RESUMEN
BACKGROUND: Neutrophil extracellular traps (NETs) consist of chromatin DNA networks that are studded with cytosolic and granular antimicrobial proteins to trap or kill an infected microorganism. A lipid emulsion, the solvent of pure propofol for intravenous application, is given to clinical patients who require intravenous feeding of fatty acids and fat for energy. Intravenous propofol is widely used to sedate critically ill patients. Both intravenous propofol and its lipid emulsion have immunomodulatory activity. However, the role of lipid emulsion of intravenous propofol on NET induction remains unclear. METHODS: In this study, neutrophils were stimulated with phorbol myristate acetate (PMA) or Escherichia coli (E. coli) in the absence or presence of intravenous propofol (Propofol-Lipuro®), its solvent lipid emulsion (Lipofundin) or pure propofol, and NETs were stained with SYTOX Green for visualization and quantification. Total HOCl was determined by measuring the taurine-chloramine complex, and intracellular HOCl was evaluated with BioTracker™ TP-HOCl 1 dye. RESULTS: PMA-induced NETs were not efficiently inhibited when Propofol-Lipuro® was added after PMA stimulation. Clinically relevant concentrations of Lipofundin exerted a significant reduction in PMA-induced NETs and total reactive oxidative species (ROS), which was comparable to that observed for Propofol-Lipuro®. Lipofundin transiently reduced intracellular HOCl production and the phosphorylation level of extracellular regulated kinase (p-ERK) but did not scavenge HOCl. Moreover, Lipofundin decreased E. coli-induced NETs in a ROS-independent pathway, similar to Propofol-Lipuro®. CONCLUSIONS: All data agree that Lipofundin, the major component of Propofol-Lipuro®, inhibits intracellular HOCl and p-ERK to suppress PMA-induced NET formation but reduces E.coli-induced NETs in a ROS-independent pathway.
Asunto(s)
Escherichia coli , Trampas Extracelulares , Neutrófilos , Fosfolípidos , Propofol , Sorbitol , Acetato de Tetradecanoilforbol , Administración Intravenosa , Combinación de Medicamentos , Emulsiones/administración & dosificación , Escherichia coli/inmunología , Quinasas MAP Reguladas por Señal Extracelular , Trampas Extracelulares/inmunología , Humanos , Ácido Hipocloroso , Neutrófilos/inmunología , Fosfolípidos/farmacología , Propofol/administración & dosificación , Propofol/antagonistas & inhibidores , Propofol/farmacología , Especies Reactivas de Oxígeno/metabolismo , Solventes , Sorbitol/farmacología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
INTRODUCTION: The formation of neutrophil extracellular traps (NETs) is a process in which several kinds of enzymes participate generating posttranslational modifications of proteins. NETs have been associated with infectious, autoimmune, and inflammatory diseases. Inhibition of several proteases reduces the formation of NETs. In the present work, we analyzed the role of several broad-acting and specific inhibitors of proteases in the formation of NETs. METHODS: Neutrophils were isolated from peripheral blood of healthy individuals by density gradient. The neutrophils were quantified and seeded into cell culture plates. Phorbol myristate acetate and A23187 were used as NETs inducers, and several specific inhibitors of proteases were used. The cells were stained for cytoskeleton or DNA. The cell-free supernatants were used to assess DNA release. Statistical analysis was carried out by a Kruskal-Wallis or ANOVA test. RESULTS: We observed marked changes in actin organization after the induction of NETs, suggesting that the cytoskeleton is being actively regulated. When we used protease inhibitors, the release of DNA was reduced, suggesting the participation of actin remodeling in the process. Further characterization of the specific proteases revealed that calpain modulates the reorganization of actin cytoskeleton and DNA release. Preservation of part of the actin cytoskeleton suggests that DNA release is not only a mechanic process associated to the chromatin decondensation; rather the process is highly regulated by active proteases that promote cytoskeleton reorganization and chromatin decondensation that culminates in DNA release. CONCLUSION: Calpain mediates the DNA release in the NET formation process by the modification of cortical actin cytoskeleton in a calcium-dependent manner.
Asunto(s)
Calpaína/metabolismo , Citoesqueleto/metabolismo , ADN/metabolismo , Trampas Extracelulares/inmunología , Neutrófilos/metabolismo , Actinas/metabolismo , Calcio/metabolismo , Células Cultivadas , Humanos , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Inhibidores de Proteasas/farmacologíaRESUMEN
INTRODUCTION: THP-1 cells, a human leukemia monocytic cell line, differentiated by phorbol myristate acetate (PMA) are widely used as surrogate of human macrophages. Differentiated THP-1 cells acquire macrophage-like characteristics including more adherence and altered cell function. Nitric oxide (NO), an intracellular messenger, is critical in regulating cell differentiation. Here we elucidated whether NO relates to PMA-induced monocyte-to-macrophage differentiation of THP-1 cells. The mutual regulation of calcium and NO was also investigated. MATERIAL & METHODS: THP-1 cells were incubated with PMA for 24 h, followed by assay of adherence, morphological change, migration or IL-1ß release. L-NG-Nitroarginine methyl ester (l-NAME, a nitric oxide synthase inhibitor) or BAPTA-AM (a calcium chelator) was added before PMA stimulation, and levels of calcium and NO were measured. Furthermore, a selective inhibitor of inducible nitric oxide synthase (iNOS) activity was employed to study the role of iNOS. RESULTS AND DISCUSSION: Effects of PMA on upregulation of adherence, lipopolysaccharide-triggered IL-1ß, and migration ability of THP-1 cells were consistent with NO concentrations. Both l-NAME and BAPTA-AM mitigated effects of PMA on THP-1 cells differentiation. BAPTA-AM decreased levels of NO, while l-NAME had no effect on calcium levels. Of note, inhibition of iNOS activity decreased PMA-triggered upregulation of NO. CONCLUSION: PMA induced differentiation of THP-1 cells partially in a NO-dependent manner. The calcium signaling may mediate PMA-triggered upregulation of NO.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Óxido Nítrico/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Humanos , Macrófagos/metabolismo , Monocitos/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo II/metabolismo , Células THP-1 , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Nutritional intervention in older dogs aims to increase lifespan and improve life quality as well as delay the development of diseases related to ageing. It is believed that active fractions of mannoproteins (AFMs) obtained through extraction and fractionation of yeast cell walls (Saccharomyces cerevisiae) may beneficially modulate the immune system. However, studies that have evaluated this component and the effects of ageing on the immune system of dogs are scarce. This study aimed to evaluate the immunological effects of AFMs in adult and elderly dogs. Three extruded iso-nutrient experimental diets were formulated: without addition of AFM (T0); with AFM at 400â¯mg/kg (T400); and with AFM at 800â¯mg/kg (T800). Thirty-six beagle dogs were used, and six experimental treatments, resulting in combinations of age (adult and elderly) and diet (T0, T400, and T800), were evaluated. On days zero, 14, and 28, blood samples were obtained for leucocyte phenotyping and phagocytosis assays. On days zero and 28, a lymphoproliferation test, quantification of reactive oxygen (H2O2) and nitrogen (NO) intermediate production, evaluation of faecal immunoglobulin A (IgA) content, and a delayed cutaneous hypersensitivity test (DCHT) were performed. Statistical analyses were performed with SAS software. Repeated measure variance analyses were performed, and means were compared by the Tukey test. Values of Pâ¯≤â¯0.05 were considered significant, and values of Pâ¯≤â¯0.10 were considered tendencies. Dogs fed T400 tended to have higher neutrophilic phagocytic activity than dogs fed T800 (Pâ¯=â¯0.073). Regarding reactive oxygen intermediates, bacterial lipopolysaccharide (LPS)-stimulated neutrophils from animals that were fed T400â¯had a tendency to produce more H2O2 than those from animals fed the control diet (Pâ¯=â¯0.093). Elderly dogs, when compared to adult dogs, had lower absolute T and B lymphocyte counts, lower auxiliary T lymphocyte counts, and higher cytotoxic T lymphocyte counts (Pâ¯<â¯0.05). A significant effect of diet, age, and time with saline inoculation was noted for the DCHT. There was no effect of diet or age on faecal IgA content in dogs. This study suggests beneficial effects of mannoproteins on the specific and nonspecific immune responses in adult and elderly dogs.
RESUMEN
Major histocompatibility complex (MHC) class II-associated invariant chain is a chaperone responsible for targeting the MHC class II dimer to the endocytic pathway, thus enabling the loading of exogenous antigens onto the MHC class II receptor. In the current study, in vivo and in vitro methods were used to investigate the regulation of the rainbow trout invariant chain proteins S25-7 and INVX, upon immune system activation. Whole rainbow trout and the macrophage/monocyte-like cell line RTS11 were treated with PMA at concentrations shown to induce IL-1ß transcripts and homotypic aggregation of RTS11. S25-7 transcript levels remained unchanged in the gill, spleen, and liver and were found to be significantly decreased in head kidney beginning 24 h post-stimulation. Meanwhile, INVX transcript levels remained unchanged in all tissues studied. Both S25-7 and INVX proteins were produced in gill and spleen tissues but their expression was unaffected by immune system stimulation. Surprisingly, neither INVX nor S25-7 protein was detected in the secondary immune organ, the head kidney. Analysis of RTS11 cultures demonstrated that both INVX and S25-7 transcript levels significantly increased at 96 h and 120 h following PMA stimulation before returning to control levels at 168 h. Meanwhile, at the protein level in RTS11, S25-7 remained unchanged while INVX had a significant decrease at 168 h post-stimulation. These results indicate that neither INVX nor S25-7 is upregulated upon immune system activation; thus, teleosts have evolved a system of immune regulation that is different than that found in mammals.
Asunto(s)
Antígenos de Diferenciación de Linfocitos B/genética , Antígenos de Diferenciación de Linfocitos B/inmunología , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Inmunomodulación/genética , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/inmunología , Inmunidad Adaptativa , Animales , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Inmunización , Macrófagos/inmunología , Macrófagos/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Especificidad de Órganos/genética , Especificidad de Órganos/inmunología , Isoformas de Proteínas , TranscriptomaRESUMEN
The neutrophil oxidative respiratory burst response is a key component of the innate immune system responsible for killing microbial pathogens. Since fish rely on the innate immune system for health, monitoring the respiratory burst activity may be an effective means of gauging fish health status. Here we report that the respiratory burst of Asian seabass neutrophils can be measured in whole blood by the dihydrorhodamine (DHR)-123 reduction assay and flow cytometry. Neutrophils responded to phorbol myristate acetate (PMA) in a concentration dependent manner with significant respiratory burst activity at 100-1000â¯nM. Other known neutrophil agonists, such as bacterial lipopolysaccharide, tumor necrosis factor, the tripeptide f-met-leu-phe and zymosan, did not induce a significant DHR reduction. Thus, the findings enable us to propose that the DHR-123 flow cytometry whole blood assay, incorporating PMA as a stimulator, would not only facilitate future studies into fish blood neutrophil research but provides a simple, rapid and reliable assay for gauging fish natural immunity status and health.
Asunto(s)
Lubina/fisiología , Citometría de Flujo/veterinaria , Inmunidad Innata , Neutrófilos/fisiología , Estallido Respiratorio/fisiología , Animales , Citometría de Flujo/métodos , Oxidación-Reducción , Rodaminas/químicaRESUMEN
PURPOSE: Diabetic retinopathy (DR) involves a proliferation of vascular endothelial cells and loss of pericytes. There is a link among the action of protein kinase C (PKC) and insulin signaling. Thus, we investigated the differences between these cells in insulin receptor (IR) phosphorylation in DR. METHODS: Retinas were removed from streptozotocin-induced diabetic or healthy rats, and IR expression levels were compared by immunoblot and immunohistochemistry. In vitro assays also were performed in order to determine the expressions of phosphorylated IR in both cells cultured under 5.5 or 25 mM glucose by immunoblot. Cell viability was determined in both cells cultured under different concentrations of phorbol myristate acetate (PMA), a PKC activator. To determine the involvement of the PI3 kinase pathway of IR, PMA with or without wortmannin-induced changes in Akt was also analyzed. RESULTS: Immunoreactivity to the IR was decreased in diabetic retina. High glucose (25 mM) increased phosphorylated IR levels in endothelial cells but not in pericytes. PMA (1 nM or higher) induced death of pericytes, while endothelial cells were increased. PMA increased phosphorylated Akt in endothelial cells and decreased in pericytes. Wortmannin suppressed the PMA-induced phosphorylation of Akt in endothelial cells. CONCLUSIONS: The different responses to 25 mM glucose and PMA were observed between retinal endothelial cells and pericytes. Thus, IR phosphorylation is likely important for retinal vascular cells to survive in diabetic retina.
Asunto(s)
Diabetes Mellitus Experimental , Retinopatía Diabética/metabolismo , Endotelio Vascular/metabolismo , Proteína Quinasa C/metabolismo , Receptor de Insulina/metabolismo , Vasos Retinianos/metabolismo , Animales , Western Blotting , Proliferación Celular , Células Cultivadas , Retinopatía Diabética/patología , Endotelio Vascular/patología , Masculino , Fosforilación , Fotomicrografía , Ratas , Ratas Wistar , Vasos Retinianos/patologíaRESUMEN
APOBEC3B (A3B) is a DNA cytosine deaminase and catalyzes cytosine deamination, resulting in mutations in genomic DNA. A3B is aberrantly expressed in a variety of cancers and considered to be a source of genomic mutations that contribute to cancer progression and metastasis. However, the mechanisms through which A3B expression is dysregulated in cancer cells are not fully elucidated. Here we report that the classical NF-κB pathway plays a crucial role in the transcriptional regulation of A3B in various cancer cells, including lymphoid malignancies. PMA, a strong activator of PKC, induces A3B at both mRNA and protein levels in cancer cell lines, and specific inhibitors of both PKC and IKK downregulate A3B expression. Using luciferase reporter and EMSA assays, we identify 3 NF-κΒ binding sites in the A3B promoter and reveal that NF-κB p65/p50 and p65/c-Rel heterodimers are important for A3B transcription. These results suggest that the classical NF-κB pathway is responsible for activation of A3B mRNA expression and further imply that inhibition of PKC and IKK might augment cancer treatment by reducing cancer progression and metastasis through downregulation of A3B expression.
Asunto(s)
Citidina Desaminasa/genética , Regulación Neoplásica de la Expresión Génica , Antígenos de Histocompatibilidad Menor/genética , FN-kappa B/metabolismo , Transducción de Señal , Sitios de Unión , Línea Celular Tumoral , Citidina Desaminasa/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Células MCF-7 , Antígenos de Histocompatibilidad Menor/metabolismo , Regiones Promotoras Genéticas , Proteína Quinasa C/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
BACKGROUND: The recent recognition that isoforms of the cellular NADPH-dependent oxidases, collectively known as the NOX protein family, participate in a wide range of physiologic and pathophysiologic processes in both the animal and plant kingdoms has stimulated interest in the identification, localization, and quantitation of their products in biological settings. Although several tools for measuring oxidants released extracellularly are available, the specificity and selectivity of the methods for reliable analysis of intracellular oxidants have not matched the enthusiasm for studying NOX proteins. SCOPE OF REVIEW: Focusing exclusively on superoxide anion and hydrogen peroxide produced by NOX proteins, this review describes the ideal probe for analysis of O2(-) and H2O2 generated extracellularly and intracellularly by NOX proteins. An overview of the components, organization, and topology of NOX proteins provides a rationale for applying specific probes for use and a context in which to interpret results and thereby construct plausible models linking NOX-derived oxidants to biological responses. The merits and shortcomings of methods currently in use to assess NOX activity are highlighted, and those assays that provide quantitation of superoxide or H2O2 are contrasted with those intended to examine spatial and temporal aspects of NOX activity. MAJOR CONCLUSIONS: Although interest in measuring the extracellular and intracellular products of the NOX protein family is great, robust analytical probes are limited. GENERAL SIGNIFICANCE: The widespread involvement of NOX proteins in many biological processes requires rigorous approaches to the detection, localization, and quantitation of the oxidants produced. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.
Asunto(s)
Peróxido de Hidrógeno/análisis , NADPH Oxidasas/metabolismo , Superóxidos/análisis , Animales , Humanos , Peróxido de Hidrógeno/metabolismo , Superóxidos/metabolismoRESUMEN
We explored the interplay between the intracellular energy sensor AMP-activated protein kinase (AMPK), extracellular signal-regulated kinase (ERK), and autophagy in phorbol myristate acetate (PMA)-induced neuronal differentiation of SH-SY5Y human neuroblastoma cells. PMA-triggered expression of neuronal markers (dopamine transporter, microtubule-associated protein 2, ß-tubulin) was associated with an autophagic response, measured by the conversion of microtubule-associated protein light chain 3 (LC3)-I to autophagosome-bound LC3-II, increase in autophagic flux, and expression of autophagy-related (Atg) proteins Atg7 and beclin-1. This coincided with the transient activation of AMPK and sustained activation of ERK. Pharmacological inhibition or RNA interference-mediated silencing of AMPK suppressed PMA-induced expression of neuronal markers, as well as ERK activation and autophagy. A selective pharmacological blockade of ERK prevented PMA-induced neuronal differentiation and autophagy induction without affecting AMPK phosphorylation. Conversely, the inhibition of autophagy downstream of AMPK/ERK, either by pharmacological agents or LC3 knockdown, promoted the expression of neuronal markers, thus indicating a role of autophagy in the suppression of PMA-induced differentiation of SH-SY5Y cells. Therefore, PMA-induced neuronal differentiation of SH-SY5Y cells depends on a complex interplay between AMPK, ERK, and autophagy, in which the stimulatory effects of AMPK/ERK signaling are counteracted by the coinciding autophagic response. Phorbol myristate acetate (PMA) induces the expression of dopamine transporter, microtubule-associated protein 2, and ß-tubulin, and subsequent neuronal differentiation of SH-SY5Y neuroblastoma cells through AMP-activated protein kinase (AMPK)-dependent activation of extracellular signal-regulated kinase (ERK). The activation of AMPK/ERK axis also induces the expression of beclin-1 and Atg7, and increases LC3 conversion, thereby triggering the autophagic response that counteracts differentiation process.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Autofagia/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Proteína 7 Relacionada con la Autofagia , Homólogo de la Proteína 1 Relacionada con la Autofagia , Línea Celular Tumoral , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Neuroblastoma/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN/fisiología , Enzimas Activadoras de Ubiquitina/metabolismoRESUMEN
BACKGROUND: Activation of ATP-gated P2X7 receptors (P2X7R) in macrophages leads to production of reactive oxygen species (ROS) by a mechanism that is partially characterized. Here we used J774 cells to identify the signaling cascade that couples ROS production to receptor stimulation. METHODS: J774 cells and mP2X7-transfected HEK293 cells were stimulated with Bz-ATP in the presence and absence of extracellular calcium. Protein inhibitors were used to evaluate the physiological role of various kinases in ROS production. In addition, phospho-antibodies against ERK1/2 and Pyk2 were used to determine activation of these two kinases. RESULTS: ROS generation in either J774 or HEK293 cells (expressing P2X7, NOX2, Rac1, p47phox and p67phox) was strictly dependent on calcium entry via P2X7R. Stimulation of P2X7R activated Pyk2 but not calmodulin. Inhibitors of MEK1/2 and c-Src abolished ERK1/2 activation and ROS production but inhibitors of PI3K and p38 MAPK had no effect on ROS generation. PKC inhibitors abolished ERK1/2 activation but barely reduced the amount of ROS produced by Bz-ATP. In agreement, the amount of ROS produced by PMA was about half of that produced by Bz-ATP. CONCLUSIONS: Purinergic stimulation resulted in calcium entry via P2X7R and subsequent activation of the PKC/c-Src/Pyk2/ERK1/2 pathway to produce ROS. This signaling mechanism did not require PI3K, p38 MAPK or calmodulin. GENERAL SIGNIFICANCE: ROS is generated in order to kill invading pathogens, thus elucidating the mechanism of ROS production in macrophages and other immune cells allow us to understand how our body copes with microbial infections.
Asunto(s)
Quinasa 2 de Adhesión Focal/metabolismo , Sistema de Señalización de MAP Quinasas , Macrófagos/metabolismo , Estrés Oxidativo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Receptores Purinérgicos P2X7/fisiología , Animales , Calcio/metabolismo , Línea Celular , Humanos , Transporte Iónico , Macrófagos/enzimología , RatonesRESUMEN
Neutrophil extracellular traps (NETs) are composed of extracellular DNA fibers with antimicrobial peptides that capture and kill microbes. NETs play a critical role in innate host defense and in autoimmune and inflammatory diseases. While the mechanism of NET formation remains unclear, reactive oxygen species (ROS) produced via activation of NADPH oxidase (Nox) are known to be an important requirement. In this study, we investigated the effect of uric acid (UA) on NET formation. UA, a well-known ROS scavenger, was found to suppress Nox-dependent ROS release in a dose-dependent manner. Low concentrations of UA significantly inhibited Nox-dependent NET formation. However, high concentrations of UA unexpectedly induced, rather than inhibited, NET formation. NETs were directly induced by UA alone in a Nox-independent manner, as revealed by experiments using control neutrophils treated with ROS inhibitors or neutrophils of patients with chronic granulomatous disease who have a congenital defect in ROS production. Furthermore, we found that UA-induced NET formation was partially mediated by NF-κB activation. Our study is the first to demonstrate the novel function of UA in NET formation and may provide insight into the management of patients with hyperuricemia.
Asunto(s)
Líquido Extracelular/inmunología , Enfermedad Granulomatosa Crónica/inmunología , NADPH Oxidasas/inmunología , Activación Neutrófila/inmunología , Neutrófilos/inmunología , Especies Reactivas de Oxígeno/inmunología , Ácido Úrico/farmacología , Adulto , Líquido Extracelular/efectos de los fármacos , Femenino , Enfermedad Granulomatosa Crónica/patología , Humanos , Masculino , Activación Neutrófila/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Neutrófilos/patología , Adulto JovenRESUMEN
Despite presence of circulating retina-specific T cells in healthy individuals, ocular immune privilege usually averts development of autoimmune uveitis. To study the breakdown of immune privilege and development of disease, we generated transgenic (Tg) mice that express a T cell receptor (TCR) specific for interphotoreceptor retinoid-binding protein (IRBP), which serves as an autoimmune target in uveitis induced by immunization. Three lines of TCR Tg mice, with different levels of expression of the transgenic R161 TCR and different proportions of IRBP-specific CD4⺠T cells in their peripheral repertoire, were successfully established. Importantly, two of the lines rapidly developed spontaneous uveitis, reaching 100% incidence by 2 and 3 months of age, respectively, whereas the third appeared "poised" and only developed appreciable disease upon immune perturbation. Susceptibility roughly paralleled expression of the R161 TCR. In all three lines, peripheral CD4⺠T cells displayed a naïve phenotype, but proliferated in vitro in response to IRBP and elicited uveitis upon adoptive transfer. In contrast, CD4⺠T cells infiltrating uveitic eyes mostly showed an effector/memory phenotype, and included Th1, Th17 as well as T regulatory cells that appeared to have been peripherally converted from conventional CD4⺠T cells rather than thymically derived. Thus, R161 mice provide a new and valuable model of spontaneous autoimmune disease that circumvents the limitations of active immunization and adjuvants, and allows to study basic mechanisms involved in maintenance and breakdown of immune homeostasis affecting immunologically privileged sites such as the eye.
Asunto(s)
Autoantígenos/inmunología , Autoinmunidad/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Retina/inmunología , Animales , Enfermedades Autoinmunes/inmunología , Linfocitos T CD4-Positivos/inmunología , Citocinas/inmunología , Proteínas del Ojo/inmunología , Humanos , Memoria Inmunológica/inmunología , Ratones , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/biosíntesis , Proteínas de Unión al Retinol/inmunología , Linfocitos T Reguladores/inmunología , Células TH1/inmunología , Células Th17/inmunología , Uveítis/inmunologíaRESUMEN
LPA1 internalization to endosomes was studied employing Förster Resonance Energy Transfer (FRET) in cells coexpressing the mCherry-lysophosphatidic acid LPA1 receptors and distinct eGFP-tagged Rab proteins. Lysophosphatidic acid (LPA)-induced internalization was rapid and decreased afterward: phorbol myristate acetate (PMA) action was slower and sustained. LPA stimulated LPA1-Rab5 interaction rapidly but transiently, whereas PMA action was rapid but sustained. Expression of a Rab5 dominant-negative mutant blocked LPA1-Rab5 interaction and receptor internalization. LPA-induced LPA1-Rab9 interaction was only observed at 60 min, and LPA1-Rab7 interaction after 5 min with LPA and after 60 min with PMA. LPA triggered immediate but transient rapid recycling (i.e., LPA1-Rab4 interaction), whereas PMA action was slower but sustained. Agonist-induced slow recycling (LPA1-Rab11 interaction) increased at 15 min and remained at this level, whereas PMA action showed early and late peaks. Our results indicate that LPA1 receptor internalization varies with the stimuli.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , Receptores del Ácido Lisofosfatídico , Receptores del Ácido Lisofosfatídico/metabolismo , Fosforilación , Acetato de Tetradecanoilforbol/farmacología , Endosomas/metabolismo , Lisofosfolípidos/farmacología , Lisofosfolípidos/metabolismoRESUMEN
OBJECTIVE: Oral lichen planus (OLP) is a T cell-mediated inflammatory condition in the oral cavity. Mucosal-associated invariant T (MAIT) cells are gaining more relevance in immune diseases because they can be activated by cytokines without T cell receptor stimulation. Herein, we tested the effect of interleukin-23 (IL-23) on the activation status of OLP MAIT cells. METHODS: Peripheral blood mononuclear cells (PBMCs) isolated from OLP patients were stimulated by IL-23 in the absence or presence of phorbol myristate acetate (PMA) and ionomycin. The activation status of MAIT cells was analyzed by flow cytometry after staining for CD3, CD4, CD8, CD161, TCR Vα7.2, and CD69. RESULTS: The fraction of MAIT cells in OLP peripheral blood was approximately 0.38% to 3.97%, and CD8+ subpopulations overwhelmed CD4+ cells. The mean percentages of OLP MAIT cells in PBMCs and CD8+MAIT cells in MAIT cells were approximately 40%. PMA and ionomycin significantly increased CD69 expression on OLP T cells, MAIT cells, and CD8+MAIT cells. Cells with enhanced activation had different responsiveness to exogenous IL-23, showing increased CD69 expression on OLP T cells, decreased CD69 on OLP CD8+MAIT cells, and no significant change on OLP MAIT cells. CONCLUSIONS: IL-23 showed different effects on the activation status of OLP MAIT cells and CD8+MAIT cells.
Asunto(s)
Interleucina-23 , Liquen Plano Oral , Células T Invariantes Asociadas a Mucosa , Humanos , Interleucina-23/farmacología , Ionomicina/farmacología , Leucocitos Mononucleares , Liquen Plano Oral/metabolismo , Células T Invariantes Asociadas a Mucosa/metabolismoRESUMEN
The respiratory burst is a rapid cellular consumption of oxygen resulting in abundant production of reactive oxygen species (ROS), most often associated with primary mediators of innate immunity, neutrophils and macrophages. These myeloid cells convert ROS into potent antimicrobial oxidants that efficiently kill pathogens. The respiratory burst also can have destructive consequences, as ROS are well known to support chronic inflammation and aberrant autoimmune responses. Interestingly, ROS perform conflicting roles in the tumor microenvironment; ROS and derived cytotoxic products can destroy cancer cells but also suppress important tumor-fighting functions of T cells or natural killer cells, or yield mutagenized proteins that can promote tumorigenesis or support tumor cell growth. Moreover, high numbers of neutrophils or macrophages in tumors are associated with poor prognosis. Therefore, accurate and quantitative assays to assess the respiratory burst are an important tool for measuring ROS production by neutrophils or cells of the monocyte/macrophage system, each recently identified in the tumor microenvironment. Described are methods to derive mouse or human models of neutrophils or macrophages, which are then used in a detailed assay to quantitatively measure ROS produced by either cell type using luminescence-enhanced reagents and a multi-well platform along with different stimulants that cause rapid ROS production.
Asunto(s)
Neutrófilos , Estallido Respiratorio , Ratones , Humanos , Animales , Especies Reactivas de Oxígeno/metabolismo , Neutrófilos/metabolismo , Macrófagos/metabolismo , Inmunidad InnataRESUMEN
Glycogen Storage Disease type 1b (GSDIb) is a genetic disorder with long term severe complications. Accumulation of the glucose analog 1,5-anhydroglucitol-6-phosphate (1,5AG6P) in neutrophils inhibits the phosphorylation of glucose in these cells, causing neutropenia and neutrophil dysfunctions. This condition leads to serious infections and inflammatory bowel disease (IBD) in GSDIb patients. We show here that dapagliflozin, an inhibitor of the renal sodium-glucose co-transporter-2 (SGLT2), improves neutrophil function in an inducible mouse model of GSDIb by reducing 1,5AG6P accumulation in myeloid cells.
RESUMEN
BACKGROUND & AIMS: Virus-specific T cell dysfunction is a common feature of HBV-related hepatocellular carcinoma (HBV-HCC). Conventional T (ConT) cells can be redirected towards viral antigens in HBV-HCC when they express an HBV-specific receptor; however, their efficacy can be impaired by liver-specific physical and metabolic features. Mucosal-associated invariant T (MAIT) cells are the most abundant innate-like T cells in the liver and can elicit potent intrahepatic effector functions. Here, we engineered ConT and MAIT cells to kill HBV expressing hepatoma cells and compared their functional properties. METHODS: Donor-matched ConT and MAIT cells were engineered to express an HBV-specific T cell receptor (TCR). Cytotoxicity and hepatocyte homing potential were investigated using flow cytometry, real-time killing assays, and confocal microscopy in 2D and 3D HBV-HCC cell models. Major histocompatibility complex (MHC) class I-related molecule (MR1)-dependent and MR1-independent activation was evaluated in an Escherichia coli THP-1 cell model and by IL-12/IL-18 stimulation, respectively. RESULTS: HBV TCR-MAIT cells demonstrated polyfunctional properties (CD107a, interferon [IFN] γ, tumour necrosis factor [TNF], and IL-17A) with strong HBV target sensitivity and liver-homing chemokine receptor expression when compared with HBV TCR-ConT cells. TCR-mediated lysis of hepatoma cells was comparable between the cell types and augmented in the presence of inflammation. Coculturing with HBV+ target cells in a 3D microdevice mimicking aspects of the liver microenvironment demonstrated that TCR-MAIT cells migrate readily towards hepatoma targets. Expression of an ectopic TCR did not affect the ability of the MAIT cells to be activated via MR1-presented bacterial antigens or IL-12/IL-18 stimulation. CONCLUSIONS: HBV TCR-MAIT cells demonstrate anti-HBV functions without losing their endogenous antimicrobial mechanisms or hepatotropic features. Our results support future exploitations of MAIT cells for liver-directed immunotherapies. LAY SUMMARY: Chronic HBV infection is a leading cause of liver cancer. T cell receptor (TCR)-engineered T cells are patients' immune cells that have been modified to recognise virus-infected and/or cancer cells. Herein, we evaluated whether mucosal-associated invariant T cells, a large population of unconventional T cells in the liver, could recognise and kill HBV infected hepatocytes when engineered with an HBV-specific TCR. We show that their effector functions may exceed those of conventional T cells currently used in the clinic, including antimicrobial properties and chemokine receptor profiles better suited for targeting liver tumours.
RESUMEN
Aberrant activation of NLRP3 inflammasome in colonic macrophages strongly associates with the occurrence and progression of ulcerative colitis. Although targeting NLRP3 inflammasome has been considered to be a potential therapy, the underlying mechanism through which pathway the intestinal inflammation is modulated remains controversial. By focusing on the flavonoid lonicerin, one of the most abundant constituents existed in a long historical anti-inflammatory and anti-infectious herb Lonicera japonica Thunb., here we report its therapeutic effect on intestinal inflammation by binding directly to enhancer of zeste homolog 2 (EZH2) histone methyltransferase. EZH2-mediated modification of H3K27me3 promotes the expression of autophagy-related protein 5, which in turn leads to enhanced autophagy and accelerates autolysosome-mediated NLRP3 degradation. Mutations of EZH2 residues (His129 and Arg685) indicated by the dynamic simulation study have found to greatly diminish the protective effect of lonicerin. More importantly, in vivo studies verify that lonicerin dose-dependently disrupts the NLRP3-ASC-pro-caspase-1 complex assembly and alleviates colitis, which is compromised by administration of EZH2 overexpression plasmid. Thus, these findings together put forth the stage for further considering lonicerin as an anti-inflammatory epigenetic agent and suggesting EZH2/ATG5/NLRP3 axis may serve as a novel strategy to prevent ulcerative colitis as well as other inflammatory diseases.
RESUMEN
Two critical functional responses of neutrophils are chemotaxis, a response driven by concentration gradients of chemokines released by infected or inflamed tissues, and production of reactive oxygen species (ROS), molecules essential to their capacity to kill pathogens. Assays to accurately test each response have been important to assess efficacies of pharmaceuticals predicted to block recruitment of neutrophils or attenuate their ROS production. Identified antagonists to neutrophil functions may help to reduce tissue damage following inflammation. Described are detailed assays to test these functions, along with steps to generate neutrophils from ex vivo-cultured murine bone marrow that produce robust responses in either assay. The first function protocol details a quantitative assay for chemotaxis that involves culture plates with dual chamber wells that separate cells from a chemokine with small pore-sized membranes. Quantitative measurements of cell numbers in the chemokine-containing chamber are performed with either fluorescence or luminescence detection reagents, which provide signals directly proportional to the numbers of migrated cells. Multiwell plates are used for rapidly testing a variety of conditions and/or chemoattractants. Described in the second function protocol is an assay to measure ROS produced by stimulated neutrophils, again using a multiwell platform for rapid, quantitative measurements of several conditions simultaneously.