMEN
Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates biological responses to endogenous and environmental chemical cues. Increasing evidence shows that the AHR plays physiological roles in regulating development, homeostasis, and function of a variety of cell lineages in the immune system. However, the role of AHR in human B cell development has not been investigated. Toward this end, an in vitro feeder-free human B cell developmental model system was employed using human cord blood CD34+ hematopoietic stem/progenitor cells. Using this model, we found that AHR activation by the high-affinity ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin significantly suppressed the generation of early B cells and pro-B cells from hematopoietic stem/progenitor cells, indicating the impairment of B cell lineage specification and commitment. Addition of an AHR antagonist reversed 2,3,7,8-tetrachlorodibenzo-p-dioxin-elicited suppression of early B and pro-B cells, suggesting a role of AHR in regulating B lymphopoiesis. Gene expression analysis revealed a significant decrease in the messenger RNA level of early B cell factor 1 (EBF1) and paired box 5, two critical transcription factors directing B cell lineage specification and commitment. Additionally, binding of the ligand-activated AHR to the putative dioxin response elements in the EBF1 promoter was demonstrated by EMSAs and chromatin immunoprecipitation analysis, suggesting transcriptional regulation of EBF1 by AHR. Taken together, this study demonstrates a role for the AHR in regulating human B cell development, and it suggests that transcriptional alterations of EBF1 by the AHR are involved in the underlying mechanism.
Asunto(s)
Linfocitos B/fisiología , Células Madre Hematopoyéticas/fisiología , Factor de Transcripción PAX5/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Transactivadores/metabolismo , Antígenos CD34/metabolismo , Diferenciación Celular , Linaje de la Célula , Células Cultivadas , Regulación de la Expresión Génica , Humanos , Linfopoyesis , Factor de Transcripción PAX5/genética , Dibenzodioxinas Policloradas/inmunología , Regiones Promotoras Genéticas/genética , Elementos de Respuesta/genética , Transactivadores/genéticaRESUMEN
Patients with HIV routinely use medicinal cannabinoids to treat neuropathic pain, anxiety, and human immunodeficiency virus (HIV)-associated wasting. However, Δ9-tetrahydrocannabinol (THC), the primary psychoactive cannabinoid in cannabis, suppresses T-cell function and secretion of interferons, both critically important in the antiviral immune response. Interferon-α (IFNα), a key cytokine in T-cell activation and peripheral control of HIV infection, can potentiate responsiveness to interleukin-7 (IL-7), a crucial homeostatic cytokine for peripheral T-cell maintenance. The objective of this investigation was to compare the response of T cells to stimulation by IFNα and IL-7 in T cells from healthy and HIV+ donors in the absence and presence of THC. To compare T-cell responses between healthy and HIV+ donors signaling through IFNα receptor, IFNα-induced expression of IL-7α receptor (IL-7Rα), cognate signaling through IL-7R, and on IL-7-mediated T-cell proliferation were measured by flow cytometry and real-time quantitative polymerase chain reaction. CD8+ T cells from HIV+ donors showed a diminished response to IFNα-induced phosphorylated signal transducer and activator of transcription-1 activation compared with CD8+ T cells from healthy donors, whereas CD4+ T cells from HIV+ donors and healthy donors were comparable. Treatment with IFNα promoted IL-7R expression and potentiated IL-7-induced STAT5 phosphorylation to augment IL-7-mediated proliferation by T cells from healthy and HIV+ donors. Finally, HIV+ donors exhibited reduced sensitivity to THC-mediated suppression by IFNα- and IL-7-mediated stimulation compared with healthy donors. These results further support THC as being immune suppressive while identifying putatively beneficial aspects of cannabinoid-based therapies in HIV+ patients.
Asunto(s)
Dronabinol/farmacología , Infecciones por VIH/inmunología , Interferón-alfa/farmacología , Activación de Linfocitos/efectos de los fármacos , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Adulto , Anciano , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/metabolismo , Estudios de Casos y Controles , Proliferación Celular/efectos de los fármacos , Interacciones Farmacológicas , Humanos , Interleucina-7/farmacología , Masculino , Persona de Mediana Edad , Fosforilación/efectos de los fármacos , Receptores de Interleucina-7/metabolismo , Factor de Transcripción STAT5/metabolismo , Transducción de Señal/efectos de los fármacos , Linfocitos T/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
2,3,7,8-Tetrachlordibenzo- p-dioxin (TCDD) is an environmental pollutant that can cause various toxic effects, including chloracne, metabolic syndrome, and immune suppression. Most of the toxicity associated with TCDD is mediated through activation of the aryl hydrocarbon receptor (AHR). Recent research has suggested the presence of a wide-range of interindividual variability in TCDD-mediated suppression of the Immunoglobulin-M (IgM) response across the human population. In an attempt to identify putative modifiers of AHR-mediated immunosuppression beyond the AHR, B cells were isolated from a panel of genetically diverse mouse strain to scan for modulators that drive interstrain differences in TCDD-mediated suppression of the IgM response. Results implicated a region of mouse Chromosome 1 near a gene encoding serine peptidase inhibitor, clade B, member 2 ( Serpinb2) whose human ortholog is plasminogen activator inhibitor 2 (PAI2). Further downstream analyses indicated that Serpinb2 is dysregulated by TCDD and, furthermore, that B cells from Serpinb2 -/- mice are significantly more sensitive to TCDD-mediated suppression as compared to littermate controls. This study suggests a protective role of Serpinb2 within TCDD-mediated immunosuppression and, furthermore, a novel function of Serpinb2-related activity in the IgM response.
Asunto(s)
Linfocitos B/efectos de los fármacos , Dibenzodioxinas Policloradas/toxicidad , Serpinas/metabolismo , Animales , Linfocitos B/citología , Linfocitos B/metabolismo , Contaminantes Ambientales/química , Contaminantes Ambientales/toxicidad , Ensayo de Immunospot Ligado a Enzimas , Inmunoglobulina M/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Filogenia , Dibenzodioxinas Policloradas/química , Sitios de Carácter Cuantitativo , Receptores de Hidrocarburo de Aril/clasificación , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Serpinas/química , Serpinas/genéticaRESUMEN
Activated carbon (AC) is an increasingly attractive remediation alternative for the sequestration of dioxins at contaminated sites globally. However, the potential for AC to reduce the bioavailability of dioxins in mammals and the residing gut microbiota has received less attention. This question was partially answered in a recent study examining 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced hallmark toxic responses in mice administered with TCDD sequestered by AC or freely available in corn oil by oral gavage. Results from that study support the use of AC to significantly reduce the bioavailability of TCDD to the host. Herein, we examined the bioavailability of TCDD sequestered to AC on a key murine gut commensal and the influence of AC on the community structure of the gut microbiota. The analysis included qPCR to quantify the expression of segmented filamentous bacteria (SFB) in the mouse ileum, which has responded to TCDD-induced host toxicity in previous studies and community structure via sequencing the 16S ribosomal RNA (rRNA) gene. The expression of SFB 16S rRNA gene and functional genes significantly increased with TCDD administered with corn oil vehicle. Such a response was absent when TCDD was sequestered by AC. In addition, AC appeared to have a minimal influence on murine gut community structure and diversity, affecting only the relative abundance of Lactobacillaceae and two other groups. Results of this study further support the remedial use of AC for eliminating bioavailability of TCDD to host and subsequent influence on the gut microbiome.
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Carbón Orgánico/administración & dosificación , Microbioma Gastrointestinal/efectos de los fármacos , Dibenzodioxinas Policloradas/administración & dosificación , Animales , Disponibilidad Biológica , Carbón Orgánico/farmacocinética , Aceite de Maíz/administración & dosificación , Aceite de Maíz/farmacocinética , Femenino , Íleon/microbiología , Lactobacillaceae/metabolismo , Ratones , Dibenzodioxinas Policloradas/farmacocinética , Dibenzodioxinas Policloradas/toxicidad , ARN Ribosómico 16S/genética , TranscriptomaRESUMEN
The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which is a strong AHR agonist, causes significant suppression of human B cell activation and differentiation. The current studies describe the identification of Src homology phosphatase 1 (SHP-1) encoded by the gene PTPN6 as a putative regulator of TCDD-mediated suppression of B cell activation. Shp-1 was initially identified through a genome-wide analysis of AHR binding in mouse B cells in the presence of TCDD. The binding of AHR to the PTPN6 promoter was further confirmed using electrophoretic mobility shift assays in which, specific binding of AHR was detected at four putative DRE sites within PTPN6 promoter. Time-course measurements performed in human B cells highlighted a significant increase in SHP-1 mRNA and protein levels in the presence of TCDD. The changes in the protein levels of SHP-1 were also observed in a TCDD concentration-dependent manner. The increase in SHP-1 levels was also seen to occur due to a change in early signaling events in the presence of TCDD. We have shown that BCL-6 regulates B cell activation by repressing activation marker CD80 in the presence of TCDD. TCDD-treatment led to a significant increase in the double positive (SHP-1hi BCL-6hi) population. Interestingly, treatment of naïve human B cells with SHP-1 inhibitor decreased BCL-6 protein levels suggesting possible regulation of BCL-6 by SHP-1 for the first time. Collectively, these results suggest that SHP-1 is regulated by AHR in the presence of TCDD and may, in part through BCL-6, regulate TCDD-mediated suppression of human B cell activation.
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Dibenzodioxinas Policloradas/toxicidad , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Receptores de Hidrocarburo de Aril/fisiología , Animales , Linfocitos B/metabolismo , Activación Enzimática , Humanos , Activación de Linfocitos , Ratones , Regiones Promotoras Genéticas , Proteína Tirosina Fosfatasa no Receptora Tipo 6/genética , ARN Mensajero/genéticaRESUMEN
Many environmental contaminants can disrupt the adaptive immune response. Exposure to the ubiquitous aryl hydrocarbon receptor (AhR) ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other agonists suppresses the antibody response. The underlying pathway mechanism by which TCDD alters B cell function is not well understood. The present study investigated the mechanism of AhR-mediated pathways and mode of suppression by which TCDD perturbs terminal differentiation of B cells to plasma cells and thereby impairs antibody production. An integrated approach combining computational pathway modeling and in vitro assays with primary mouse B cells activated by lipopolysaccharide was employed. We demonstrated that suppression of the IgM response by TCDD occurs in an all-or-none (binary) rather than graded mode: i.e., it reduces the number of IgM-secreting cells in a concentration-dependent manner without affecting the IgM content in individual plasma cells. The mathematical model of the gene regulatory circuit underpinning B cell differentiation revealed that two previously identified AhR-regulated pathways, inhibition of signaling protein AP-1 and activation of transcription factor Bach2, could account for the all-or-none mode of suppression. Both pathways disrupt the operation of a bistable-switch circuit that contains transcription factors Bcl6, Prdm1, Pax5, and Bach2 and regulates B cell fate. The model further predicted that by transcriptionally activating Bach2, TCDD might delay B cell differentiation and increase the likelihood of isotype switching, thereby altering the antibody repertoire. In conclusion, the present study revealed the mode and specific pathway mechanisms by which the environmental immunosuppressant TCDD suppresses B cell differentiation.
Asunto(s)
Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Modelos Inmunológicos , Dibenzodioxinas Policloradas/toxicidad , Inmunidad Adaptativa/efectos de los fármacos , Inmunidad Adaptativa/inmunología , Animales , Linfocitos B/inmunología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/antagonistas & inhibidores , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/inmunología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Simulación por Computador , Femenino , Citometría de Flujo , Inmunoglobulina M/inmunología , Ratones , Ratones Endogámicos C57BL , Distribución Aleatoria , Receptores de Hidrocarburo de Aril/inmunología , Factor de Transcripción AP-1/antagonistas & inhibidores , Factor de Transcripción AP-1/inmunologíaRESUMEN
In vitro models to study simultaneous development of different human immune cells and hematopoietic lineages are lacking. We identified and characterized, using single-cell methods, an in vitro stromal cell-free culture system of human hematopoietic stem and progenitor cell (HSPC) differentiation that allows concurrent development of multiple immune cell lineages. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor influencing many biological processes in diverse cell types. Using this in vitro model, we found that AHR activation by the highly specific AHR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin, drives differentiation of human umbilical cord blood-derived CD34+ HSPCs toward monocytes and granulocytes with a significant decrease in lymphoid and megakaryocyte lineage specification that may lead to reduced immune competence. To our knowledge, we also discovered for the first time, using single-cell modalities, that AHR activation decreased the expression of BCL11A and IRF8 in progenitor cells, which are critical genes involved in hematopoietic lineage specification processes at both transcriptomic and protein levels. Our in vitro model of hematopoiesis, coupled with single-cell tools, therefore allows for a better understanding of the role played by AHR in modulating hematopoietic differentiation.
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Células Madre Hematopoyéticas , Receptores de Hidrocarburo de Aril , Humanos , Células Madre Hematopoyéticas/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Ligandos , Hematopoyesis , Diferenciación CelularRESUMEN
Innate-like B cells (ILBs) are a heterogeneous population B cells which participate in innate and adaptive immune responses. This diverse subset of B cells is characterized by the expression of CD5 and has been shown to secrete high levels of immunoglobulin M (IgM) in the absence of infection or vaccination. Further, CD5+ ILBs have been shown to express high basal levels of lymphocyte specific protein tyrosine kinase (LCK) and programmed cell death protein-1 (PD-1), which are particularly sensitive to stimulation by interferon gamma (IFNγ). Previous studies have demonstrated that activation of the aryl hydrocarbon receptor (AHR), a cytosolic ligand-activated transcription factor, results in suppressed IgM responses and is dependent on LCK. A recent study showed that CD5+ ILBs are particularly sensitive to AHR activation as evidenced by a significant suppression of the IgM response compared to CD5- B cells, which were refractory. Therefore, the objective of this study was to further investigate the role of LCK and PD-1 signaling in AHR-mediated suppression of CD5+ ILBs. In addition, studies were conducted to establish whether IFNγ alters the levels of LCK and PD-1 in CD5+ ILBs. We found that AHR activation led to a significant upregulation of total LCK and PD-1 proteins in CD5+ ILBs, which correlated with suppression of IgM. Interestingly, treatment with recombinant IFNγ reduced LCK protein levels and reversed AHR-mediated IgM suppression in CD5+ ILBs in a similar manner as LCK inhibitors. Collectively, these results support a critical role for LCK and PD-1 in AHR-mediated suppression of the IgM response in human CD5+ ILBs.
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Linfocitos B , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Dibenzodioxinas Policloradas , Receptor de Muerte Celular Programada 1 , Receptores de Hidrocarburo de Aril , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Humanos , Inmunoglobulina M/metabolismo , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Receptores de Hidrocarburo de Aril/metabolismoRESUMEN
Cannabis is well established as possessing immune modulating activity. The objective of this study was to evaluate the anti-inflammatory properties of selected cannabis-derived terpenes and cannabinoids. Based on their activity in cannabis-chemovar studies, α-pinene, trans-nerolidol, D-limonene, linalool and phytol were the selected terpenes evaluated. The cannabinoid compounds evaluated included cannabidivarin, cannabidiol, cannabinol, cannabichromene, cannabigerol and delta-9-tetrahydrocannabinol. Human PBMC were pretreated with each compound, individually, at concentrations extending from 0.001 to 10 µM and then stimulated with CpG (plasmacytoid dendritic cell), LPS (monocytes), or anti-CD3/CD28 (T cells). Proliferation, activation marker expression, cytokine production and phagocytosis, were quantified. Of the 21 responses assayed for each compound, cannabinoids showed the greatest immune modulating activity compared to their vehicle control. Delta-9-tetrahydrocannabinol possessed the greatest activity affecting 11 immune parameters followed by cannabidivarin, cannabigerol, cannabichromene, cannabinol and cannabidiol. α-Pinene showed the greatest immune modulating activity from the selected group of terpenes, followed by linalool, phytol, trans-nerolidol. Limonene had no effect on any of the parameters tested. Overall, these studies suggest that selected cannabis-derived terpenes displayed minimal immunological activity, while cannabinoids exhibited a broader range of activity. Compounds possessing anti-inflammatory effects may be useful in decreasing inflammation associated with a range of disorders, including neurodegenerative disorders.
Asunto(s)
Cannabidiol , Cannabinoides , Cannabis , Humanos , Terpenos/farmacología , Dronabinol/farmacología , Cannabinol , Leucocitos Mononucleares , Cannabinoides/farmacología , FitolRESUMEN
Suppression of the primary antibody response is particularly sensitive to suppression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice; however, surprisingly little is known concerning the effects of TCDD on humoral immunity or B cell function in humans. Results from a limited number of previous studies, primarily employing in vitro activation models, suggested that human B cell effector function is suppressed by TCDD. The present study sought to extend these findings by investigating, in primary human B cells, the effects of TCDD on several critical stages leading to antibody secretion including activation and plasmacytic differentiation using an in vitro CD40 ligand activation model. These studies revealed important differences in the response of human and mouse B cells to TCDD, the most striking being altered expression of plasmacytic differentiation regulators, B lymphocyte-induced maturation protein 1 and paired box protein 5, in mouse but not human B cells. The activation of human B cells was profoundly impaired by TCDD, as evidenced by decreased expression of activation markers CD80, CD86, and CD69. The impaired activation correlated with decreased cell viability, which prevented the progression of human B cells toward plasmacytic differentiation. TCDD treatment also attenuated the early activation of mitogen-activated protein kinases (MAPK) and Akt signaling in human B cells. Collectively, the present study provided experimental evidence for novel mechanisms by which TCDD impairs the effector function of primary human B cells.
Asunto(s)
Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Ligando de CD40/fisiología , Dibenzodioxinas Policloradas/toxicidad , Animales , Ligando de CD40/antagonistas & inhibidores , Ligando de CD40/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Distribución AleatoriaRESUMEN
Exposure to the aryl hydrocarbon receptor (AHR) agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters B-cell differentiation and suppresses antibody production. Previous genomic studies in mouse B cells identified Bach2 as a direct target of the AHR. Bach2 is known to repress expression of Prdm1, a key transcription factor involved in B-cell differentiation, by binding to Maf elements (MAREs) in the regulatory regions of the gene. Chromatin immunoprecipitation followed by quantitative PCR in TCDD-treated lipopolysaccharide (LPS)-activated B cells showed increased binding of the AHR within the first intron in the Bach2 gene. The binding was further confirmed by electrophoretic mobility shift assay (EMSA). TCDD also induced expression of Bach2 in activated as well as resting B cells from 2 to 24h post-treatment in a time- and concentration-dependent manner. Expression of Prdm1 was decreased by TCDD at 24h and was consistent with repression by Bach2. Increased DNA binding activity to the intron 5 MARE with increasing TCDD concentrations was observed by EMSA. Supershifts identified the presence of Bach2 in the DNA binding complex associated with the intron 5 MARE of Prdm1. Functional validation of the role of Bach2 in the suppression of B-cell differentiation by TCDD was performed using RNA interference (RNAi). Knockdown of Bach2 showed approximately 40% reversal in the TCDD-induced suppression of IgM secretion when compared to controls. The results suggest that the transcriptional regulation of Bach2 by the AHR is one of the mechanisms involved in the suppression of B-cell differentiation by TCDD.
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Linfocitos B/efectos de los fármacos , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Diferenciación Celular/efectos de los fármacos , Inhibidores de Crecimiento/fisiología , Dibenzodioxinas Policloradas/toxicidad , Receptores de Hidrocarburo de Aril/fisiología , Animales , Linfocitos B/citología , Linfocitos B/fisiología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/fisiología , Diferenciación Celular/fisiología , Línea Celular Tumoral , Técnicas de Inactivación de Genes , Inhibidores de Crecimiento/toxicidad , Ratones , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiologíaRESUMEN
Dioxins, including 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD), produce a wide range of toxic effects in mammals. Most, if not all, of these toxic effects are regulated by the aryl hydrocarbon receptor (AHR). The AHR is a ligand activated transcription factor that has been shown to interact with numerous proteins capable of influencing the receptor's function. The ability of secondary proteins to alter AHR-mediated transcriptional events, a necessary step for toxicity, led us to determine whether additional interacting proteins could be identified. To this end, we have employed tandem affinity purification (TAP) of the AHR in Hepa1c1c7 cells. TAP of the AHR, followed by mass spectrometry (MS) identified ATP5α1, a subunit of the ATP synthase complex, as a strong AHR interactor in the absence of ligand. The interaction was lost upon exposure to TCDD. The association was confirmed by co-immunoprecipitation in multiple cell lines. In addition, cell fractionation experiments showed that a fraction of the AHR is found in the mitochondria. To ascribe a potential functional role to the AHR:ATP5α1 interaction, TCDD was shown to induce a hyperpolarization of the mitochondrial membrane in an AHR-dependent and transcription-independent manner. These results suggest that a fraction of the total cellular AHR pool is localized to the mitochondria and contributes to the organelle's homeostasis.
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Mitocondrias/fisiología , Proteínas Mitocondriales/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Factores de Acoplamiento de la Fosforilación Oxidativa/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Complejos de ATP Sintetasa/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Línea Celular Tumoral , Homeostasis/fisiología , Ratones , Ratones Noqueados , Mitocondrias/efectos de los fármacos , Datos de Secuencia Molecular , Dibenzodioxinas Policloradas/metabolismo , Dibenzodioxinas Policloradas/toxicidad , Unión Proteica/fisiología , Subunidades de Proteína/metabolismoRESUMEN
Xenobiotic-mediated activation of the aryl hydrocarbon receptor (AHR) is immunotoxic in a number of immune cell types, with the B cell being a well-established sensitive target. Recent advances have provided evidence that the B cell repertoire is a heterogeneous population, with subpopulations exhibiting vastly different cellular and functional phenotypes. Recent work from our laboratory identified the T cell specific kinase lck as being differentially regulated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which is a potent activator of AHR. While LCK is primarily expressed in T cells, a subset of CD5+ B cells also express LCK. CD5 positivity describes a broad class of B lymphocytes termed innate-like B cells (ILBs) that are critical mediators of innate immunity through constitutive secretion of polyvalent natural immunoglobulin M (IgM). We hypothesized that CD5+ ILBs may be sensitive to AHR-mediated immunotoxicity. Indeed, when CD5+ B cells were isolated from the CD19+ pool and treated with TCDD, they showed increased suppression of the CD40 ligand-induced IgM response compared to CD5- B cells. Further, characterization of the CD5+ population indicated increased basal expression of AHR, AHR repressor (AHRR), and cytochrome p450 family 1 member a1 (CYP1A1). Indeed the levels of AHR-mediated suppression of the IgM response from individual donors strongly correlated with the percentage of the B cell pool that was CD5+, suggesting that CD5+ B cells are more sensitive to AHR-mediated impairment. Together these data highlight the sensitive nature of CD5+ ILBs to AHR activation and provide insight into mechanisms associated with AHR activation in human B cells.
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Subgrupos de Linfocitos B/efectos de los fármacos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/agonistas , Antígenos CD5/metabolismo , Inmunidad Innata/efectos de los fármacos , Inmunoglobulina M/metabolismo , Dibenzodioxinas Policloradas/toxicidad , Receptores de Hidrocarburo de Aril/agonistas , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Antígenos CD5/genética , Células Cultivadas , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Humanos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Fenotipo , Proteína 2 Ligando de Muerte Celular Programada 1/genética , Proteína 2 Ligando de Muerte Celular Programada 1/metabolismo , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/metabolismo , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
Cannabidiol (CBD) is a major non-euphoric cannabis-derived compound that has become popular in its over-the-counter use. CBD possesses low affinity for cannabinoid receptors, while the primary molecular target(s) by which it mediates biological activity remain poorly defined. Individuals commonly self-medicate using CBD products with little knowledge of its specific immunopharmacological effects on the human immune system; however, research has established primarily in rodent models that CBD possesses immune modulating properties. The objective of this study was to evaluate whether CBD modulates the innate immune response by human primary monocytes activated through toll-like receptors (TLR) 1-9. Monocytes were activated through each TLR and treated with CBD (0.5-10 µM) for 22 h. Monocyte secretion profiles for 13 immune mediators were quantified including: IL-4, IL-2, IP-10, IL-1ß, TNFα, MCP-1, IL-17a, IL-6, IL-10, IFNγ, IL-12p70, IL-8, and TGF-ß1. CBD treatment significantly suppressed secretion of proinflammatory cytokine IL-1ß by monocytes activated through most TLRs, apart from TLRs 3 and 8. Additionally, CBD treatment induced significant modulation of IL-6 production by monocytes activated through most TLRs, except for TLRs 1 and 3. Most other monocyte-derived factors assayed were refractory to CBD modulation. Overall, CBD selectively altered monocyte-derived IL-1ß and IL-6 when activated through most TLRs. This study is of particular importance as it provides a direct and comprehensive assessment of the effects of CBD on TLR-activated primary human monocytes at a time when CBD containing products are being widely used by the public.
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Cannabidiol/farmacología , Citocinas/inmunología , Factores Inmunológicos/farmacología , Monocitos/efectos de los fármacos , Cannabidiol/administración & dosificación , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Inmunidad Innata/efectos de los fármacos , Factores Inmunológicos/administración & dosificación , Interleucina-1beta/inmunología , Interleucina-6/inmunología , Monocitos/inmunología , Receptores Toll-Like/inmunologíaRESUMEN
Translocator protein 18 kDa (TSPO) is a well-known outer mitochondrial membrane protein and it is widely used as a biomarker of neuroinflammation and brain injury. Although it is thought that TSPO plays key roles in a multitude of host cell functions, including steroid biosynthesis, apoptosis, generation of reactive oxygen species, and proliferation, some of these functions have recently been questioned. Here, we report the unexpected finding that circulating immune cells differentially express basal levels of TSPO on their cell surface, with a high percentage of monocytes and neutrophils expressing cell surface TSPO. In vitro stimulation of monocytes with LPS significantly increases the frequency of cells with surface TSPO expression in the absence of altered gene expression. Importantly, the LPS increase in TSPO cell surface expression in monocytes appears to be selective for LPS because two other distinct monocyte activators failed to increase the frequency of cells with surface TSPO. Finally, when we quantified immune cell TSPO surface expression in antiretroviral therapy-treated HIV+ donors, a chronic inflammatory disease, we found significant increases in the frequency of TSPO surface localization, which could be pharmacologically suppressed with ∆9 -tetrahydrocannabinol. These findings suggest that cell surface TSPO in circulating leukocytes could serve as a peripheral blood-based biomarker of inflammation.