RESUMEN
INTRODUCTION: Interferon-γ levels are increased upon viral infections and during inflamm-aging. Resistance to infections due to Escherichia coli (E. coli), a major cause of bacteriaemia and sepsis, is impaired in aged individuals, partly due to altered phagocytic capacity and cytokine release of immune cells. Here, we analyzed the effect of IFN-γ on phagocytosis of E. coli K1 and release of proinflammatory cytokines by macrophages in resting condition and upon stimulation with different bacterial Toll-like receptor (TLR) agonists. METHODS: Primary peritoneal macrophages from C57BL/6 mice were exposed to medium or stimulated with agonists of TLR4 (LPS), 1/2 (Pam3CSK4), and 9 (CpG-DNA) in the presence and absence of IFN-γ (100 U/ml) for 24 h. TNF-α, IL-6, and KC were measured in the cell culture supernatant by ELISA. Macrophages were exposed to viable E. coli K1. After 90 min, intracellular phagozytosed bacteria were quantified by quantitative plating. RESULTS: Macrophages treated with LPS 1 µg/ml in the presence of IFN-γ ingested more than 10-fold lower numbers of E. coli than macrophages treated with LPS alone. Phagocytosis of E. coli by macrophages in resting condition or upon stimulation with Pam3CSK4 or CpG was not significantly affected by IFN-γ. Cytokine release was differentially modulated by IFN-γ, with reduced KC release by TLR-stimulated macrophages in the presence of IFN-γ being the most striking effect. CONCLUSIONS: In vitro, IFN-γ reduces the phagocytosis of E. coli by LPS-stimulated macrophages and differentially modulates cytokine release of macrophages activated by different bacterial TLR agonists. Elevated levels of IFN-γ might lead to reduced bacterial clearance and worse outcome of bacterial infections, e.g., in aged individuals and after viral infections and other inflammatory events.
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BACKGROUND: Individuals with impaired immunity are more susceptible to infections than immunocompetent subjects. No vaccines are currently available to induce protection against E. coli meningoencephalitis. This study evaluated the potential of poly(I:C) pre-treatment to induce trained immunity. Poly(I:C) was administered as a non-specific stimulus of innate immune responses to protect immunocompetent and neutropenic wild-type mice from a subsequent challenge by the intracranial injection of E. coli K1. METHODS: Three days prior to infection, mice received an intraperitoneal injection of poly(I:C) or vehicle. Kaplan-Meier survival curves were analyzed. In short-term experiments, bacterial titers and the inflammatory response were characterized in the blood, cerebellum, and spleen homogenates. NK cell subpopulations in the brain and spleen were analyzed by flow cytometry. Numbers of microglia and activation scores were evaluated by histopathology. RESULTS: Pre-treatment with 200 µg poly(I:C) increased survival time, reduced mortality, and enhanced bacterial clearance in the blood, cerebellum, and spleen at early infection in neutropenic mice. Poly(I:C)-mediated protection correlated with an augmented number of NK cells (CD45+NK1.1+CD3-) and Iba-1+ microglial cells and a higher production of IFN-γ in the brain. In the spleen, levels of CCL5/RANTES and IFN-γ were increased and sustained in surviving poly(I:C)-treated animals for 14 days after infection. In immunocompetent animals, survival time was not significantly prolonged in poly(I:C)-treated animals although poly(I:C) priming reduced brain bacterial concentrations compared with vehicle-injected animals at early infection. CONCLUSIONS: Pre-treatment with the viral TLR3 agonist poly(I:C) modulated innate immune responses and strengthened the resistance of neutropenic mice against E. coli K1 meningoencephalitis.
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Inmunidad Innata/efectos de los fármacos , Huésped Inmunocomprometido/inmunología , Meningitis por Escherichia coli/inmunología , Poli I-C/farmacología , Animales , Inmunidad Innata/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Neutropenia/inmunología , Poli I-C/inmunología , Receptor Toll-Like 3/agonistas , Receptor Toll-Like 3/efectos de los fármacosRESUMEN
Geriatric animal models are crucial for a better understanding and an improved therapy of age-related diseases. We observed a high mortality of aged mice after anesthesia with a standard dose of ketamine/xylazine, an anesthetic regimen frequently used in laboratory veterinary medicine. C57BL/6-N mice at the age of 2.14 ± 0.23 months (young mice) and 26.31 ± 2.15 months (aged mice) were anesthetized by intraperitoneal injection of 2 mg ketamine and 0.2 mg xylazine. 4 of 26 aged mice (15.4%) but none of 26 young mice died within 15 min after injection of the anesthetics. The weight of aged mice was significantly higher than that of young mice (32.8 ± 5.4 g versus 23.2 ± 3.4 g, p < 0.0001). Thus, aged mice received lower doses of anesthetics in relation to their body weight which are within the lower range of doses recommended in the literature or even beneath. There were no differences between deceased and surviving aged mice concerning their sex, weight and their motor performance prior to anesthesia. Our data clearly show an age-related increase of mortality upon anesthesia with low standard doses of ketamine/xylazine. Assessment of weight and motor performance did not help to predict vulnerability of aged mice to the anesthetics. Caution is necessary when this common anesthetic regimen is applied in aged mice: lower doses or the use of alternative anesthetics should be considered to avoid unexpected mortality. The present data from our geriatric mouse model strongly corroborate an age-adjusted reduction of anesthetic doses to reduce anesthesia-related mortality in aged individuals.
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Easy-to-achieve interventions to promote healthy longevity are desired to diminish the incidence and severity of infections, as well as associated disability upon recovery. The dietary supplement palmitoylethanolamide (PEA) exerts anti-inflammatory and neuroprotective properties. Here, we investigated the effect of prophylactic PEA on the early immune response, clinical course, and survival of old mice after intracerebral E. coli K1 infection. Nineteen-month-old wild type mice were treated intraperitoneally with two doses of either 0.1 mg PEA/kg in 250 µl vehicle solution (n = 19) or with 250 µl vehicle solution only as controls (n = 19), 12 h and 30 min prior to intracerebral E. coli K1 infection. The intraperitoneal route was chosen to reduce distress in mice and to ensure exact dosing. Survival time, bacterial loads in cerebellum, blood, spleen, liver, and microglia counts and activation scores in the brain were evaluated. We measured the levels of IL-1ß, IL-6, MIP-1α, and CXCL1 in cerebellum and spleen, as well as of bioactive lipids in serum in PEA- and vehicle-treated animals 24 h after infection. In the absence of antibiotic therapy, the median survival time of PEA-pre-treated infected mice was prolonged by 18 h compared to mice of the vehicle-pre-treated infected group (P = 0.031). PEA prophylaxis delayed the onset of clinical symptoms (P = 0.037). This protective effect was associated with lower bacterial loads in the spleen, liver, and blood compared to those of vehicle-injected animals (P ≤ 0.037). PEA-pre-treated animals showed diminished levels of pro-inflammatory cytokines and chemokines in spleen 24 h after infection, as well as reduced serum concentrations of arachidonic acid and of one of its metabolites, 20-hydroxyeicosatetraenoic acid. In the brain, prophylactic PEA tended to reduce bacterial titers and attenuated microglial activation in aged infected animals (P = 0.042). Our findings suggest that prophylactic PEA can counteract infection associated detrimental responses in old animals. Accordingly, PEA treatment slowed the onset of infection symptoms and prolonged the survival of old infected mice. In a clinical setting, prophylactic administration of PEA might extend the potential therapeutic window where antibiotic therapy can be initiated to rescue elderly patients.
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Antiinflamatorios no Esteroideos/uso terapéutico , Escherichia coli/metabolismo , Etanolaminas/uso terapéutico , Inflamación/dietoterapia , Meningitis por Escherichia coli/dietoterapia , Meningitis por Escherichia coli/prevención & control , Ácidos Palmíticos/uso terapéutico , Envejecimiento/inmunología , Amidas , Animales , Cerebelo/microbiología , Citocinas/metabolismo , Suplementos Dietéticos , Modelos Animales de Enfermedad , Estimación de Kaplan-Meier , Meningitis por Escherichia coli/metabolismo , Ratones , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Bazo/microbiología , Estadísticas no Paramétricas , Tasa de SupervivenciaRESUMEN
In order to elucidate the causes for the increased mortality of aged patients with bacterial central nervous system (CNS) infections, we compared the course of Streptococcus pneumoniae (S. pneumoniae) meningitis in aged and young mice. Aged (21.2 ± 3.1 months, n = 40) and young (3.2 ± 0.9 months, n = 42) C57BL/6N and B6/SJL mice were infected by intracerebral injection of 50-70 CFU S. pneumoniae serotype 3 and monitored for 15 days. Aged and young mice did not differ concerning mortality (35% versus 38%), weight loss, development of clinical symptoms, bacterial concentrations in cerebellum and spleen as well as the number of leukocytes infiltrating the CNS. In contrast to results from our geriatric mouse model of Escherichia coli (E. coli) meningitis, where aged mice showed a higher mortality and an impaired elimination of bacteria, we did not find any differences between aged and young mice after intracerebral infection with S. pneumoniae serotype 3. This indicates that the increased susceptibility of aged mice to bacterial CNS infections is pathogen-specific: It appears less prominent in infections caused by hardly phagocytable pathogens with thick capsules like S. pneumoniae serotype 3, where the age-related decline of the phagocytic capacity of microglia and macrophages has a minor influence on the disease course.
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BACKGROUND: Bacterial meningitis is associated with high mortality and long-term neurological sequelae. Increasing the phagocytic activity of microglia could improve the resistance of the CNS against infections. We studied the influence of activin A, a member of the TGF-ß family with known immunoregulatory and neuroprotective effects, on the functions of microglial cells in vitro. METHODS: Primary murine microglial cells were treated with activin A (0.13 ng/ml-13 µg/ml) alone or in combination with agonists of TLR2, 4, and 9. Phagocytosis of Escherichia coli K1 as well as release of TNF-α, IL-6, CXCL1, and NO was assessed. RESULTS: Activin A dose-dependently enhanced the phagocytosis of Escherichia coli K1 by microglial cells activated by agonists of TLR2, 4, and 9 without further increasing NO and proinflammatory cytokine release. Cell viability of microglial cells was not affected by activin A. CONCLUSIONS: Priming of microglial cells with activin A could increase the elimination of bacteria in bacterial CNS infections. This preventive strategy could improve the resistance of the brain to infections, particularly in elderly and immunocompromised patients.
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Activinas/farmacología , Citocinas/metabolismo , Lipopolisacáridos/farmacología , Microglía/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Receptores Toll-Like/agonistas , Animales , Animales Recién Nacidos , Encéfalo/citología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Escherichia coli/fisiología , Humanos , Recién Nacido , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico/metabolismo , Lectinas de Plantas/metabolismo , Receptores Toll-Like/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Streptococcus pneumoniae is the most common cause of bacterial meningitis in adults and is characterized by high lethality and substantial cognitive disabilities in survivors. Here, we have studied the capacity of an established therapeutic agent, magnesium, to improve survival in pneumococcal meningitis by modulating the neurological effects of the major pneumococcal pathogenic factor, pneumolysin. EXPERIMENTAL APPROACH: We used mixed primary glial and acute brain slice cultures, pneumolysin injection in infant rats, a mouse meningitis model and complementary approaches such as Western blot, a black lipid bilayer conductance assay and live imaging of primary glial cells. KEY RESULTS: Treatment with therapeutic concentrations of magnesium chloride (500 mg·kg-1 in animals and 2 mM in cultures) prevented pneumolysin-induced brain swelling and tissue remodelling both in brain slices and in animal models. In contrast to other divalent ions, which diminish the membrane binding of pneumolysin in non-therapeutic concentrations, magnesium delayed toxin-driven pore formation without affecting its membrane binding or the conductance profile of its pores. Finally, magnesium prolonged the survival and improved clinical condition of mice with pneumococcal meningitis, in the absence of antibiotic treatment. CONCLUSIONS AND IMPLICATIONS: Magnesium is a well-established and safe therapeutic agent that has demonstrated capacity for attenuating pneumolysin-triggered pathogenic effects on the brain. The improved animal survival and clinical condition in the meningitis model identifies magnesium as a promising candidate for adjunctive treatment of pneumococcal meningitis, together with antibiotic therapy.
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Cloruro de Magnesio/administración & dosificación , Meningitis Neumocócica/tratamiento farmacológico , Streptococcus pneumoniae/efectos de los fármacos , Estreptolisinas/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/microbiología , Modelos Animales de Enfermedad , Femenino , Cloruro de Magnesio/farmacología , Meningitis Neumocócica/microbiología , Ratones , Ratones Endogámicos C57BL , Neuroglía/efectos de los fármacos , Neuroglía/microbiología , Ratas , Ratas Sprague-Dawley , Streptococcus pneumoniae/aislamiento & purificación , Tasa de SupervivenciaRESUMEN
BACKGROUND: The adaptive immune system has been considered to play a minimal role in the early host response during bacterial meningitis. METHODS: We investigated the progression and outcome of pneumococcal meningitis in Rag1-/- mice lacking functional B and T cells by assessing overall and symptom-free survival, bacteriological and histological studies, as well as flow cytometry and measurements of proinflammatory mediators. RESULTS: The intracerebral injection of S. pneumoniae D39 induced the recruitment of B and T cells (CD4+, γδ and natural killer) into the brain of wild-type mice. Mice with no functional B and T cells developed clinical symptoms and succumbed to the infection earlier than the wild-type group. In the CNS, Rag1-/- mice showed lower levels of interleukin 1ß, reduced microglial proliferation, and impaired granulocyte recruitment with an earlier spread of pneumococci into the bloodstream, compared with wild-type mice. Lack of B and T cells resulted in a severe impairment of bacterial clearance in blood, spleen, and liver and an exaggerated systemic inflammatory response. CONCLUSIONS: B and T cells are important effector cells delaying the spread of pneumococci from the brain to the systemic circulation and shaping the immune response, thereby prolonging the survival of the host in the absence of antibiotic treatment.
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Inmunidad Adaptativa , Encéfalo/inmunología , Meningitis Neumocócica/inmunología , Meningitis Neumocócica/fisiopatología , Streptococcus pneumoniae/inmunología , Animales , Linfocitos B/inmunología , Encéfalo/microbiología , Encéfalo/ultraestructura , Citocinas/biosíntesis , Interleucina-1beta/inmunología , Células Asesinas Naturales , Meningitis Neumocócica/microbiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/inmunología , Bazo/microbiología , Linfocitos T/inmunologíaRESUMEN
BACKGROUND: Pneumococcal proteins involved in the resistance against oxidative stress are present in all strains and therefore are potential antigens that could be suitable for new therapies and/or vaccines. Their role in the pathogenesis of pneumococcal meningitis has not been addressed. METHODS: We investigated the individual contributions of extracellular thioredoxin lipoproteins (Etrx1 and Etrx2) and the intracellular and extracellular methionine sulfoxide reductases (SpMsrAB1 and SpMsrAB2, respectively) in the progression and outcome of pneumococcal meningitis, using Kaplan-Meier survival curves, bacteriological and histological studies, and measurements of proinflammatory mediators. RESULTS: The absence of Etrx1, Etrx2, or SpMsrAB1 moderately attenuated virulence as compared to the wild-type strain but did not significantly affect bacterial growth in the brain and bloodstream. Loss of function of SpMsrAB2 alone, both Etrx proteins, or both SpMsrAB proteins resulted in a less severe course of infection, with low numbers of animals dying of infection, a lower risk of associated meningeal inflammation, and reduced bacterial densities in the cerebellum, blood, and spleen. CONCLUSIONS: Our data support the importance of the extracellular redox repair system in virulence and its potential as a target for the design of new antimicrobials and vaccine formulations against Streptococcus pneumoniae.
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Meningitis Neumocócica/patología , Metionina Sulfóxido Reductasas/metabolismo , Streptococcus pneumoniae/patogenicidad , Tiorredoxinas/metabolismo , Factores de Virulencia/metabolismo , Animales , Sangre/microbiología , Encéfalo/microbiología , Modelos Animales de Enfermedad , Femenino , Eliminación de Gen , Meningitis Neumocócica/inmunología , Metionina Sulfóxido Reductasas/genética , Ratones Endogámicos C57BL , Oxidación-Reducción , Estrés Oxidativo , Bazo , Streptococcus pneumoniae/genética , Análisis de Supervivencia , Tiorredoxinas/genética , Virulencia , Factores de Virulencia/genéticaRESUMEN
Microglia, innate immune cells of the CNS, sense infection and damage through overlapping receptor sets. Toll-like receptor (TLR) 4 recognizes bacterial lipopolysaccharide (LPS) and multiple injury-associated factors. We show that its co-receptor CD14 serves three non-redundant functions in microglia. First, it confers an up to 100-fold higher LPS sensitivity compared to peripheral macrophages to enable efficient proinflammatory cytokine induction. Second, CD14 prevents excessive responses to massive LPS challenges via an interferon ß-mediated feedback. Third, CD14 is mandatory for microglial reactions to tissue damage-associated signals. In mice, these functions are essential for balanced CNS responses to bacterial infection, traumatic and ischemic injuries, since CD14 deficiency causes either hypo- or hyperinflammation, insufficient or exaggerated immune cell recruitment or worsened stroke outcomes. While CD14 orchestrates functions of TLR4 and related immune receptors, it is itself regulated by TLR and non-TLR systems to thereby fine-tune microglial damage-sensing capacity upon infectious and non-infectious CNS challenges.
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Lesiones Encefálicas/inmunología , Isquemia Encefálica/inmunología , Infecciones por Escherichia coli/metabolismo , Receptores de Lipopolisacáridos/metabolismo , Microglía/inmunología , Accidente Cerebrovascular/inmunología , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Encéfalo/inmunología , Encéfalo/patología , Lesiones Encefálicas/complicaciones , Lesiones Encefálicas/patología , Isquemia Encefálica/patología , Células Cultivadas , Modelos Animales de Enfermedad , Escherichia coli , Infecciones por Escherichia coli/complicaciones , Infecciones por Escherichia coli/patología , Retroalimentación Fisiológica/fisiología , Infarto de la Arteria Cerebral Media , Interferón beta/metabolismo , Receptores de Lipopolisacáridos/genética , Lipopolisacáridos/toxicidad , Macrófagos/inmunología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Neuroinmunomodulación , Accidente Cerebrovascular/patología , Receptor Toll-Like 4/agonistas , Receptor Toll-Like 4/antagonistas & inhibidores , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismoRESUMEN
The outcome of bacterial meningitis critically depends on the rapid initiation of bactericidal antibiotic therapy and adequate management of septic shock. In community-acquired meningitis, the choice of an optimum initial empirical antibiotic regimen depends on the regional resistance patterns. Pathogens resistant to antibacterials prevail in nosocomial bacterial meningitis. Dexamethasone is recommended as adjunctive therapy for community-acquired meningitis in developed countries. In comatose patients, aggressive measures to lower intracranial pressure <20 mmHg (in particular, external ventriculostomy, osmotherapy and temporary hyperventilation) were effective in a case-control study. Although many experimental approaches were protective in animal models, none of them has been proven effective in patients. Antibiotics, which are bactericidal but do not lyse bacteria, and inhibitors of matrix metalloproteinases or complement factor C5 appear the most promising therapeutic options. At present, vaccination is the most efficient method to reduce disease burden. Palmitoylethanolamide appears promising to enhance the resistance of the brain to infections.
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Antibacterianos/clasificación , Antibacterianos/uso terapéutico , Infección Hospitalaria/tratamiento farmacológico , Dexametasona/uso terapéutico , Meningitis Bacterianas/tratamiento farmacológico , Anticuerpos Monoclonales/uso terapéutico , Países Desarrollados , Humanos , Vacunación , Vitaminas/uso terapéuticoRESUMEN
The putative protein tyrosine kinase (PTK) inhibitor tyrphostin AG126 has proven beneficial in various models of inflammatory disease. Yet molecular targets and cellular mechanisms remained enigmatic. We demonstrate here that AG126 treatment has beneficial effects in experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. AG126 alleviates the clinical symptoms, diminishes encephalitogenic Th17 differentiation, reduces inflammatory CNS infiltration as well as microglia activation and attenuates myelin damage. We show that AG126 directly inhibits Bruton's tyrosine kinase (BTK), a PTK associated with B cell receptor and Toll-like receptor (TLR) signaling. However, BTK inhibition cannot account for the entire activity spectrum. Effects on TLR-induced proinflammatory cytokine expression in microglia involve AG126 hydrolysis and conversion of its dinitrile side chain to malononitrile (MN). Notably, while liberated MN can subsequently mediate critical AG126 features, full protection in EAE still requires delivery of intact AG126. Its anti-inflammatory potential and especially interference with TLR signaling thus rely on a dual mechanism encompassing BTK and a novel MN-sensitive target. Both principles bear great potential for the therapeutic management of disturbed innate and adaptive immune functions.
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Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Fármacos Neuroprotectores/farmacología , Tirfostinos/farmacología , Agammaglobulinemia Tirosina Quinasa , Animales , Células Cultivadas , Citocinas/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Encefalomielitis Autoinmune Experimental/fisiopatología , Femenino , Hidrólisis , Factores Inmunológicos/química , Factores Inmunológicos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/efectos de los fármacos , Microglía/fisiología , Factor 88 de Diferenciación Mieloide/metabolismo , Fármacos Neuroprotectores/química , Nitrilos/química , Nitrilos/metabolismo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Bazo/citología , Bazo/efectos de los fármacos , Bazo/fisiopatología , Células Th17/efectos de los fármacos , Células Th17/patología , Células Th17/fisiología , Tirfostinos/químicaRESUMEN
Despite the development of new antibiotic agents, mortality of pneumococcal meningitis remains high. In addition, meningitis results in severe long-term morbidity, most prominently cognitive deficits. Granulocyte colony-stimulating factor (G-CSF) stimulates proliferation and differentiation of hematopoietic progenitor cells and increases the number of circulating neutrophil granulocytes. This study investigated the effect of adjuvant G-CSF treatment on cognitive function after pneumococcal meningitis. C57BL/6 mice were infected by subarachnoid injection of Streptococcus pneumoniae serotype 3 and treated with ceftriaxone and G-CSF subcutaneously or ceftriaxone alone for 5 days. Clinical scores, motor performance, and mortality during bacterial meningitis were unaffected by adjuvant G-CSF treatment. No effect of G-CSF treatment on production of proinflammatory cytokines or activation of microglia or astrocytes was observed. The G-CSF treatment did, however, result in hippocampal neurogenesis and improved spatial learning performance 6 weeks after meningitis. These results suggest that G-CSF might offer a new adjuvant therapeutic approach in bacterial meningitis to reduce long-term cognitive deficits.
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Trastornos del Conocimiento/tratamiento farmacológico , Trastornos del Conocimiento/etiología , Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Hipocampo/efectos de los fármacos , Meningitis Neumocócica/complicaciones , Neurogénesis/efectos de los fármacos , Células Madre Adultas/efectos de los fármacos , Animales , Antibacterianos/uso terapéutico , Ceftriaxona/uso terapéutico , Diferenciación Celular/efectos de los fármacos , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Meningitis Neumocócica/tratamiento farmacológico , Ratones , Ratones Endogámicos C57BL , Actividad Motora/efectos de los fármacos , Fosforilación/efectos de los fármacos , Streptococcus pneumoniae/patogenicidad , Factores de TiempoRESUMEN
Incidence and mortality of bacterial meningitis are strongly increased in aged compared to younger adults demanding new strategies to improve prevention and therapy of bacterial central nervous system (CNS) infections the elderly. Here, we established a geriatric mouse model for an intracerebral E. coli infection which reflects the clinical situation in aged patients: After intracerebral challenge with E. coli K1, aged mice showed a higher mortality, a faster development of clinical symptoms, and a more pronounced weight loss. Elimination of bacteria and systemic inflammatory response were impaired in aged mice, however, the number of infiltrating leukocytes and microglial cells in the CNS of aged and young mice did not differ substantially. In vitro, primary microglial cells and peritoneal macrophages from aged mice phagocytosed less E. coli and released less NO and cyto-/chemokines compared to cells from young mice both without activation and after stimulation by agonists of TLR 2, 4, and 9. Our results suggest that the age-related decline of microglia and macrophage functions plays an essential role for the higher susceptibility of aged mice to intracerebral infections. Strategies to improve the phagocytic potential of aged microglial cells and macrophages appear promising for prevention and treatment of CNS infections in elderly patients.
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Encefalopatías/microbiología , Escherichia coli/aislamiento & purificación , Macrófagos/microbiología , Meningitis Bacterianas/microbiología , Microglía/microbiología , Factores de Edad , Animales , Encefalopatías/inmunología , Modelos Animales de Enfermedad , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/microbiología , Macrófagos/inmunología , Meningitis Bacterianas/inmunología , Ratones , Microglía/inmunologíaRESUMEN
In healthy individuals, infections of the central nervous system (CNS) are comparatively rare. Based on the ability of microglial cells to phagocytose and kill pathogens and on clinical findings in immunocompromised patients with CNS infections, we hypothesize that an intact microglial function is crucial to protect the brain from infections. Phagocytosis of pathogens by microglial cells can be stimulated by agonists of receptors of the innate immune system. Enhancing this pathway to increase the resistance of the brain to infections entails the risk of inducing collateral damage to the nervous tissue. The diversity of microglial cells opens avenue to selectively stimulate sub-populations responsible for the defence against pathogens without stimulating sub-populations which are responsible for collateral damage to the nervous tissue. Palmitoylethanolamide (PEA), an endogenous lipid, increased phagocytosis of bacteria by microglial cells in vitro without a measurable proinflammatory effect. It was tested clinically apparently without severe side effects. Glatiramer acetate increased phagocytosis of latex beads by microglia and monocytes, and dimethyl fumarate enhanced elimination of human immunodeficiency virus from infected macrophages without inducing a release of proinflammatory compounds. Therefore, the discovery of compounds which stimulate the elimination of pathogens without collateral damage of neuronal structures appears an achievable goal. PEA and, with limitations, glatiramer acetate and dimethyl fumarate appear promising candidates.
RESUMEN
BACKGROUND: Palmitoylethanolamide (PEA), an endogenous lipid and a congener of anandamide, possesses a wide range of effects related to metabolic and cellular homeostasis including anti-inflammatory and neuroprotective properties. METHODS: In vitro, we studied the ability of macrophages to phagocytose Escherichia coli K1 after stimulation with increasing doses of PEA. In vivo, wild-type mice were treated with PEA intraperitoneally 12 hours and 30 minutes before infection. Meningoencephalitis or sepsis was induced by intracerebral or intraperitoneal infection with E. coli K1. RESULTS: Stimulation of macrophages with PEA for 30 minutes increased the phagocytosis of E. coli K1 without inducing the release of TNFα or CXCL1. Intracellular killing of E. coli K1 was higher in PEA-stimulated than in unstimulated peritoneal macrophages and microglial cells. Pre-treatment with PEA significantly increased survival of mice challenged intracerebrally or intraperitoneally with E. coli K1. This effect was associated with a decreased production of CXCL1, IL-1ß and IL-6 in homogenates of spleen and cerebellum in mice treated with PEA. CONCLUSIONS: Our observations suggest that these protective effects of PEA in mice can increase the resistance to bacterial infections without the hazard of collateral damage by excessive stimulation of phagocytes.
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Antiinflamatorios no Esteroideos/farmacología , Endocannabinoides/uso terapéutico , Infecciones por Escherichia coli/prevención & control , Etanolaminas/uso terapéutico , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Microglía/efectos de los fármacos , Ácidos Palmíticos/uso terapéutico , Amidas , Animales , Animales Recién Nacidos , Encéfalo/citología , Células Cultivadas , Cerebelo/microbiología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Endocannabinoides/farmacología , Escherichia coli/fisiología , Infecciones por Escherichia coli/etiología , Infecciones por Escherichia coli/metabolismo , Etanolaminas/farmacología , Ratones , Ratones Endogámicos C57BL , PPAR alfa/metabolismo , Ácidos Palmíticos/farmacología , Fagocitosis/efectos de los fármacos , Bazo/microbiología , Estadísticas no ParamétricasRESUMEN
Meningitis and meningoencephalitis caused by Escherichia coli are associated with high rates of mortality and neurological sequelae. A high prevalence of neurological disorders has been observed in geriatric populations at risk of hypovitaminosis D. Vitamin D has potent effects on human immunity, including induction of antimicrobial peptides (AMPs) and suppression of T-cell proliferation, but its influence on microglial cells is unknown. The purpose of the present study was to determine the effects of vitamin D deficiency on the phagocytosis rate, intracellular killing, and immune response of murine microglial cultures after stimulation with the Toll-like receptor (TLR) agonists tripalmitoyl-S-glyceryl-cysteine (TLR1/2), poly(I·C) (TLR3), lipopolysaccharide (TLR4), and CpG oligodeoxynucleotide (TLR9). Upon stimulation with high concentrations of TLR agonists, the release of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) was decreased in vitamin D-deficient compared to that in vitamin D-sufficient microglial cultures. Phagocytosis of E. coli K1 after stimulation of microglial cells with high concentrations of TLR3, -4, and -9 agonists and intracellular killing of E. coli K1 after stimulation with high concentrations of all TLR agonists were lower in vitamin D-deficient microglial cells than in the respective control cells. Our observations suggest that vitamin D deficiency may impair the resistance of the brain against bacterial infections.
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Escherichia coli/fisiología , Inmunidad Innata/fisiología , Meningitis por Escherichia coli/fisiopatología , Microglía/fisiología , Fagocitosis/fisiología , Deficiencia de Vitamina D , Vitamina D/fisiología , Análisis de Varianza , Animales , Calcifediol/sangre , Supervivencia Celular , Células Cultivadas , Quimiocinas/metabolismo , Recuento de Colonia Microbiana , Citocinas/metabolismo , Modelos Animales de Enfermedad , Lipopolisacáridos/farmacología , Meningitis por Escherichia coli/inmunología , Ratones , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Microglía/microbiología , Óxido Nítrico/metabolismo , Receptores Toll-Like/agonistas , Deficiencia de Vitamina D/inmunologíaRESUMEN
BACKGROUND: Prophylaxis with unmethylated cytosine phosphate guanidine (CpG) oligodeoxynucleotides (ODN) protects against several systemic experimental infections. Escherichia coli is a major cause of Gram-negative neonatal bacterial meningitis and also causes meningitis and meningoencephalitis in older and immunocompromised patients. METHODS: Wild-type (wt) and Toll-like receptor 9 (TLR9)-deficient mice were rendered neutropenic by intraperitoneal administration of the anti-Ly-6G monoclonal antibody. Immunocompetent and neutropenic mice received intraperitoneal CpG ODN or vehicle 72 h prior to induction of E. coli K1 meningoencephalitis. RESULTS: Pre-treatment with CpG ODN significantly increased survival of neutropenic wt mice from 33% to 75% (P = 0.0003) but did not protect neutropenic TLR9-/- mice. The protective effect of CpG ODN was associated with an enhanced production of interleukin (IL)-12/IL-23p40 with sustained increased levels in serum and spleen at least for 17 days after conditioning compared to buffer-treated animals. CpG-treated neutropenic wt mice showed reduced bacterial concentrations and increased recruitment of Ly6ChighCCR2+ monocytes in brain and spleen 42 h after infection. The levels of macrophage inflammatory protein 1α (MIP-1α) and interferon gamma (IFN-γ) in spleen were higher 42 h after infection in CpG-treated compared to buffer-treated neutropenic animals. In immunocompetent mice, prophylaxis with CpG ODN did not significantly increase survival compared to the buffer group (60% vs. 45%, P = 0.2). CONCLUSIONS: These findings suggest that systemic administration of CpG ODN may help to prevent bacterial CNS infections in immunocompromised individuals.
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Infecciones por Escherichia coli/prevención & control , Guanidina/química , Oligodesoxirribonucleótidos/uso terapéutico , Animales , Antígenos CD/metabolismo , Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/microbiología , Sistema Nervioso Central/patología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Esquema de Medicación , Escherichia coli/fisiología , Citometría de Flujo , Meningoencefalitis/prevención & control , Ratones , Ratones Noqueados , Bazo/microbiología , Bazo/patología , Receptor Toll-Like 9/deficienciaRESUMEN
BACKGROUND: Pneumococcal virulence factors common to all serotypes, such as choline-binding proteins (CBPs), are promising therapeutic targets in pneumococcal infections. We studied the effect of a choline dendrimer with maximized binding affinity/specificity for CBPs on microglia-mediated pneumococcal phagocytosis. METHODS: Pneumoccocal cultures were exposed to dendrimers containing 8 choline end groups or amino groups as controls, either from the beginning of bacterial growth or at the late exponential phase. The effect of long/short co-incubation was assessed in terms of bacterial morphological changes and increase in bacterial uptake by primary microglial cultures. RESULTS: Inhibiting CBPs by micromolar concentrations of a choline dendrimer caused the formation of long pneumococcal chains that were readily phagocytosed by microglia. Enhanced phagocytosis was dendrimer dose-dependent. Long bacteria-dendrimer co-incubation (14 h) resulted in a higher bacterial uptake than short co-incubation (2 h; p < 0.001). CONCLUSIONS: Multivalent dendrimers containing choline end groups are promising antimicrobial agents for the management of pneumococcal diseases.
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Antiinfecciosos/farmacología , Colina/química , Dendrímeros/farmacología , Microglía/efectos de los fármacos , Microglía/microbiología , Fagocitosis/efectos de los fármacos , Streptococcus pneumoniae/metabolismo , Animales , Antiinfecciosos/química , Proteínas Bacterianas/metabolismo , Células Cultivadas , Colina/metabolismo , Dendrímeros/química , Ratones , Ratones Endogámicos C57BL , Microglía/citología , Microglía/fisiología , Unión ProteicaRESUMEN
BACKGROUND: Toll-Like receptors (TLRs) belong to the family of pattern-recognition receptors with a crucial function of recognising pathogen-associated molecular patterns (PAMPs). Cryptococcal meningitis is a potentially fatal disease with a high mortality and risk of neurological sequelae. METHODS: We studied the ability of microglial cells to increase the phagocytosis of cryptococci after stimulation with agonists of TLR1/2, TLR3, TLR4 and TLR9. RESULTS: Stimulation of murine microglial cells with these TLR agonists for 24 h increased the phagocytosis of encapsulated Cryptococcus neoformans. Stimulation increased the release of TNF-α, CXCL1 (KC), IL-6, IL-10 and MIP-2, which indicated the activation of microglial cells. Unstimulated and TLR agonist-stimulated MyD88-deficient cells showed a reduced ability to phagocytose cryptococci compared to their wild-type counterpart. Intracellular killing of cryptococci was also increased in TLR-stimulated cells compared to unstimulated microglial cells. CONCLUSION: Our observation suggests that stimulation of microglial cells by TLR agonists can increase the resistance of the brain against CNS infections caused by Cryptococcus neoformans. This may be of interest when an immunocompromised patient is unable to eliminate Cryptococcus neoformans despite antifungal therapy.