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1.
Adv Exp Med Biol ; 1109: 53-65, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30523589

RESUMO

The microvascular pericyte was identified in 1873 by the French scientist Charles Benjamin Rouget and originally called the Rouget cell (Rouget.Sciences 88:916-8, 1879). However, it was not until the early 1900s that Rouget's work was confirmed, and the Rouget cell renamed the pericyte by virtue of its peri-endothelial location (Dore. Brit J Dermatol 35:398-404, 1923; Zimmermann. Z Anat Entwicklungsgesch 68:3-109, 1923). Over the years a large number of publications have emerged, but the pericyte has remained a truly enigmatic cell. This is due, in part, by the paucity of easy and reliable methods to isolate and characterize the cell as well as its heterogeneity and pluripotent characteristics. However, more recent advances in molecular genetics and development of novel cell isolation and imaging techniques have enable scientists to more readily define pericyte function. This chapter will discuss general approaches to the isolation, characterization, and propagation of primary pericytes in the establishment of cell lines. We will attempt to dispel misinterpretations about the pericyte that cloud the literature.


Assuntos
Técnicas de Cultura de Células , Separação Celular , Pericitos/citologia , Linhagem Celular , Humanos
2.
Adv Exp Med Biol ; 1109: C1, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30875064

RESUMO

The author has spotted an error on page 61, in the middle of the second paragraph from bottom. The content has been revised and the corrected version is as follows.

3.
Adv Exp Med Biol ; 923: 37-42, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27526122

RESUMO

The microvascular pericyte is an important regulatory cell that maintains tissue homeostasis. One of the mechanisms by which pericytes maintain tissue homeostasis is through the induction of endogenous adaptative changes to stress signals. These adaptations include migration, differentiation and induction of angiogenesis. We have investigated pericyte responses to hypoxic stress (1 % O2) and have reported that pericytes adapt to hypoxia, in part, through changes in endogenous and released microRNAs (miRNAs). Of those miRNAs, Let-7d plays an important role. We exposed pericytes to hypoxia with and without basic fibroblast growth factor (bFGF) in stem cell medium. The expression of Let-7d in pericyte-derived neurospheres was determined. Evidence of differentiation was determined by immunocytochemistry. Hypoxia enhanced pericyte spheres were positive for Let-7d. The transcription factor Sox2, a marker of cell differentiation, was also induced in pericytic spheres. Taken together, our results suggest that pericyte expression of Let-7d in response to hypoxia and bFGF is involved in pericyte differentiation. Thus, for the first time, we propose a pathway for induction of pericyte differentiation. Modulation of this pathway in pericytes may be an important target in tissue repair.


Assuntos
Diferenciação Celular , MicroRNAs/metabolismo , Oxigênio/metabolismo , Pericitos/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Hipóxia Celular , Fator 2 de Crescimento de Fibroblastos/farmacologia , Perfilação da Expressão Gênica , MicroRNAs/genética , Pericitos/efeitos dos fármacos , Cultura Primária de Células , Ratos , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais , Esferoides Celulares , Fatores de Tempo , Regulação para Cima
4.
J Neuroinflammation ; 13: 13, 2016 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-26785841

RESUMO

BACKGROUND: In the brain, chronic inflammatory activity may lead to compromised delivery of oxygen and glucose suggesting that therapeutic approaches aimed at restoring metabolic balance may be useful. In vivo exposure to chronic mild normobaric hypoxia (10 % oxygen) leads to a number of endogenous adaptations that includes vascular remodeling (angioplasticity). Angioplasticity promotes tissue survival. We have previously shown that induction of adaptive angioplasticity modulates the disease pattern in myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). In the present study, we define mechanisms by which adaptation to low oxygen functionally ameliorates the signs and symptoms of EAE and for the first time show that tissue hypoxia may fundamentally alter neurodegenerative disease. METHODS: C57BL/6 mice were immunized with MOG, and some of them were kept in the hypoxia chambers (day 0) and exposed to 10 % oxygen for 3 weeks, while the others were kept at normoxic environment. Sham-immunized controls were included in both hypoxic and normoxic groups. Animals were sacrificed at pre-clinical and peak disease periods for tissue collection and analysis. RESULTS: Exposure to mild hypoxia decreased histological evidence of inflammation. Decreased numbers of cluster of differentiation (CD)4+ T cells were found in the hypoxic spinal cords associated with a delayed Th17-specific cytokine response. Hypoxia-induced changes did not alter the sensitization of peripheral T cells to the MOG peptide. Exposure to mild hypoxia induced significant increases in anti-inflammatory IL-10 levels and an increase in the number of spinal cord CD25+FoxP3+ T-regulatory cells. CONCLUSIONS: Acclimatization to mild hypoxia incites a number of endogenous adaptations that induces an anti-inflammatory milieu. Further understanding of these mechanisms system may pinpoint possible new therapeutic targets to treat neurodegenerative disease.


Assuntos
Adaptação Biológica/fisiologia , Encefalomielite Autoimune Experimental/patologia , Hipóxia/fisiopatologia , Oxigênio/administração & dosagem , Medula Espinal/patologia , Linfócitos T Reguladores/metabolismo , Animais , Antígenos CD/metabolismo , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/induzido quimicamente , Feminino , Adjuvante de Freund/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Interferon gama/metabolismo , Interleucina-17/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Glicoproteína Mielina-Oligodendrócito/toxicidade , Infiltração de Neutrófilos , Fragmentos de Peptídeos/toxicidade , Fatores de Tempo
5.
Glia ; 62(9): 1452-62, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24829092

RESUMO

Lymphoid chemokines are crucial for the development and maintenance of lymphoid organs, but their ectopic expression in non-lymphoid tissues is implicated in both local response to infection and chronic organ-specific autoimmunity. Production of one such chemokine, C-X-C motif ligand 13 (CXCL13), within the central nervous system (CNS) has been linked to the pathogenesis of multiple sclerosis (MS), although little is known about factors controlling its expression in different neural cell types and across a range of disease states. We provoked acute neuroinflammation in experimental animals without causing any associated demyelination using neuroadapted Sindbis virus (NSV) to better understand the sources and regulators of this chemokine in the CNS. We found that mice genetically deficient in the transcription factor, interferon (IFN) regulatory factor-7 (IRF7), made significantly higher CXCL13 protein levels in the CNS compared with wild-type (WT) controls. Microglia proved to be the main producer of CXCL13 in the brain during infection of both WT and IRF7(-/-) mice, and primary microglia cultured in vitro generated CXCL13 following stimulation with either virus particles or synthetic Toll-like receptor (TLR) ligands. Microglia cultured from IRF7(-/-) mice selectively overproduced CXCL13, and manipulation of extracellular type-I IFN levels demonstrated the existence of a negative feedback loop whereby type-I IFN receptor signaling specifically suppressed microglial CXCL13 release. Since IFN-ß is used to treat patients with relapsing-remitting MS and yet acts through unknown mechanisms, we speculate that suppressed lymphoid chemokine production by microglia could contribute to its therapeutic effects.


Assuntos
Encéfalo/imunologia , Quimiocina CXCL13/metabolismo , Fator Regulador 7 de Interferon/metabolismo , Interferon Tipo I/metabolismo , Microglia/metabolismo , Infecções por Alphavirus/imunologia , Animais , Células Cultivadas , Quimiocina CXCL13/genética , Modelos Animais de Doenças , Encefalite Viral/imunologia , Encefalomielite/imunologia , Fator Regulador 7 de Interferon/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuroimunomodulação/fisiologia , Sindbis virus , Receptores Toll-Like/metabolismo
6.
J Neurol Sci ; 333(1-2): 88-92, 2013 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-23810780

RESUMO

While the pathologic events associated with multiple sclerosis (MS), diffuse axonal injury, cognitive damage, and white matter plaques, have been known for some time, their etiology is unknown and therapeutic efforts are still somewhat disappointing. This may be due to a lack of fundamental knowledge on how to maintain tissue homeostasis and buffer the brain from secondary injury. Maintenance of homeostasis in the brain is the result of regulatory adjustments by cellular constituents of the neurovascular unit (pericytes, endothelial cells, astrocytes, and neurons) that include induction of adaptive vascular remodeling. Results from our laboratory and others suggest that aspects of stress induced adaptation are seen in MS and in the murine model of experimental autoimmune encephalomyelitis (EAE), vascular remodeling is ineffective and biometabolic balance is disrupted. In murine white matter, capillary density is 1/2 that observed in gray matter thus disruption of vascular homeostasis will have a profound impact on tissue integrity. We therefore hypothesized that restoration of microvascular angiodynamics would augment tissue plasticity mitigating the extent of secondary injury and sparing cognitive decline in patients with MS. To test this hypothesis, we have performed preclinical studies and characterized changes in angiodynamics in myelin oligodendrocyte glycoprotein (MOG) peptide (35-55)-induced EAE in C57BL/6 mice with or without concomitant exposure to chronic mild low oxygen. We have reported that exposure to chronic mild low oxygen ameliorated clinical disease in EAE. While the mechanisms of protection are unclear, results suggest that normobaric hypoxia stabilizes the stress response, promotes physiological angiogenesis, and is neuroprotective.


Assuntos
Encéfalo/irrigação sanguínea , Encefalomielite Autoimune Experimental/terapia , Esclerose Múltipla/terapia , Neovascularização Fisiológica , Indutores da Angiogênese , Animais , Encéfalo/imunologia , Encéfalo/patologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Homeostase , Humanos , Hipóxia , Camundongos , Modelos Biológicos , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Medula Espinal/imunologia , Medula Espinal/patologia
7.
ASN Neuro ; 4(4): 207-21, 2012 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-22471445

RESUMO

Microglia express multiple TLRs (Toll-like receptors) and provide important host defence against viruses that invade the CNS (central nervous system). Although prior studies show these cells become activated during experimental alphavirus encephalitis in mice to generate cytokines and chemokines that influence virus replication, tissue inflammation and neuronal survival, the specific PRRs (pattern recognition receptors) and signalling intermediates controlling microglial activation in this setting remain unknown. To investigate these questions directly in vivo, mice ablated of specific TLR signalling molecules were challenged with NSV (neuroadapted Sindbis virus) and CNS viral titres, inflammatory responses and clinical outcomes followed over time. To approach this problem specifically in microglia, the effects of NSV on primary cells derived from the brains of wild-type and mutant animals were characterized in vitro. From the standpoint of the virus, microglial activation required viral uncoating and an intact viral genome; inactivated virus particles did not elicit measurable microglial responses. At the level of the target cell, NSV triggered multiple PRRs in microglia to produce a broad range of inflammatory mediators via non-overlapping signalling pathways. In vivo, disease survival was surprisingly independent of TLR-driven responses, but still required production of type-I IFN (interferon) to control CNS virus replication. Interestingly, the ER (endoplasmic reticulum) protein UNC93b1 facilitated host survival independent of its known effects on endosomal TLR signalling. Taken together, these data show that alphaviruses activate microglia via multiple PRRs, highlighting the complexity of the signalling networks by which CNS host responses are elicited by these infections.


Assuntos
Infecções por Alphavirus , Citocinas/metabolismo , Regulação para Baixo/fisiologia , Imunidade Inata/imunologia , Transdução de Sinais/imunologia , Infecções por Alphavirus/complicações , Infecções por Alphavirus/imunologia , Infecções por Alphavirus/patologia , Análise de Variância , Animais , Animais Recém-Nascidos , Antígeno CD11b/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Modelos Animais de Doenças , Encefalite Viral/etiologia , Encefalite Viral/imunologia , Encefalite Viral/patologia , Citometria de Fluxo , Regulação da Expressão Gênica/imunologia , Humanos , Interferon Tipo I/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/imunologia , Microglia/metabolismo , Monócitos/metabolismo , Mutação/genética , Receptor 3 Toll-Like/deficiência
8.
Brain Behav Immun ; 25(5): 922-31, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20933590

RESUMO

Cases of progressive multifocal leukoencephalopathy can occur in patients treated with the B cell depleting anti-CD20 antibody, rituximab, highlighting the importance of B cell surveillance of the central nervous system (CNS). The lymphoid chemokine, CXCL13, is critical for B cell recruitment and functional organization of peripheral lymphoid tissues, and CXCL13 levels are often elevated in the inflamed CNS. To more directly investigate the role of CXCL13 in CNS B cell migration, its role in animal models of infectious and inflammatory demyelinating disease was examined. During acute alphavirus encephalitis where viral clearance depends on the local actions of anti-viral antibodies, CXCL13 levels and B cell numbers increased in brain tissue over time. Surprisingly, however, CXCL13-deficient animals showed normal CNS B cell recruitment, unaltered CNS virus replication and clearance, and intact peripheral anti-viral antibody responses. During experimental autoimmune encephalomyelitis (EAE), CNS levels of CXCL13 increased as symptoms emerged and equivalent numbers of B cells were identified among the CNS infiltrates of CXCL13-deficient mice compared to control animals. However, CXCL13-deficient mice did not sustain pathogenic anti-myelin T cell responses, consistent with their known propensity to develop more self-limited EAE. These data show that CXCL13 is dispensable for CNS B cell recruitment in both models. The disease course is unaffected by CXCL13 in a CNS infection paradigm that depends on a pathogen-specific B cell response, while it is heightened and prolonged by CXCL13 when myelin-specific CD4+ T cells drive CNS pathology. Thus, CXCL13 could be a therapeutic target in certain neuroinflammatory diseases, but not by blocking B cell recruitment to the CNS.


Assuntos
Infecções por Alphavirus/imunologia , Linfócitos B/fisiologia , Quimiocina CXCL13/fisiologia , Quimiotaxia de Leucócito/fisiologia , Encefalite Viral/imunologia , Encefalomielite Autoimune Experimental/imunologia , Sindbis virus/imunologia , Infecções por Alphavirus/fisiopatologia , Animais , Astrócitos/imunologia , Astrócitos/virologia , Encéfalo/imunologia , Encéfalo/virologia , Células Cultivadas , Encefalite Viral/fisiopatologia , Encefalomielite Autoimune Experimental/fisiopatologia , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Microglia/imunologia , Microglia/virologia , Reação em Cadeia da Polimerase , Células Th1/imunologia , Células Th1/fisiologia , Células Th17/imunologia , Células Th17/fisiologia
9.
J Neuroimmunol ; 218(1-2): 83-93, 2010 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-19906446

RESUMO

Brain abscesses are mainly caused by either direct or indirect inoculation of gram positive bacteria including Stapylococcus aureus (S. aureus) or Streptococcus species into the central nervous system. In the present study, we aimed to compare potential changes in brain abscess pathogenesis induced by two different strains of S. aureus, namely the laboratory strain RN6390 and the clinical isolate Reynolds. Although the Reynolds strain was expected to be more resistant to eradication by the host, due to the existence of a polysaccharide capsule, and subsequently to be more virulent, instead we found parenchymal damage and mortality rates to be more prominent following RN6390 infection. In contrast, the Reynolds strain proliferated faster and induced early expression of the chemokine CXCL2, matrix metalloproteinase-9 (MMP-9), and complement 3a and C5. Furthermore, there were early and more abundant infiltration of PMNs, T cells and erythrocyte extravasation in brain abscesses induced by the Reynolds strain. However, several immune parameters were not different between the two strains during the later stages of the disease. These results suggest that capsular S. aureus can modulate innate immunity and complement system activation differently than the acapsular strain RN6390, and the early changes induced by Reynolds strain may have an important impact on survival.


Assuntos
Cápsulas Bacterianas/imunologia , Abscesso Encefálico/imunologia , Abscesso Encefálico/microbiologia , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Animais , Western Blotting , Quimiocina CXCL2/imunologia , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Metaloproteinase 9 da Matriz/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Infiltração de Neutrófilos/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Staphylococcus aureus
10.
Curr Top Microbiol Immunol ; 336: 41-61, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19688327

RESUMO

Brain abscesses arise from a localized parenchymal infection, typically elicited by pyogenic bacteria such as Staphylococcus aureus. Despite improvements in detection and treatment strategies, brain abscesses continue to occur, with an increased prevalence in developing countries and immune-compromised patients. Adding to the seriousness of these infections is the recent emergence of antibiotic-resistant strains of bacteria, which are becoming more commonly associated with brain abscesses. Recent studies using a mouse experimental brain abscess model have revealed a complex role for Toll-like receptors (TLRs) in disease pathogenesis. Interestingly, TLR2 has limited impact on the innate immune response during the acute stage of brain abscess formation induced by S. aureus but influences adaptive immunity. In contrast, mice deficient in MyD88, a central adapter molecule for the majority of TLRs in addition to the IL-1R and IL-18R, demonstrate severe defects in innate immunity coupled with exaggerated tissue destruction. It is envisioned that understanding the roles for TLRs in both resident CNS glia as well as infiltrating immune cells will provide insights into how the immune response to bacterial infection can be tailored to achieve effective pathogen destruction without inducing excessive bystander damage of surrounding noninfected brain parenchyma. A discussion of recent findings in this field is presented along with outstanding questions and the concept of a pathogen-necrosis-autoantigen triad for the amplification of TLR signaling is introduced.


Assuntos
Abscesso Encefálico/imunologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia , Receptor 2 Toll-Like/imunologia , Animais , Abscesso Encefálico/microbiologia , Humanos , Infecções Estafilocócicas/microbiologia
11.
ASN Neuro ; 1(2)2009 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-19570030

RESUMO

MyD88 KO (knockout) mice are exquisitely sensitive to CNS (central nervous system) infection with Staphylococcus aureus, a common aetiological agent of brain abscess, exhibiting global defects in innate immunity and exacerbated tissue damage. However, since brain abscesses are typified by the involvement of both activated CNS-resident and infiltrating immune cells, in our previous studies it has been impossible to determine the relative contribution of MyD88-dependent signalling in the CNS compared with the peripheral immune cell compartments. In the present study we addressed this by examining the course of S. aureus infection in MyD88 bone marrow chimaera mice. Interestingly, chimaeras where MyD88 was present in the CNS, but not bone marrow-derived cells, mounted pro-inflammatory mediator expression profiles and neutrophil recruitment equivalent to or exceeding that detected in WT (wild-type) mice. These results implicate CNS MyD88 as essential in eliciting the initial wave of inflammation during the acute response to parenchymal infection. Microarray analysis of infected MyD88 KO compared with WT mice revealed a preponderance of differentially regulated genes involved in apoptotic pathways, suggesting that the extensive tissue damage characteristic of brain abscesses from MyD88 KO mice could result from dysregulated apoptosis. Collectively, the findings of the present study highlight a novel mechanism for CNS-resident cells in initiating a protective innate immune response in the infected brain and, in the absence of MyD88 in this compartment, immunity is compromised.

12.
J Immunol ; 182(11): 7119-30, 2009 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-19454709

RESUMO

TLR2 plays a pivotal role in recognizing Staphylococcus aureus, a common etiologic agent of CNS parenchymal infections, such as brain abscess. We previously reported that brain abscesses of TLR2 knockout (KO) mice exhibited elevated IL-17 levels, suggesting the presence of an alternative pathway available to respond to S. aureus infection that may involve Th17 cells. Both CD4(+) and CD8(+) T cell infiltrates were elevated in brain abscesses of TLR2 KO mice at days 3, 7, and 14 postinfection compared with wild-type animals. Intracellular cytokine staining revealed a significant increase in the frequency of IL-17-producing Th17 cells in TLR2 KO mice with relatively few IFN-gamma-positive cells. gammadelta T cells were also a source of IL-17 in brain abscesses. Microglia, astrocytes, and macrophages were shown to express both IL-17RA and IL-17RC. Despite receptor expression, IL-17 was relatively ineffective at eliciting glial activation, whereas the cytokine augmented the ability of TNF-alpha to induce CXCL2 and CCL2 expression by macrophages. Based on the ability of IL-17 to elicit the release of chemokines and other proinflammatory mediators, we propose that the exaggerated IL-17 response that occurs in TLR2 KO mice functions in a compensatory manner to control brain abscess pathogenesis, with cells other than glia as targets for IL-17 action. This is supported by our findings in which innate immune infiltrates were not significantly different between TLR2 KO and wild-type mice in conjunction with the lack of prolonged alterations in the synthesis of other proinflammatory molecules during the course of infection.


Assuntos
Interleucina-17/biossíntese , Linfócitos T Auxiliares-Indutores/imunologia , Receptor 2 Toll-Like/deficiência , Animais , Abscesso Encefálico , Quimiotaxia de Leucócito , Citocinas/análise , Imunidade Inata , Camundongos , Camundongos Knockout , Receptores de Interleucina-17 , Linfócitos T/fisiologia
13.
J Immunol ; 181(6): 3841-9, 2008 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-18768838

RESUMO

Astrocytes participate in CNS innate immune responses as evident by their ability to produce a wide array of inflammatory mediators upon exposure to diverse stimuli. Although we have established that astrocytes use TLR2 to signal inflammatory mediator production in response to Staphylococcus aureus, a common etiological agent of CNS infections, the signal transduction pathways triggered by this pathogen and how TLR2 expression is regulated remain undefined. Three disparate inhibitors that block distinct steps in the NF-kappaB pathway, namely SC-514, BAY 11-7082, and caffeic acid phenethyl ester, attenuated NO, TNF-alpha, and CXCL2 release from S. aureus-activated astrocytes. Among these proinflammatory mediators, autocrine/paracrine TNF-alpha was pivotal for augmenting TLR2 expression, since receptor levels were not elevated in astrocytes isolated from TNF-alpha knockout mice upon bacterial exposure. Since TLR2 is critical for signaling astrocytic cytokine production in response to S. aureus, we evaluated the effect of TNF-alpha loss on proinflammatory mediator release. Interestingly, among the molecules assayed, only NO production was significantly attenuated in TNF-alpha knockout astrocytes compared with wild-type cells. Similar results were obtained following LPS treatment, suggesting that TNF-alpha is an important regulator of astrocytic TLR2 expression and NO release in response to diverse microbial stimuli. In addition, NF-kappaB inhibitors attenuated TNF-alpha-induced TLR2 expression in astrocytes. Overall, this study suggests that two important anti-bacterial effector molecules, TLR2 and NO, are regulated, in part, by NF-kappaB-dependent autocrine/paracrine effects of TNF-alpha in astrocytes.


Assuntos
Astrócitos/imunologia , Astrócitos/metabolismo , NF-kappa B/fisiologia , Transdução de Sinais/imunologia , Receptor 2 Toll-Like/biossíntese , Fator de Necrose Tumoral alfa/fisiologia , Adjuvantes Imunológicos/deficiência , Adjuvantes Imunológicos/genética , Adjuvantes Imunológicos/fisiologia , Animais , Astrócitos/microbiologia , Comunicação Autócrina/imunologia , Células Cultivadas , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/metabolismo , Comunicação Parácrina/imunologia , Transdução de Sinais/genética , Staphylococcus aureus/imunologia , Staphylococcus aureus/metabolismo , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/fisiologia , Fator de Necrose Tumoral alfa/deficiência , Fator de Necrose Tumoral alfa/genética
14.
PPAR Res ; 2008: 453120, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18584038

RESUMO

Microglia and astrocytes express numerous members of the Toll-like receptor (TLR) family that are pivotal for recognizing conserved microbial motifs expressed by a wide array of pathogens. Despite the critical role for TLRs in pathogen recognition, when dysregulated these pathways can also exacerbate CNS tissue destruction. Therefore, a critical balance must be achieved to elicit sufficient immunity to combat CNS infectious insults and downregulate these responses to avoid pathological tissue damage. We performed a comprehensive survey on the efficacy of various PPAR-gamma agonists to modulate proinflammatory mediator release from primary microglia and astrocytes in response to numerous TLR ligands relevant to CNS infectious diseases. The results demonstrated differential abilities of select PPAR-gamma agonists to modulate glial activation. For example, 15d-PGJ(2) and pioglitazone were both effective at reducing IL-12 p40 release by TLR ligand-activated glia, whereas CXCL2 expression was either augmented or inhibited by 15d-PGJ(2), effects that were dependent on the TLR ligand examined. Pioglitazone and troglitazone demonstrated opposing actions on microglial CCL2 production that were TLR ligand-dependent. Collectively, this information may be exploited to modulate the host immune response during CNS infections to maximize host immunity while minimizing inappropriate bystander tissue damage that is often characteristic of such diseases.

15.
J Immunol ; 180(7): 5004-16, 2008 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-18354226

RESUMO

Brain abscesses result from a pyogenic parenchymal infection commonly initiated by Gram-positive bacteria such as Staphylococcus aureus. Although the host immune response elicited following infection is essential for effective bacterial containment, this response also contributes to the significant loss of brain parenchyma by necrosis that may be reduced by modulating the inflammatory response. Ciglitazone, a PPAR-gamma agonist with anti-inflammatory properties, was evaluated for its ability to influence the course of brain abscess development when treatment was initiated 3 days following infection. Interestingly, abscess-associated bacterial burdens were significantly lower following ciglitazone administration, which could be explained, in part, by the finding that ciglitazone enhanced S. aureus phagocytosis by microglia. In addition, ciglitazone attenuated the expression of select inflammatory mediators during brain abscess development including inducible NO synthase, TNF-alpha, IL-1beta, CXCL2, and CCL3. Unexpectedly, ciglitazone also accelerated brain abscess encapsulation, which was typified by the heightened expression of fibronectin and alpha-smooth muscle actin-positive myofibroblasts. Collectively, through its ability to attenuate excessive inflammation and accelerate abscess encapsulation, ciglitazone may effectively sequester brain abscesses and limit bacterial dissemination.


Assuntos
Abscesso Encefálico/tratamento farmacológico , Abscesso Encefálico/metabolismo , PPAR gama/agonistas , PPAR gama/metabolismo , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/metabolismo , Tiazolidinedionas/uso terapêutico , Animais , Abscesso Encefálico/microbiologia , Abscesso Encefálico/patologia , Parede Celular/metabolismo , Fibronectinas/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/microbiologia , Inflamação/patologia , Ligantes , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Viabilidade Microbiana/efeitos dos fármacos , Microglia/efeitos dos fármacos , Microglia/imunologia , Óxido Nítrico Sintase Tipo II/metabolismo , PPAR gama/imunologia , Fagocitose/efeitos dos fármacos , Proteoglicanas/metabolismo , Sensibilidade e Especificidade , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/patologia , Staphylococcus aureus/fisiologia , Tiazolidinedionas/síntese química , Tiazolidinedionas/química
16.
Am J Pathol ; 171(4): 1199-214, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17717149

RESUMO

Minocycline exerts beneficial immune modulatory effects in several noninfectious neurodegenerative disease models; however, its potential to influence the host immune response during central nervous system bacterial infections, such as brain abscess, has not yet been investigated. Using a minocycline-resistant strain of Staphylococcus aureus to dissect the antibiotic's bacteriostatic versus immune modulatory effects in a mouse experimental brain abscess model, we found that minocycline significantly reduced mortality rates within the first 24 hours following bacterial exposure. This protection was associated with a transient decrease in the expression of several proinflammatory mediators, including interleukin-1beta and CCL2 (MCP-1). Minocycline was also capable of protecting the brain parenchyma from necrotic damage as evident by significantly smaller abscesses in minocycline-treated mice. In addition, minocycline exerted anti-inflammatory effects when administered as late as 3 days following S. aureus infection, which correlated with a significant decrease in brain abscess size. Finally, minocycline was capable of partially attenuating S. aureus-dependent microglial and astrocyte activation. Therefore, minocycline may afford additional therapeutic benefits extending beyond its antimicrobial activity for the treatment of central nervous system infectious diseases typified by a pathogenic inflammatory component through its ability to balance beneficial versus detrimental inflammation.


Assuntos
Antibacterianos/uso terapêutico , Abscesso Encefálico/tratamento farmacológico , Encefalite/tratamento farmacológico , Minociclina/uso terapêutico , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Animais , Antibacterianos/farmacologia , Abscesso Encefálico/imunologia , Abscesso Encefálico/patologia , Quimiocina CCL2/metabolismo , Encefalite/imunologia , Encefalite/microbiologia , Interleucina-1beta/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Minociclina/farmacologia , Neuroglia/efeitos dos fármacos , Neuroglia/imunologia , Infecções Estafilocócicas/imunologia , Receptor 2 Toll-Like/antagonistas & inibidores , Receptor 2 Toll-Like/metabolismo
17.
J Neuroinflammation ; 4: 10, 2007 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-17374157

RESUMO

BACKGROUND: It is well appreciated that obtaining sufficient numbers of primary microglia for in vitro experiments has always been a challenge for scientists studying the biological properties of these cells. Supplementing culture medium with granulocyte-macrophage colony-stimulating factor (GM-CSF) partially alleviates this problem by increasing microglial yield. However, GM-CSF has also been reported to transition microglia into a dendritic cell (DC)-like phenotype and consequently, affect their immune properties. METHODS: Although the concentration of GM-CSF used in our protocol for mouse microglial expansion (0.5 ng/ml) is at least 10-fold less compared to doses reported to affect microglial maturation and function (>/= 5 ng/ml), in this study we compared the responses of microglia derived from mixed glial cultures propagated in the presence/absence of low dose GM-CSF to establish whether this growth factor significantly altered the immune properties of microglia to diverse bacterial stimuli. These stimuli included the gram-positive pathogen Staphylococcus aureus (S. aureus) and its cell wall product peptidoglycan (PGN), a Toll-like receptor 2 (TLR2) agonist; the TLR3 ligand polyinosine-polycytidylic acid (polyI:C), a synthetic mimic of viral double-stranded RNA; lipopolysaccharide (LPS) a TLR4 agonist; and the TLR9 ligand CpG oligonucleotide (CpG-ODN), a synthetic form of bacteria/viral DNA. RESULTS: Interestingly, the relative numbers of microglia recovered from mixed glial cultures following the initial harvest were not influenced by GM-CSF. However, following the second and third collections of the same mixed cultures, the yield of microglia from GM-CSF-supplemented flasks was increased two-fold. Despite the ability of GM-CSF to expand microglial numbers, cells propagated in the presence/absence of GM-CSF demonstrated roughly equivalent responses following S. aureus and PGN stimulation. Specifically, the induction of tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2 (MIP-2/CXCL2), and major histocompatibility complex (MHC) class II, CD80, CD86 expression by microglia in response to S. aureus were similar regardless of whether cells had been exposed to GM-CSF during the mixed culture period. In addition, microglial phagocytosis of intact bacteria was unaffected by GM-CSF. In contrast, upon S. aureus stimulation, CD40 expression was induced more prominently in microglia expanded in GM-CSF. Analysis of microglial responses to additional pathogen-associate molecular patterns (PAMPs) revealed that low dose GM-CSF did not significantly alter TNF-alpha or MIP-2 production in response to the TLR3 and TLR4 agonists polyI:C or LPS, respectively; however, cells expanded in the presence of GM-CSF produced lower levels of both mediators following CpG-ODN stimulation. CONCLUSION: We demonstrate that low levels of GM-CSF are sufficient to expand microglial numbers without significantly affecting their immunological responses following activation of TLR2, TLR4 or TLR3 signaling. Therefore, low dose GM-CSF can be considered as a reliable method to achieve higher microglial yields without introducing dramatic activation artifacts.


Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Mediadores da Inflamação/metabolismo , Microglia/metabolismo , Microglia/patologia , Receptores de Reconhecimento de Padrão/fisiologia , Animais , Células Cultivadas , Técnicas de Cocultura/métodos , Relação Dose-Resposta Imunológica , Camundongos , Camundongos Endogâmicos C57BL , Microglia/microbiologia , Transdução de Sinais/fisiologia , Staphylococcus aureus/imunologia , Receptor 2 Toll-Like/fisiologia , Receptor 3 Toll-Like/fisiologia , Receptor 4 Toll-Like/fisiologia
18.
J Immunol ; 178(7): 4528-37, 2007 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-17372011

RESUMO

Brain abscesses form in response to a parenchymal infection by pyogenic bacteria, with Staphylococcus aureus representing a common etiologic agent of human disease. Numerous receptors that participate in immune responses to bacteria, including the majority of TLRs, the IL-1R, and the IL-18R, use a common adaptor molecule, MyD88, for transducing activation signals leading to proinflammatory mediator expression and immune effector functions. To delineate the importance of MyD88-dependent signals in brain abscesses, we compared disease pathogenesis using MyD88 knockout (KO) and wild-type (WT) mice. Mortality rates were significantly higher in MyD88 KO mice, which correlated with a significant reduction in the expression of several proinflammatory mediators, including but not limited to IL-1beta, TNF-alpha, and MIP-2/CXCL2. These changes were associated with a significant reduction in neutrophil and macrophage recruitment into brain abscesses of MyD88 KO animals. In addition, microglia, macrophages, and neutrophils isolated from the brain abscesses of MyD88 KO mice produced significantly less TNF-alpha, IL-6, MIP-1alpha/CCL3, and IFN-gamma-induced protein 10/CXCL10 compared with WT cells. The lack of MyD88-dependent signals had a dramatic effect on the extent of tissue injury, with significantly larger brain abscesses typified by exaggerated edema and necrosis in MyD88 KO animals. Interestingly, despite these striking changes in MyD88 KO mice, bacterial burdens did not significantly differ between the two strains at the early time points examined. Collectively, these findings indicate that MyD88 plays an essential role in establishing a protective CNS host response during the early stages of brain abscess development, whereas MyD88-independent pathway(s) are responsible for pathogen containment.


Assuntos
Abscesso Encefálico/imunologia , Fator 88 de Diferenciação Mieloide/fisiologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus , Animais , Abscesso Encefálico/genética , Abscesso Encefálico/patologia , Citocinas/metabolismo , Imunidade/genética , Macrófagos/imunologia , Camundongos , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Neutrófilos/imunologia , Transdução de Sinais , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/patologia
19.
Glia ; 55(1): 104-17, 2007 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-17029244

RESUMO

Gap junctions establish direct intercellular conduits between adjacent cells and are formed by the hexameric organization of protein subunits called connexins (Cx). It is unknown whether the proinflammatory milieu that ensues during CNS infection with S. aureus, one of the main etiologic agents of brain abscess in humans, is capable of eliciting regional changes in astrocyte homocellular gap junction communication (GJC) and, by extension, influencing neuron homeostasis at sites distant from the primary focus of infection. Here we investigated the effects of S. aureus and its cell wall product peptidoglycan (PGN) on Cx43, Cx30, and Cx26 expression, the main Cx isoforms found in astrocytes. Both bacterial stimuli led to a time-dependent decrease in Cx43 and Cx30 expression; however, Cx26 levels were elevated following bacterial exposure. Functional examination of dye coupling, as revealed by single-cell microinjections of Lucifer yellow, demonstrated that both S. aureus and PGN inhibited astrocyte GJC. Inhibition of protein synthesis with cyclohexamide (CHX) revealed that S. aureus directly modulates, in part, Cx43 and Cx30 expression, whereas Cx26 levels appear to be regulated by a factor(s) that requires de novo protein production; however, CHX did not alter the inhibitory effects of S. aureus on astrocyte GJC. The p38 MAPK inhibitor SB202190 was capable of partially restoring the S. aureus-mediated decrease in astrocyte GJC to that of unstimulated cells, suggesting the involvement of p38 MAPK-dependent pathway(s). These findings could have important implications for limiting the long-term detrimental effects of abscess formation in the brain which may include seizures and cognitive deficits.


Assuntos
Astrócitos/metabolismo , Encéfalo/metabolismo , Encéfalo/microbiologia , Conexinas/metabolismo , Junções Comunicantes/metabolismo , Staphylococcus aureus/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Encéfalo/fisiopatologia , Abscesso Encefálico/metabolismo , Abscesso Encefálico/fisiopatologia , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura , Conexina 26 , Conexina 30 , Conexina 43/efeitos dos fármacos , Conexina 43/genética , Conexina 43/metabolismo , Conexinas/efeitos dos fármacos , Conexinas/genética , Junções Comunicantes/efeitos dos fármacos , Isoquinolinas , Camundongos , Camundongos Endogâmicos C57BL , Peptidoglicano/metabolismo , Peptidoglicano/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/fisiopatologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
20.
J Immunol ; 176(11): 6802-11, 2006 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-16709840

RESUMO

Microglia, the innate immune effector cells of the CNS parenchyma, express TLR that recognize conserved motifs of microorganisms referred to as pathogen-associated molecular patterns (PAMP). All TLRs identified to date, with the exception of TLR3, use a common adaptor protein, MyD88, to transduce activation signals. Recently, we reported that microglial activation in response to the Gram-positive bacterium Staphylococcus aureus was not completely attenuated following TLR2 ablation, suggesting the involvement of additional receptors. To assess the functional role of alternative TLRs in microglial responses to S. aureus and its cell wall product peptidoglycan as well as the Gram-negative PAMP LPS, we evaluated primary microglia from MyD88 knockout (KO) and wild-type mice. The induction of TNF-alpha, IL-12 p40, and MIP-2 (CXCL2) expression by S. aureus- and peptidoglycan-stimulated microglia was MyD88 dependent, as revealed by the complete inhibition of cytokine production in MyD88 KO cells. In addition, the expression of additional pattern recognition receptors, including TLR9, pentraxin-3, and lectin-like oxidized LDL receptor-1, was regulated, in part, via a MyD88-dependent manner as demonstrated by the attenuated expression of these receptors in MyD88 KO microglia. Microglial activation was only partially inhibited in LPS-stimulated MyD88 KO cells, suggesting the involvement of MyD88-independent pathways. Collectively, these findings reveal the complex mechanisms for microglia to respond to diverse bacterial pathogens, which occur via both MyD88-dependent and -independent pathways.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Microglia/imunologia , Microglia/microbiologia , Receptores de Reconhecimento de Padrão/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Adjuvantes Imunológicos/biossíntese , Adjuvantes Imunológicos/fisiologia , Animais , Células Cultivadas , Quimiocina CXCL2 , Quimiocinas/biossíntese , Indução Enzimática/imunologia , Mediadores da Inflamação/metabolismo , Interleucina-12/biossíntese , Subunidade p40 da Interleucina-12 , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/enzimologia , Microglia/metabolismo , Fator 88 de Diferenciação Mieloide , Óxido Nítrico Sintase Tipo II/biossíntese , Peptidoglicano/farmacologia , Subunidades Proteicas/biossíntese , Receptores de Reconhecimento de Padrão/biossíntese , Transdução de Sinais/imunologia , Staphylococcus aureus/imunologia , Staphylococcus aureus/patogenicidade , Fator de Necrose Tumoral alfa/biossíntese
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