RESUMO
The goal of the innate immune system is containment of a pathogen at the site of infection prior to the initiation of an effective adaptive immune response. However, effector mechanisms must be kept in check to combat the pathogen while simultaneously limiting undesirable destruction of tissue resulting from these actions. Here we demonstrate that innate immune effector cells contain a peripheral poxvirus infection, preventing systemic spread of the virus. These innate immune effector cells are comprised primarily of CD11bâºLy6CâºLy6Gâ» monocytes that accumulate initially at the site of infection, and are then supplemented and eventually replaced by CD11bâºLy6CâºLy6G⺠cells. The phenotype of the CD11bâºLy6CâºLy6G⺠cells resembles neutrophils, but the infiltration of neutrophils typically occurs prior to, rather than following, accumulation of monocytes. Indeed, it appears that the CD11bâºLy6CâºLy6G⺠cells that infiltrated the site of VACV infection in the ear are phenotypically distinct from the classical description of both neutrophils and monocyte/macrophages. We found that CD11bâºLy6CâºLy6G⺠cells produce Type I interferons and large quantities of reactive oxygen species. We also observed that depletion of Ly6G⺠cells results in a dramatic increase in tissue damage at the site of infection. Tissue damage is also increased in the absence of reactive oxygen species, although reactive oxygen species are typically thought to be damaging to tissue rather than protective. These data indicate the existence of a specialized population of CD11bâºLy6CâºLy6G⺠cells that infiltrates a site of virus infection late and protects the infected tissue from immune-mediated damage via production of reactive oxygen species. Regulation of the action of this population of cells may provide an intervention to prevent innate immune-mediated tissue destruction.
Assuntos
Imunidade Inata , Interferon Tipo I/biossíntese , Monócitos/imunologia , Neutrófilos/imunologia , Vaccinia virus/imunologia , Vacínia/imunologia , Animais , Antígenos Ly/análise , Antígeno CD11b/análise , Interferon Tipo I/imunologia , Macrófagos/imunologia , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/virologia , Neutrófilos/virologia , Espécies Reativas de Oxigênio/metabolismo , Vacínia/virologia , Vaccinia virus/patogenicidadeRESUMO
BACKGROUND: Most of the known functions of microglia, including neurotoxic and neuroprotective properties, are attributed to morphologically-activated microglia. Resting, ramified microglia are suggested to primarily monitor their environment including synapses. Here, we show an active protective role of ramified microglia in excitotoxicity-induced neurodegeneration. METHODS: Mouse organotypic hippocampal slice cultures were treated with N-methyl-D-aspartic acid (NMDA) to induce excitotoxic neuronal cell death. This procedure was performed in slices containing resting microglia or slices that were chemically or genetically depleted of their endogenous microglia. RESULTS: Treatment of mouse organotypic hippocampal slice cultures with 10-50 µM N-methyl-D-aspartic acid (NMDA) induced region-specific excitotoxic neuronal cell death with CA1 neurons being most vulnerable, whereas CA3 and DG neurons were affected less. Ablation of ramified microglia severely enhanced NMDA-induced neuronal cell death in the CA3 and DG region rendering them almost as sensitive as CA1 neurons. Replenishment of microglia-free slices with microglia restored the original resistance of CA3 and DG neurons towards NMDA. CONCLUSIONS: Our data strongly suggest that ramified microglia not only screen their microenvironment but additionally protect hippocampal neurons under pathological conditions. Morphological activation of ramified microglia is thus not required to influence neuronal survival.
Assuntos
Hipocampo/citologia , Microglia/fisiologia , N-Metilaspartato/toxicidade , Degeneração Neural/patologia , Neurotoxinas/toxicidade , Animais , Antígeno CD11b/genética , Morte Celular/efeitos dos fármacos , Ácido Clodrônico/toxicidade , Relação Dose-Resposta a Droga , Proteína Glial Fibrilar Ácida/metabolismo , Hipocampo/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/citologia , Microglia/efeitos dos fármacos , Degeneração Neural/terapia , Neurônios/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Fosfopiruvato Hidratase/metabolismoRESUMO
BACKGROUND & AIMS: Natural killer (NK) cells are innate immune effector cells first characterized by their ability to lyse susceptible tumor cells. Recent studies demonstrated their role in initiating and modulating adaptive immunity. NK cells represent a larger percentage of the lymphoid population in liver than other organs, suggesting that hepatic NK cells express some unique function. Here, we examined the response of NK cells to liver injury that occurs in a mouse model of biliary obstruction. METHODS: Bile duct ligations (BDL) were performed in mice previously depleted or not depleted of NK cells. NK cell activation, interleukin (IL)-6 mRNA expression and protein production by Kupffer cells, and the ability of exogenous IL-6 to ameliorate liver injury in NK cell-depleted mice, were determined. RESULTS: The number of activated hepatic NK cells increased markedly following BDL. Activation was suppressed in mice rendered Kupffer cell-depleted prior to ligation. Increased liver injury occurred in NK cell-depleted mice correlating with a reduction in IL-6 production. Purified Kupffer cells, obtained from NK cell-depleted or anti-interferon (IFN)-γ monoclonal antibody-pretreated mice following BDL, produced less IL-6 in culture than did Kupffer cells derived from control animals. In culture, hepatic NK cells derived from BDL mice stimulated IFN-γ-dependent IL-6 production by Kupffer cells; splenic NK cells obtained from the same animals had a negligible effect. Treatment with recombinant murine IL-6 reduced liver injury in BDL, NK cell-depleted mice. CONCLUSIONS: Hepatic NK cells suppress cholestatic liver injury by stimulating Kupffer cell-dependent IL-6 production.
Assuntos
Colestase/imunologia , Colestase/prevenção & controle , Interleucina-6/biossíntese , Células Matadoras Naturais/imunologia , Células de Kupffer/imunologia , Imunidade Adaptativa , Animais , Colestase/etiologia , Modelos Animais de Doenças , Feminino , Interferon gama/metabolismo , Interleucina-6/deficiência , Interleucina-6/genética , Interleucina-6/farmacologia , Ativação Linfocitária , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Recombinantes/farmacologiaRESUMO
CD8(+) T cells are essential for long-term, vaccine-induced resistance against intracellular pathogens. Here we show that natural antibodies, acting in concert with complement, are endogenous adjuvants for the generation of protective CD8(+) T cells after vaccination against visceral leishmaniasis. IL-4 was crucial for the priming of vaccine-specific CD8(+) T cells, and we defined the primary source of IL-4 as a CD11b(+)CD11c(lo) phagocyte. IL-4 secretion was not observed in antibody-deficient mice and could be reconstituted with serum from normal, but not Btk immune-deficient, mice. Similarly, no IL-4 response or CD8(+) T-cell priming was seen in C1qa(-/-) mice. These results identify a new pathway by which immune complex-mediated complement activation can regulate T-cell-mediated immunity. We propose that this function of natural antibodies could be exploited when developing new vaccines for infectious diseases.
Assuntos
Adjuvantes Imunológicos , Anticorpos/imunologia , Linfócitos T CD8-Positivos/imunologia , Proteínas do Sistema Complemento/imunologia , Vacinas/imunologia , Animais , Antígenos de Protozoários/genética , Antígenos de Protozoários/metabolismo , Antígeno CD11b/imunologia , Antígeno CD11c/imunologia , Linfócitos T CD8-Positivos/metabolismo , Ativação do Complemento , Proteínas do Sistema Complemento/genética , Interleucina-12/metabolismo , Interleucina-4/genética , Interleucina-4/imunologia , Interleucina-4/metabolismo , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/prevenção & controle , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Fagócitos/imunologia , Fagócitos/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
See page 841 To explore systemic utilization of Epstein-Barr virus (EBV)-specific transcriptionally targeted adenoviruses, three vectors were constructed to examine kinetics, specificity, and biodistribution: adv.oriP.luc, expressing luciferase under EBV-specific control; adv.SV40luc, expressing luciferase constitutively; and adv.oriP.E1A.oriP.luc, a conditionally replicating adenovirus, expressing both luciferase and E1A. Bioluminescence imaging (BLI) was conducted on tumor-bearing severe combined immunodeficient (SCID) mice (C666-1, EBV-positive human nasopharyngeal cancer) treated intravenously (i.v.) with 3 × 108 infectious units (ifu) of the adenoviral vectors. At 72 hours, adv.oriPluc demonstrated an 8.4-fold higher tumor signal than adv.SV40luc; adv.oriP.E1A.oriP.luc was 26.7-fold higher; however, a significant liver signal was also observed, necessitating further action to improve biodistribution. Several compounds were examined to this end, including norepinephrine, serotonin, clodronate liposomes, and STI571, to determine whether any of these measures could improve adenoviral biodistribution. Each of these interventions was assessed using BLI in mice i.v. injected with adv.oriP.luc. STI571 achieved the highest increase in tumor-to-liver ratio (TLR; 6.6-fold), which was associated with a 59% reduction in tumor interstitial fluid pressure (IFP) along with a decrease in platelet-derived growth factor-ß receptor (PDGFßR) activation. This study reports the favorable modulation by STI571 of the biodistribution of adenoviral vectors, providing a potential approach to improving therapeutic outcome.
RESUMO
Gliomas represent the most frequent type of human brain tumor, and their strong invasiveness is a significant clinical problem. Microglia, the immunocompetent cells of the brain, contribute significantly to the tumor and are potential interaction partners of the glioma cells. We studied the impact of the presence of microglia on tumor cell invasion in cultured brain slices. To selectively deplete microglia, the slices were treated with clodronate-filled liposomes. When glioma cells were injected into slices devoid of endogenous microglia, the invasiveness of the tumors was significantly decreased as compared with controls. Inoculation of exogenous microglia together with glioma cells into cultured brain slices increased the infiltrative behavior of the tumor depending on the microglia/glioma cell ratio. Cell culture experiments revealed that soluble factors released from glioma cells strongly stimulate metalloprotease-2 activity in microglia. In the brain slices inoculated with glioma cells, increased activity of metalloprotease-2 was directly correlated with the abundance of microglia. Our data indicate that glioma cells stimulate microglial cells to increase breakdown of extracellular matrix and thereby promote tumor invasiveness.
Assuntos
Neoplasias Encefálicas/patologia , Glioma/patologia , Metaloproteases/metabolismo , Microglia/enzimologia , Invasividade Neoplásica/patologia , Animais , Antimetabólitos/administração & dosagem , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Ácido Clodrônico/administração & dosagem , Imunofluorescência , Glioma/metabolismo , Humanos , Lipossomos , Masculino , Camundongos , Microglia/efeitos dos fármacos , Microscopia Confocal , Técnicas de Cultura de ÓrgãosRESUMO
Atrophic age-related macular degeneration (AMD) is associated with the subretinal accumulation of mononuclear phagocytes (MPs). Their role in promoting or inhibiting retinal degeneration is unknown. We here show that atrophic AMD is associated with increased intraocular CCL2 levels and subretinal CCR2(+) inflammatory monocyte infiltration in patients. Using age- and light-induced subretinal inflammation and photoreceptor degeneration in Cx3cr1 knockout mice, we show that subretinal Cx3cr1 deficient MPs overexpress CCL2 and that both the genetic deletion of CCL2 or CCR2 and the pharmacological inhibition of CCR2 prevent inflammatory monocyte recruitment, MP accumulation and photoreceptor degeneration in vivo. Our study shows that contrary to CCR2 and CCL2, CX3CR1 is constitutively expressed in the retina where it represses the expression of CCL2 and the recruitment of neurotoxic inflammatory CCR2(+) monocytes. CCL2/CCR2 inhibition might represent a powerful tool for controlling inflammation and neurodegeneration in AMD.
Assuntos
Degeneração Macular/imunologia , Monócitos/imunologia , Células Fotorreceptoras de Vertebrados/imunologia , Receptores CCR2/imunologia , Receptores de Quimiocinas/deficiência , Animais , Receptor 1 de Quimiocina CX3C , Quimiocina CCL2/imunologia , Feminino , Humanos , Inflamação/genética , Inflamação/imunologia , Degeneração Macular/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infiltração de Neutrófilos , Receptores CCR2/genética , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/imunologiaRESUMO
The acute inflammatory response that follows spinal cord injury (SCI) contributes to secondary injury that results in the expansion of the lesion and further loss of neurologic function. A cascade of receptor-mediated signaling events after SCI leads to activation of innate immune responses including the migration of microglia and active recruitment of circulating leukocytes. Because conventional techniques do not always distinguish macrophages derived from CNS-resident microglia from blood-derived monocytes, the role that each macrophage type performs cannot be assessed unambiguously in these processes. We demonstrate that, in the normal and spinal cord-injured lys-EGFP-ki transgenic mouse, enhanced green fluorescent protein (EGFP) is expressed only in mature hematopoietic granulomyelomonocytic cells and not in microglia. This allowed us to assess the temporal and spatial relationships between microglia-derived and hematogenous macrophages as well as neutrophils during a period of 6 weeks after clip compression SCI. Within the lesion, EGFP-positive monocyte-derived macrophages were found at the epicenter surrounded by EGFP-negative-activated microglia and microglia-derived macrophages. Neutrophils were not present when EGFP-positive monocyte-derived macrophages were depleted, indicating that neutrophil persistence in the lesion depended on the presence of these monocytes. Thus, these 2 distinct macrophage populations can be independently identified and tracked, thereby allowing their roles in acute and chronic stages of SCI-associated inflammation to be defined.
Assuntos
Macrófagos/patologia , Microglia/patologia , Traumatismos da Medula Espinal/patologia , Animais , Antígenos CD1/metabolismo , Antígenos de Diferenciação/metabolismo , Antígenos Ly/metabolismo , Ácido Clodrônico/administração & dosagem , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Lipossomos/administração & dosagem , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/metabolismo , Muramidase/genética , Muramidase/metabolismo , Neutrófilos/metabolismo , Neutrófilos/patologia , Peroxidase/metabolismo , Traumatismos da Medula Espinal/prevenção & controle , Fatores de TempoRESUMO
The precise role that macrophages play in both influenza-induced pathology and the host's cytokine-mediated response to infection remains largely unknown. We examined the effects of lung macrophage depletion on susceptibility to influenza virus (H1N1, A/PR/8/34) infection and how this relates to the inflammatory cytokine response in the lungs. ICR mice were administered 100 µL of clodronate (CL(2)MDP) or PBS-encapsulated liposomes via an intranasal route 2 days before infection. Then, mice were intranasally inoculated with influenza virus and monitored for morbidity, mortality, and symptom severity for 21 days. Additional mice were sacrificed at 2 and 5 days postinfection, and lung tissue was analyzed for viral replication and for gene expression and protein concentration of interleukin-1ß (IL-1ß), IL-6, and TNF-α. Macrophage depletion increased morbidity, mortality, and symptom severity (P < 0.05) and viral replication at 2 and 5 days postinfection (P < 0.05). IL-1ß, IL-6, and TNF-α mRNA was greater at day 2 (P < 0.05) and IL-6 and TNF-α was greater at day 5 postinfection (P < 0.05) in macrophage depleted mice. Macrophage depletion increased protein concentration of IL-1ß and IL-6 at day 2 postinfection (P < 0.05). These data suggest that macrophages play a necessary role in controlling susceptibility to influenza virus and the host's cytokine-mediated response to influenza infection.