RESUMEN
Glomerulonephritis is an acquired serious glomerular disease, which involves the interplay of many factors such as cytokines, chemokines, inflammatory cells, and heparan sulfate (HS). We previously showed that blocking of inflammatory heparan sulfate domains on cultured glomerular endothelium by specific anti-HS single chain antibodies reduced polymorphonuclear cell (PMN) adhesion and chemokine binding. We hypothesized that injection of anti-HS antibodies in PMN-driven experimental glomerulonephritis should reduce glomerular influx of PMNs and thereby lead to a better renal outcome. In contrast to our hypothesis, co-injection of anti-HS antibodies did not alter the final outcome of anti-glomerular basement membrane (anti-GBM)-induced glomerulonephritis. Glomerular PMN influx, normally peaking 2 hours after induction of glomerulonephritis with anti-GBM IgG was not reduced by co-injection of anti-HS antibodies. Four days after induction of glomerulonephritis, albuminuria, renal function, glomerular hyalinosis and fibrin deposition were similar in mice treated and not treated with anti-HS antibodies. Interestingly, we observed transient effects in mice co-injected with anti-HS antibodies compared to mice that did not receive anti-HS antibodies: (i) a decreased renal function 2 hours and 1 day after induction of glomerulonephritis; (ii) an increased albuminuria after 2 hours and 1 day; (iii) an increased glomerular fibrin deposition after 1 day; (iv) a reduced glomerular macrophage influx after 1 day; (v) a sustained glomerular presence of PMNs at day 1 and 4, accompanied by an increased renal expression of IL-6, CXCL1, ICAM-1, L-selectin, CD11b and NF-κB. The mechanism underlying these observations induced by anti-HS antibodies remains unclear, but may be explained by a temporarily altered glycocalyx and/or altered function of PMNs due to the binding of anti-HS antibodies. Nevertheless, the evaluated anti-HS antibodies do not show therapeutic potential in anti-GBM-induced glomerulonephritis. Future research should evaluate other strategies to target HS domains involved in inflammatory processes during glomerulonephritis.
Asunto(s)
Glomerulonefritis/metabolismo , Glomérulos Renales/metabolismo , Anticuerpos de Cadena Única/farmacología , Animales , Antígeno CD11b/biosíntesis , Quimiocina CXCL1/biosíntesis , Fibrina/metabolismo , Regulación de la Expresión Génica , Glomerulonefritis/patología , Glomerulonefritis/prevención & control , Heparitina Sulfato , Molécula 1 de Adhesión Intercelular/biosíntesis , Interleucina-6/biosíntesis , Glomérulos Renales/patología , Selectina L/biosíntesis , RatonesRESUMEN
Chronic psychosocial stress is a risk factor for the development of numerous disorders, of which most are associated with chronic low-grade inflammation. Given the immunosuppressive effects of glucocorticoids (GC), one underlying mechanism might be the development of stress-induced GC resistance in certain immune cell subpopulations. In line with this hypothesis, male mice exposed to the chronic subordinate colony housing (CSC, 19 days) model develop GC resistance of in vitro lipopolysaccharide (LPS)-stimulated splenocytes, splenomegaly and an increased percentage of splenic CD11b+ cells. Here male C57BL/6N mice were euthanized at different days during CSC, and following 30 days of single housing after stressor termination to assess when CSC-induced splenic GC resistance starts to develop and whether this is a transient effect. Moreover, splenic CD11b, GC receptor (GR) and/or macrophage migration inhibiting factor (MIF) protein levels were quantified at respective days. While mild forms of CSC-induced GC resistance, increased splenic CD11b expression and/or splenomegaly were detectable on days 8 and 9 of CSC, more severe forms took until days 15 and 16 to develop, but normalized almost completely within 30 days following stressor termination (day 51). In contrast, splenic GR expression was decreased in CSC versus single-housed control (SHC) mice at all days assessed. While MIF expression was increased on days 15 and 16 of CSC, it was decreased in CSC versus SHC mice on day 20 despite persisting splenomegaly, increased CD11b expression and functional GC resistance. In summary, our data indicate that GC resistance and CD11b+ cell-mediated splenomegaly develop gradually and in parallel over time during CSC exposure and are transient in nature. Moreover, while we can exclude that CSC-induced reduction in splenic GR expression is sufficient to induce functional GC resistance, the role of MIF in CD11b+ cell-mediated splenomegaly and GC resistance requires further investigation.
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Cortisona/farmacología , Glucocorticoides/farmacología , Leucocitos/fisiología , Bazo/citología , Estrés Psicológico/inmunología , Conducta Agonística , Animales , Mordeduras y Picaduras , Antígeno CD11b/biosíntesis , Antígeno CD11b/genética , Enfermedad Crónica , Cortisona/sangre , Aglomeración , Resistencia a Medicamentos , Vivienda para Animales , Oxidorreductasas Intramoleculares/biosíntesis , Oxidorreductasas Intramoleculares/genética , Leucocitos/efectos de los fármacos , Lipopolisacáridos/farmacología , Factores Inhibidores de la Migración de Macrófagos/biosíntesis , Factores Inhibidores de la Migración de Macrófagos/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Tamaño de los Órganos , Receptores de Glucocorticoides/biosíntesis , Receptores de Glucocorticoides/genética , Bazo/patología , TerritorialidadRESUMEN
The parenchymal microglia possess different morphological characteristics in cerebral physiological and pathological conditions; thus, visualizing these cells is useful as a means of further investigating parenchymal microglial function. Annexin A3 (ANXA3) is expressed in microglia, but it is unknown whether it can be used as a marker protein for microglia and its physiological function. Here, we compared the distribution and morphology of parenchymal microglia labeled by ANXA3, cluster of differentiation 11b (CD11b), and ionized calcium-binding adaptor molecule 1 (Iba1) and measured the expression of ANXA3 in nonparenchymal macrophages (meningeal and perivascular macrophages). We also investigated the spatiotemporal expression of ANXA3, CD11b, and Iba1 in vivo and in vitro and the cellular function of ANXA3 in microglia. We demonstrated that ANXA3-positive cells were abundant and evenly distributed throughout the whole brain tissue and spinal cord of adult rats. The morphology and distribution of ANXA3-labeled microglia were quite similar to those labeled by the microglial-specific markers CD11b and Iba1 in the central nervous system (CNS). ANXA3 was expressed in the cytoplasm of microglia, and its expression was significantly increased in activated microglia. ANXA3 was almost undetectable in the nonparenchymal macrophages. Meanwhile, the protein and mRNA expression levels of ANXA3 in different regions of the CNS were different from those of CD11b and Iba1. Moreover, knockdown of ANXA3 inhibited the proliferation and migration of microglia, while overexpression of ANXA3 enhanced these activities. This study confirms that ANXA3 may be a novel marker for parenchymal microglia in the CNS of adult rats and enriches our understanding of ANXA3 from expression patterns to physiological function.
Asunto(s)
Anexina A3/análisis , Sistema Nervioso Central/citología , Microglía/química , Proteínas del Tejido Nervioso/análisis , Animales , Anexina A3/biosíntesis , Anexina A3/genética , Biomarcadores , Antígeno CD11b/biosíntesis , Antígeno CD11b/genética , Proteínas de Unión al Calcio/biosíntesis , Proteínas de Unión al Calcio/genética , Ciclo Celular , Movimiento Celular , Células Cultivadas , Técnicas de Silenciamiento del Gen , Vectores Genéticos , Infarto de la Arteria Cerebral Media/patología , Lentivirus , Macrófagos/química , Proteínas de Microfilamentos/biosíntesis , Proteínas de Microfilamentos/genética , Especificidad de Órganos , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , TransfecciónRESUMEN
Background Integrin αM (CD11b), which is encoded by the Integrin Subunit Alpha M (ITGAM) gene, is not only a surface marker of monocytes but also an essential adhesion molecule. In this study, we investigated the effect of CD11b on experimental abdominal aortic aneurysm and the potential underlying mechanisms. Methods and Results The incidence of abdominal aortic aneurysm was not significantly lower in ITGAM(-/-) mice than in control mice. Nevertheless, knockout of CD11b reduced the maximum abdominal aortic diameter, macrophage infiltration, matrix metalloproteinase-9 expression, and elastin and collagen degradation. Additionally, lower expression of IL-6 was found in both the peripheral blood and abdominal aortas of ITGAM(-/-) mice, indicating a biological correlation between CD11b and the inflammatory response in abdominal aortic aneurysm. In vitro, the number of ITGAM(-/-) bone marrow-derived macrophages (BMDMs) that adhered to endothelial cells was significantly lower than the number of wild-type BMDMs. Moreover, the CD11b monoclonal antibody and CD11b agonist leukadherin-1 decreased and increased the number of adherent wild-type BMDMs, respectively. Through RNA sequencing, genes associated with leukocyte transendothelial migration were found to be downregulated in ITGAM(-/-) BMDMs. Furthermore, immunoprecipitation-mass spectrometry analysis predicted that the Akt pathway might be responsible for the impaired transmigratory ability of ITGAM(-/-) BMDMs. The reduced activation of Akt was then confirmed, and the Akt agonist SC79 partially rescued the transendothelial migratory function of ITGAM(-/-) BMDMs. Conclusions CD11b might promote the development and progression of abdominal aortic aneurysm by mediating the endothelial cells adhesion and transendothelial migration of circulating monocytes/macrophages.
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Aorta Abdominal/metabolismo , Aneurisma de la Aorta Abdominal/genética , Antígeno CD11b/genética , Regulación de la Expresión Génica , ARN/genética , Animales , Aorta Abdominal/patología , Aneurisma de la Aorta Abdominal/metabolismo , Aneurisma de la Aorta Abdominal/patología , Antígeno CD11b/biosíntesis , Células Cultivadas , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
γδ T cells are a numerically significant subset of immune cells in ruminants, where they may comprise up to 70 % of all peripheral blood mononuclear cells (PBMCs) in young animals and 25 % in adults. These cells can be activated through traditional TCR-dependent mechanisms, or alternatively in a TCR-independent manner by pattern recognition receptors and have been shown to uptake antigen, as well as process and present it to αß T cells. We have identified a novel CD11b+ subset of γδ T cells in normal sheep peripheral blood. An increase in the frequency of these cells in sheep peripheral blood in response to immunization with an experimental recombinant subunit Rift Valley fever (RVF) vaccine was observed. However, injection of the vaccine adjuvant ISA-25VG alone without the recombinant RVF virus antigens demonstrated the same effect, pointing to an antigen-independent innate immune function of CD11b+ γδ T cells in response to the adjuvant. In vitro studies showed repeatable increases of CD11b-, CD14-, CD86-, CD40-, CD72-, and IFNγ- expressing γδ T cells in PBMCs after 24 h of incubation in the absence of a mitogen. Moreover, the majority of these myeloid-like γδ T cells were demonstrated to process exogenous antigen even in the absence of mitogen. ConA activation increased CD25- and MHCII- expression in γδ T cells, but not the myeloid associated receptors CD14 or CD11b or co-stimulatory molecules such as CD86 and CD40. Considering the role of CD11b and CD14 in the activation of innate immunity, we hypothesize that this subpopulation of sheep γδ T cells may function as innate antigen presenting and pro-inflammatory cells during immune responses. The results presented here also suggest that stress molecules and/or damage-associated molecular patterns may be involved in triggering antigen presenting and pro-inflammatory functions of γδ T cells, given their appearance in vitro in the absence of specific stimulation. Taken together, these data suggest that the early appearance of γδ T cells following adjuvant administration and their possible role in early activation of αß T cell subsets may non-specifically contribute to augmented innate immunity and may promote strong initiation of the adaptive immune response to vaccines in general.
Asunto(s)
Linfocitos Intraepiteliales/inmunología , Fiebre del Valle del Rift/inmunología , Vacunas Virales/inmunología , Animales , Presentación de Antígeno , Antígenos de Diferenciación/biosíntesis , Antígeno CD11b/biosíntesis , Proliferación Celular , Células Cultivadas , Femenino , Inmunidad Innata , Inmunogenicidad Vacunal , Inmunofenotipificación , Subunidad alfa del Receptor de Interleucina-2/biosíntesis , Células Mieloides/inmunología , Fiebre del Valle del Rift/prevención & control , OvinosRESUMEN
Immune system components also regulate synapse formation and refinement in neurodevelopment. The complement pathway, associated with cell lysis and phagocytosis, is implicated in synaptic elimination. Aberrant adolescent synaptic pruning may underpin schizophrenia onset; thus, changes in cortical complement activity during human development are of major interest. Complement is genetically linked to schizophrenia via increased C4 copy number variants, but the developmental trajectory of complement expression in the human brain is undetermined. As complement increases during periods of active synaptic engulfment in rodents, we hypothesized that complement expression would increase during postnatal development in humans, particularly during adolescence. Using human postmortem prefrontal cortex, we observed that complement activator (C1QB and C3) transcripts peaked in early neurodevelopment, and were highest in toddlers, declining in teenagers (all ANCOVAs between F = 2.41 -3.325, p = .01-0.05). We found that C4 protein was higher at 1-5 years (H = 16.378, p = .012), whereas C3 protein levels were unchanged with age. The microglial complement receptor subunit CD11b increased in mRNA early in life and peaked in the toddler brain (ANCOVA: pH, F = 4.186, p = .003). Complement inhibitors (CD46 and CD55) increased at school age, but failed to decrease like complement activators (both ANCOVAs, F > 4.4, p < .01). These data suggest the activation of complement in the human prefrontal cortex occurs between 1 and 5 years. We did not find evidence of induction of complement factors during adolescence and instead found increased or sustained levels of complement inhibitor mRNA at maturation. Dysregulation of these typical patterns of complement may predispose the brain to neurodevelopmental disorders such as autism or schizophrenia.
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Envejecimiento/metabolismo , Química Encefálica/fisiología , Encéfalo/crecimiento & desarrollo , Proteínas del Sistema Complemento/metabolismo , Adolescente , Adulto , Antígeno CD11b/biosíntesis , Antígeno CD11b/genética , Antígeno CD56/genética , Antígeno CD56/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Niño , Preescolar , Vía Clásica del Complemento/genética , ADN/biosíntesis , ADN/genética , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Lactante , Recién Nacido , Masculino , Proteína Cofactora de Membrana/genética , Proteína Cofactora de Membrana/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Corteza Prefrontal/crecimiento & desarrollo , Corteza Prefrontal/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Adulto JovenRESUMEN
Understanding the molecular mechanisms of graphene oxide (GO)-based biomaterials is important for logical biomedical applications. Previous studies have revealed biointeractions between GO and immune effector cells, but the effects on neutrophils, crucial cells in the immune system, have not been thoroughly discussed. In this study, GO nanoformulations were synthesized with different functional groups, including GO, GO-carboxylated (GO-COOH), and PEGylated GO (GO-PEG), with different surface features, which were elucidated using imaging methods and surface-sensitive quantitative spectroscopic techniques, including atomic force microscopy (AFM), transmission electron microscopy (TEM), and X-ray photoemission spectroscopy (XPS). The GO-based nanoformulations elicited reactive oxygen species (ROS) generation and neutrophil extracellular trap (NET) formation in human neutrophils. Nanoformulated GO stimulates NET development via the formation of ROS. An endocytosis study revealed that nanoformulated GO facilitated internalization by neutrophils via macropinocytosis and actin-dependent phagocytosis. Importantly, calcium mobilization and phosphorylation proteins such as mitogen-activated protein kinases (extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38) and AKT were involved in the activation of neutrophils. These findings offer the first verification that nanoformulated GO exhibits direct effects on human neutrophils.
Asunto(s)
Materiales Biocompatibles/farmacología , Grafito/farmacología , Nanopartículas/química , Neutrófilos/efectos de los fármacos , Adulto , Materiales Biocompatibles/síntesis química , Materiales Biocompatibles/química , Antígeno CD11b/biosíntesis , Grafito/síntesis química , Grafito/química , Humanos , Activación Neutrófila/efectos de los fármacos , Activación Neutrófila/inmunología , Neutrófilos/inmunología , Tamaño de la Partícula , Especies Reactivas de Oxígeno/inmunología , Propiedades de Superficie , Adulto JovenRESUMEN
BACKGROUND AND AIMS: Atherosclerosis is characterized by lipid deposition, monocyte infiltration and foam cell formation in the artery wall. Translocator protein (TSPO) is abundantly expressed in lipid rich tissues. Recently, TSPO has been identified as a potential diagnostic tool in cardiovascular disease. The purpose of this study was to determine if the TSPO ligand, 18F-PBR111, can identify early atherosclerotic lesions and if TSPO expression can be used to identify distinct macrophage populations during lesion progression. METHODS: ApoE-/- mice were maintained on a high-fat diet for 3 or 12 weeks. C57BL/6J mice maintained on chow diet served as controls. Mice were administered 18F-PBR111 intravenously and PET/CT imaged. After euthanasia, aortas were isolated, fixed and optically cleared. Cleared aortas were immunostained with DAPI, and fluorescently labelled with antibodies to TSPO, the tissue resident macrophage marker F4/80 and the monocyte-derived macrophage marker CD11b. TSPO expression and the macrophage markers were visualised in fatty streaks and established plaques by light sheet microscopy. RESULTS: While tissue resident F4/80 + macrophages were evident in the arteries of animals without atherosclerosis, no CD11b + macrophages were observed in these animals. In contrast, established plaques had high CD11b and low F4/80 expression. A â¼3-fold increase in the uptake of 18F-PBR111 was observed in the aortas of atherosclerotic mice relative to controls. CONCLUSIONS: Imaging of TSPO expression is a new approach for studying atherosclerotic lesion progression and inflammatory cell infiltration. The TSPO ligand, 18F-PBR111, is a potential clinical diagnostic tool for the detection and quantification of atherosclerotic lesion progression in humans.
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Aterosclerosis/sangre , Aterosclerosis/diagnóstico , Antígeno CD11b/fisiología , Macrófagos , Receptores de GABA/fisiología , Animales , Antígeno CD11b/biosíntesis , Progresión de la Enfermedad , Diagnóstico Precoz , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Piridinas/administración & dosificación , Receptores de GABA/biosíntesisRESUMEN
Macranthol is a lignans natural product isolated from Illicium dunnianum Tutch. Our previous studies have shown that BDNF dependent signaling pathway activation was involved in the antidepressant-like effects of macranthol. However, it is not clear whether neuro-inflammation suppression is involved in the effects of macranthol. Therefore, the aim of this present study was to determine whether macranthol affected the neuro-inflammation system in lipopolysaccharide (LPS)-induced mice by measuring pro-inflammatory cytokines and CD11b. Macranthol was orally administrated for successive seven days before a single LPS injection. The behavioral evaluation showed that macranthol prevented LPS-induced depressive-like deficits both in sucrose preference test and forced swimming test. The elevation of serum and prefrontal cortex pro-inflammatory cytokines including interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) was decreased by macranthol pretreatment. In addition, LPS induced the elevation of CD11b in the prefrontal cortex, which was also inhibited by macranthol. Last but not the least, the immunofluorescence found that the number of positive iba-1 cells was also decreased by macranthol. These findings suggest that macranthol could alleviate depressive-like behaviors in mice induced by LPS that are mediated, at least by suppressing microglia-related neuro-inflammation in the prefrontal cortex.
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Alquenos/farmacología , Depresión/prevención & control , Encefalitis/prevención & control , Lipopolisacáridos/antagonistas & inhibidores , Fenoles/farmacología , Corteza Prefrontal/efectos de los fármacos , Animales , Peso Corporal/efectos de los fármacos , Antígeno CD11b/biosíntesis , Antígeno CD11b/genética , Proteínas de Unión al Calcio/metabolismo , Línea Celular , Citocinas/biosíntesis , Depresión/psicología , Encefalitis/psicología , Illicium/química , Lipopolisacáridos/toxicidad , Masculino , Ratones , Ratones Endogámicos ICR , Proteínas de Microfilamentos/metabolismo , Actividad Motora/efectos de los fármacos , Sacarosa/farmacología , Natación/psicologíaRESUMEN
PURPOSE OF REVIEW: The aim of this review is to discuss recent developments in our understanding of how systemic lupus erythematosus (SLE)-associated genes contribute to autoimmunity. RECENT FINDINGS: Gene-function studies have revealed mechanisms through which SLE-associated alleles of IFIH1, TNFAIP3, IRF5, and PRDM1 likely contribute to the development of autoimmunity. Novel research has identified Mac-1 (encoded by ITGAM), CaMK4, and iRhom2 as plausible therapeutic targets in lupus nephritis. SUMMARY: The work discussed in this review has broad implications for our understanding of the pathogenesis of SLE and for the development of novel therapeutic strategies.
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Antígeno CD11b/genética , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Terapia Genética/métodos , Lupus Eritematoso Sistémico/genética , ARN/genética , Antígeno CD11b/biosíntesis , Humanos , Lupus Eritematoso Sistémico/metabolismo , Lupus Eritematoso Sistémico/terapiaRESUMEN
Interleukin-17 receptor B (IL-17RB), a member of the IL-17 receptor family activated by IL-17B/IL-17E, has been shown to be involved in inflammatory diseases. However, the regulation and function of IL-17RB in Helicobacter pylori (H. pylori) infection, especially in the early-phase is still unknown. Here, we found that gastric IL-17RB mRNA and protein were decreased in gastric mucosa of both patients and mice infected with H. pylori. In vitro experiments show that IL-17RB expression was down regulated via PI3K/AKT pathway on gastric epithelial cells (GECs) stimulated with H. pylori in a cagA-involved manner, while in vivo studies showed that the effect was partially dependent on cagA expression. IL-17E was also decreased during the early-phase of H. pylori infection, and provision of exogenous IL-17E resulted in increased CD11b+CD11c- myeloid cells accumulation and decreased bacteria colonization within the gastric mucosa. In the early-phase of H. pylori infection, IL-17E-IL-17RB promoted gastric epithelial cell-derived CXCL1/2/5/6 to attract CD11b+CD11c- myeloid cells, and also contributed to host defense by promoting the production of antibacterial protein Reg3a. This study defines a negative regulatory network involving IL-17E, GECs, IL-17RB, CD11b+CD11c- myeloid cells, and Reg3a in the early-phase of H. pylori infection, which results in an impaired host defense within the gastric microenvironment, suggesting IL-17RB as a potential early intervening target in H. pylori infection.
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Antígeno CD11b/inmunología , Antígeno CD11c/inmunología , Mucosa Gástrica/inmunología , Infecciones por Helicobacter/inmunología , Helicobacter pylori/aislamiento & purificación , Células Mieloides/inmunología , Receptores de Interleucina-17/inmunología , Animales , Antígenos CD11/biosíntesis , Antígenos CD11/inmunología , Antígeno CD11b/biosíntesis , Antígeno CD11b/sangre , Antígeno CD11c/biosíntesis , Infecciones por Helicobacter/sangre , Infecciones por Helicobacter/genética , Helicobacter pylori/genética , Humanos , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genética , ARN Mensajero/inmunología , Receptores de Interleucina-17/biosíntesis , Receptores de Interleucina-17/genéticaRESUMEN
Clinical studies indicate that psychosocial stress contributes to adverse chronic pain outcomes in patients, but it is unclear how this is initiated or amplified by stress. Repeated social defeat (RSD) is a mouse model of psychosocial stress that activates microglia, increases neuroinflammatory signaling, and augments pain and anxiety-like behaviors. We hypothesized that activated microglia within the spinal cord facilitate increased pain sensitivity following RSD. Here we show that mechanical allodynia in male mice was increased with exposure to RSD. This stress-induced behavior corresponded with increased mRNA expression of several inflammatory genes, including IL-1ß, TNF-α, CCL2, and TLR4 in the lumbar spinal cord. While there were several adhesion and chemokine-related genes increased in the lumbar spinal cord after RSD, there was no accumulation of monocytes or neutrophils. Notably, there was evidence of microglial activation selectively within the nociceptive neurocircuitry of the dorsal horn of the lumbar cord. Elimination of microglia using the colony stimulating factor 1 receptor antagonist PLX5622 from the brain and spinal cord prevented the development of mechanical allodynia in RSD-exposed mice. Microglial elimination also attenuated RSD-induced IL-1ß, CCR2, and TLR4 mRNA expression in the lumbar spinal cord. Together, RSD-induced allodynia was associated with microglia-mediated inflammation within the dorsal horn of the lumbar spinal cord.SIGNIFICANCE STATEMENT Mounting evidence indicates that psychological stress contributes to the onset and progression of adverse nociceptive conditions. We show here that repeated social defeat stress causes increased pain sensitivity due to inflammatory signaling within the nociceptive circuits of the spinal cord. Studies here mechanistically tested the role of microglia in the development of pain by stress. Pharmacological ablation of microglia prevented stress-induced pain sensitivity. These findings demonstrate that microglia are critical mediators in the induction of pain conditions by stress. Moreover, these studies provide a proof of principle that microglia can be targeted as a therapeutic strategy to mitigate adverse pain conditions.
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Dolor Crónico/fisiopatología , Dolor Crónico/psicología , Inflamación/psicología , Microglía , Medio Social , Enfermedades de la Médula Espinal/psicología , Estrés Psicológico/psicología , Animales , Ansiedad/psicología , Conducta Animal , Antígeno CD11b/biosíntesis , Antígeno CD11b/genética , Dolor Crónico/genética , Regulación de la Expresión Génica/genética , Hiperalgesia/fisiopatología , Hiperalgesia/psicología , Inflamación/genética , Inflamación/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Compuestos Orgánicos/farmacología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/antagonistas & inhibidores , Médula Espinal , Enfermedades de la Médula Espinal/genética , Enfermedades de la Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal , Estrés Psicológico/genéticaRESUMEN
Acetate, an agonist for the free fatty acid receptor 2 (FFA2R/GPR43), triggers an increase in the cytosolic concentration of free Ca2+ in neutrophils without any assembly of the superoxide generating NADPH-oxidase. We show that the phenylacetamide compound 58 (Cmp 58; (S)-2-(4-chlorophenyl)-3,3-dimethyl-N-(5-phenylthiazol-2-yl)butanamide), lacking a direct activating effect on neutrophils, acts as a positive FFA2R modulator that turns acetate into a potent activating agonist that triggers an assembly of the NADPH-oxidase. The NADPH-oxidase activity could be further increased in neutrophils treated with the pro-inflammatory cytokine TNF-α. Many neutrophil chemoattractant receptors are stored in secretory organelles but no FFA2R mobilization was induced in neutrophils treated with TNF-α. The receptor selectivity was demonstrated through the inhibition of the neutrophil response induced by the combined action of acetate and Cmp 58 by the FFA2R antagonist CATPB. Receptor modulators that positively co-operate with natural FFA2R agonists and prime neutrophils in their response to such agonists, may serve as good tools for further unraveling the physiological functions of FFA2R and its involvement in various diseases. In this study, we show that neutrophils primed with a presumed allosteric FFA2R modulator produce increased amounts of reactive oxygen species when activated by receptor specific agonists.
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Acetanilidas/farmacología , NADPH Oxidasas/metabolismo , Neutrófilos/efectos de los fármacos , Receptores de Superficie Celular/agonistas , Superóxidos/metabolismo , Tiazoles/farmacología , Acetatos/farmacología , Regulación Alostérica , Antígeno CD11b/biosíntesis , Antígeno CD11b/genética , Señalización del Calcio , Células Cultivadas , Ciclopropanos/farmacología , Activación Enzimática/efectos de los fármacos , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/antagonistas & inhibidores , Humanos , Neutrófilos/enzimología , Oligopéptidos/farmacología , Péptidos Cíclicos/farmacología , Toxina del Pertussis/farmacología , Receptores Acoplados a Proteínas G/fisiología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
AIMS: Painful diabetic neuropathy (PDN) is one of the most frequent complications of diabetes and the current therapies have limited efficacy. This study aimed to study the neuroprotective effect of duloxetine, a serotonin noradrenaline reuptake inhibitor (SNRI), in a mouse model of diabetic neuropathy. MAIN METHODS: Nine weeks after developing of PDN, mice were treated with either saline or duloxetine (15 or 30â¯mg/kg) for four weeks. The effect of duloxetine was assessed in terms of pain responses, histopathology of sciatic nerve and spinal cord, sciatic nerve growth factor (NGF) gene expression and on the spinal expression of astrocytes (glial fibrillary acidic protein, GFAP) and microglia (CD11b). KEY FINDINGS: The present results highlighted that duloxetine (30â¯mg/kg) increased the withdrawal threshold in von-Frey test. In addition, both doses of duloxetine prolonged the licking time and latency to jump in the hot-plate test. Moreover, duloxetine administration downregulated the spinal expression of both CD11b and GFAP associated with enhancement in sciatic mRNA expression of NGF. SIGNIFICANCE: The current results highlighted that duloxetine provided peripheral and central neuroprotective effects in neuropathic pain is, at least in part, related to its downregulation in spinal astrocytes and microglia. Further, this neuroprotective effect was accompanied by upregulation of sciatic expression of NGF.
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Antidepresivos/uso terapéutico , Neuropatías Diabéticas/tratamiento farmacológico , Clorhidrato de Duloxetina/uso terapéutico , Neuroglía/efectos de los fármacos , Fármacos Neuroprotectores/uso terapéutico , Animales , Astrocitos/metabolismo , Antígeno CD11b/biosíntesis , Antígeno CD11b/genética , Regulación de la Expresión Génica/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/biosíntesis , Masculino , Ratones , Neuralgia/tratamiento farmacológico , Dimensión del Dolor/efectos de los fármacos , Nervio Ciático/patología , Síndrome de la Serotonina/metabolismo , Médula Espinal/metabolismo , Columna Vertebral/citologíaRESUMEN
Docosahexaenoic acid (DHA, 22:6 n-3) is an essential omega-3 (ω-3) long chain polyunsaturated fatty acid of neuronal membranes involved in normal growth, development, and function. DHA has been proposed to reduce deleterious effects in neurodegenerative processes. Even though, some inconsistencies in findings from clinical and pre-clinical studies with DHA could be attributed to the presence of phytanic acid (PhA) in standard DHA treatments. Thus, the aim of our study was to analyze and compare the effects of a low PhA-concentrated DHA with a standard PhA-concentrated DHA under different neurotoxic conditions in BV-2 activated microglial cells. To this end, mouse microglial BV-2 cells were stimulated with either lipopolysaccharide (LPS) or hydrogen peroxide (H2O2) and co-incubated with DHA 50 ppm of PhA (DHA (PhA:50)) or DHA 500 ppm of PhA (DHA (PhA:500)). Cell viability, superoxide anion (O2-) production, Interleukin 6 (L-6), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), glutathione peroxidase (GtPx), glutathione reductase (GtRd), Caspase-3, and the brain-derived neurotrophic factor (BDNF) protein expression were explored. Low PhA-concentrated DHA protected against LPS or H2O2-induced cell viability reduction in BV-2 activated cells and O2- production reduction compared to DHA (PhA:500). Low PhA-concentrated DHA also decreased COX-2, IL-6, iNOS, GtPx, GtRd, and SOD-1 protein expression when compared to DHA (PhA:500). Furthermore, low PhA-concentrated DHA increased BDNF protein expression in comparison to DHA (PhA:500). The study provides data supporting the beneficial effect of low PhA-concentrated DHA in neurotoxic injury when compared to a standard PhA-concentrated DHA in activated microglia.
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Ácidos Docosahexaenoicos/farmacología , Microglía/efectos de los fármacos , Neuroprotección , Fármacos Neuroprotectores/farmacología , Ácido Fitánico/farmacología , Animales , Antioxidantes/metabolismo , Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Antígeno CD11b/biosíntesis , Línea Celular , Supervivencia Celular/efectos de los fármacos , Ácidos Docosahexaenoicos/uso terapéutico , Peróxido de Hidrógeno/farmacología , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Lipopolisacáridos/farmacología , Ratones , Microglía/metabolismo , Fármacos Neuroprotectores/uso terapéutico , Síndromes de Neurotoxicidad/tratamiento farmacológico , Concentración Osmolar , Ácido Fitánico/uso terapéutico , Superóxidos/metabolismoRESUMEN
CXCR2 has emerged as a therapeutic target for not only peripheral inflammatory diseases but also neurological abnormalities in the central nervous system (CNS). Herein, we describe the discovery of a novel 1-cyclopentenyl-3-phenylurea series as potent and CNS penetrant CXCR2 antagonists. Extensive SAR studies, wherein molecules' property forecast index (PFI) was carefully optimized for overall balanced developability profiles, led to the discovery of the advanced lead compound 68 with a desirable PFI. Compound 68 demonstrated good in vitro pharmacology with excellent selectivity over CXCR1 and other chemokine receptors. Rat and dog pharmacokinetics (PK) revealed good oral bioavailability, high oral exposure, and desirable elimination half-life of the compound in both species. In addition, the compound demonstrated dose-dependent efficacy in the in vivo pharmacology neutrophil infiltration "air pouch" model in rodents after oral administration. Further, compound 68 is a CNS penetrant molecule with high unbound fraction in brain tissue.
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Encéfalo/metabolismo , Compuestos de Fenilurea/síntesis química , Compuestos de Fenilurea/farmacología , Receptores de Interleucina-8B/antagonistas & inhibidores , Animales , Disponibilidad Biológica , Antígeno CD11b/biosíntesis , Cristalografía por Rayos X , Perros , Relación Dosis-Respuesta a Droga , Semivida , Humanos , Células de Riñón Canino Madin Darby , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Infiltración Neutrófila/efectos de los fármacos , Compuestos de Fenilurea/farmacocinética , Ratas , Relación Estructura-ActividadRESUMEN
Manipulation of costimulatory and surface molecules that shape the extent of immune responses by Leishmania is suggested as one of the mechanisms of evading the host's defences. The experiments reported here were designed to evaluate the expressions of CD11b, CD11c, CD14, CD18, CD54, CD80, CD86, CD206, MHC class II and TLR-2 (Toll-like receptor 2) in human macrophages infected with L. amazonensis. Phenotypic evaluation revealed a negative modulation in CD11b, CD11c, CD14, CD18, CD54 and MHC class II molecules, depending on the level of infection. The results showed that as early as 1 hour after infection no reduction in marker expression occurs, whereas after 24 hours, downregulation of these molecules was observed in macrophages. No significant changes were observed in the expressions of CD80, CD86, CD206 and TLR2. Evidence of the differential modulation of markers expression and that after parasite uptake no reduction in surface marker expression occurs indicates that parasite internalization is not involved in the phenomena of down-modulation.
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Antígenos CD/biosíntesis , Leishmania mexicana/inmunología , Leishmaniasis Cutánea/inmunología , Macrófagos/inmunología , Macrófagos/parasitología , Adulto , Animales , Antígenos CD/inmunología , Antígenos de Superficie/biosíntesis , Antígenos de Superficie/inmunología , Antígeno B7-1/biosíntesis , Antígeno CD11b/biosíntesis , Células Cultivadas , Antígenos de Histocompatibilidad Clase II/biosíntesis , Humanos , Leishmaniasis Cutánea/parasitología , Ratones , Ratones Endogámicos BALB C , Receptor Toll-Like 2/biosíntesisRESUMEN
Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.
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Sistema Nervioso Central/citología , Citometría de Flujo/métodos , Macrófagos/inmunología , Microglía/inmunología , Animales , Biomarcadores/análisis , Antígeno CD11b/biosíntesis , Sistema Nervioso Central/inmunología , Antígenos Comunes de Leucocito/biosíntesis , Macrófagos/citología , Ratones , Microglía/citologíaRESUMEN
Treatment with the TLR4 agonist MPLA augments innate resistance to common bacterial pathogens. However, the cellular and molecular mechanisms by which MPLA augments innate immunocyte functions are not well characterized. This study examined the importance of MyD88- and TRIF-dependent signaling for leukocyte mobilization, recruitment, and activation following administration of MPLA. MPLA potently induced MyD88- and TRIF-dependent signaling. A single injection of MPLA caused rapid mobilization and recruitment of neutrophils, a response that was largely mediated by the chemokines CXCL1 and -2 and the hemopoietic factor G-CSF. Rapid neutrophil recruitment and chemokine production were regulated by both pathways although the MyD88-dependent pathway showed some predominance. In further studies, multiple injections of MPLA potently induced mobilization and recruitment of neutrophils and monocytes. Neutrophil recruitment after multiple injections of MPLA was reliant on MyD88-dependent signaling, but effective monocyte recruitment required activation of both pathways. MPLA treatment induced expansion of myeloid progenitors in bone marrow and upregulation of CD11b and shedding of L-selectin by neutrophils, all of which were attenuated in MyD88- and TRIF-deficient mice. These results show that MPLA-induced neutrophil and monocyte recruitment, expansion of bone marrow progenitors and augmentation of neutrophil adhesion molecule expression are regulated by both the MyD88- and TRIF-dependent pathways.
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Proteínas Adaptadoras del Transporte Vesicular/fisiología , Inmunidad Innata , Lípido A/análogos & derivados , Monocitos/inmunología , Factor 88 de Diferenciación Mieloide/fisiología , Neutrófilos/inmunología , Receptor Toll-Like 4/agonistas , Proteínas Adaptadoras del Transporte Vesicular/deficiencia , Animales , Antígeno CD11b/biosíntesis , Antígeno CD11b/genética , Quimiocina CXCL1/fisiología , Quimiocina CXCL2/fisiología , Quimiotaxis de Leucocito/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos/fisiología , Selectina L/metabolismo , Lípido A/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Factor 88 de Diferenciación Mieloide/deficiencia , Mielopoyesis/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Receptores de Interleucina-8B/fisiología , Transducción de Señal , Receptor Toll-Like 4/fisiologíaRESUMEN
BACKGROUND: Bone marrow-derived mesenchymal stem cells (BMMSCs) transplantation has been considered as a promising milestone in liver fibrosis treatment. However, low amounts of homing are a major obstacle. We aimed to investigate the role of melatonin pretreatment in BMMSC homing into experimental liver fibrosis. METHODS: BMMSCs were obtained, grown, propagated and preconditioned with 5 µM melatonin and analyzed for multipotency and immunophenotypic features at passage three. The cells were labelled with CM-Dil and infused into the rats received the i.p. injection of carbon tetrachloride (CCl4) for five weeks to induce liver fibrosis. Animals were divided into two groups: One group received BMMSCs, whereas the other group received melatonin-pretreated BMMSCs (MT-BMMSCs). After cell injection at 72 h, animals were sacrificed, and the liver tissues were assessed for further evaluations: fibrosis using Masson's trichrome and hematoxylin and eosin staining and homing using fluorescent microscopy and flow cytometry. RESULTS: BMMSCs and MT-BMMSCs expressed a high level of CD44 but low levels of CD11b, CD45 and CD34 (for all P≤0.05) and were able to differentiate into adipocytes and Schwann cells. CCl4 induction resulted in extensive collagen deposition, tissue disruption and fatty accumulation with no obvious difference between the two groups. There was a significant increase in homing of MT-BMMSCs in both florescent microscopy (P≤0.001) and flow cytometry (P≤0.01) assays, as compared with non-treated BMMSCs. CONCLUSION: This study indicates the improved homing potential of BMMSCs in pretreatment with melatonin. Therefore, this strategy may represent an applied approach for improving the stem cell therapy of liver fibrosis.