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1.
J Virol ; 91(22)2017 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-28878079

RESUMO

West Nile virus (WNV) is a neurotropic flavivirus that can cause significant neurological disease. Mouse models of WNV infection demonstrate that a proinflammatory environment is induced within the central nervous system (CNS) after WNV infection, leading to entry of activated peripheral immune cells. We utilized ex vivo spinal cord slice cultures (SCSC) to demonstrate that anti-inflammatory mechanisms may also play a role in WNV-induced pathology and/or recovery. Microglia are a type of macrophage that function as resident CNS immune cells. Similar to mouse models, infection of SCSC with WNV induces the upregulation of proinflammatory genes and proteins that are associated with microglial activation, including the microglial activation marker Iba1 and CC motif chemokines CCL2, CCL3, and CCL5. This suggests that microglia assume a proinflammatory phenotype in response to WNV infection similar to the proinflammatory (M1) activation that can be displayed by other macrophages. We now show that the WNV-induced expression of these and other proinflammatory genes was significantly decreased in the presence of minocycline, which has antineuroinflammatory properties, including the ability to inhibit proinflammatory microglial responses. Minocycline also caused a significant increase in the expression of anti-inflammatory genes associated with alternative anti-inflammatory (M2) macrophage activation, including interleukin 4 (IL-4), IL-13, and FIZZ1. Minocycline-dependent alterations to M1/M2 gene expression were associated with a significant increase in survival of neurons, microglia, and astrocytes in WNV-infected slices and markedly decreased levels of inducible nitric oxide synthase (iNOS). These results demonstrate that an anti-inflammatory environment induced by minocycline reduces viral cytotoxicity during WNV infection in ex vivo CNS tissue.IMPORTANCE West Nile virus (WNV) causes substantial morbidity and mortality, with no specific therapeutic treatments available. Antiviral inflammatory responses are a crucial component of WNV pathology, and understanding how they are regulated is important for tailoring effective treatments. Proinflammatory responses during WNV infection have been extensively studied, but anti-inflammatory responses (and their potential protective and reparative capabilities) following WNV infection have not been investigated. Minocycline induced the expression of genes associated with the anti-inflammatory (M2) activation of CNS macrophages (microglia) in WNV-infected SCSC while inhibiting the expression of genes associated with proinflammatory (M1) macrophage activation and was protective for multiple CNS cell types, indicating its potential use as a therapeutic reagent. This ex vivo culture system can uniquely address the ability of CNS parenchymal cells (neurons, astrocytes, and microglia) to respond to minocycline and to modulate the inflammatory environment and cytotoxicity in response to WNV infection without peripheral immune cell involvement.


Assuntos
Anti-Inflamatórios/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Minociclina/farmacologia , Coluna Vertebral/imunologia , Febre do Nilo Ocidental/tratamento farmacológico , Vírus do Nilo Ocidental/imunologia , Animais , Biomarcadores , Proteínas de Ligação ao Cálcio/imunologia , Citocinas/imunologia , Macrófagos/patologia , Camundongos , Proteínas dos Microfilamentos/imunologia , Microglia/imunologia , Microglia/patologia , Microglia/virologia , Óxido Nítrico Sintase Tipo II/imunologia , Coluna Vertebral/patologia , Coluna Vertebral/virologia , Febre do Nilo Ocidental/imunologia , Febre do Nilo Ocidental/patologia
2.
J Virol ; 88(22): 13005-14, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25165111

RESUMO

UNLABELLED: West Nile virus (WNV) is a neurotropic flavivirus that causes significant neuroinvasive disease involving the brain and/or spinal cord. Experimental mouse models of WNV infection have established the importance of innate and adaptive immune responses in controlling the extent and severity of central nervous system (CNS) disease. However, differentiating between immune responses that are intrinsic to the CNS and those that are dependent on infiltrating inflammatory cells has proven difficult. We used a murine ex vivo spinal cord slice culture (SCSC) model to determine the innate immune processes specific to the CNS during WNV infections. By 7 days after ex vivo infection of SCSCs, the majority of neurons and a substantial percentage of astrocytes were infected with WNV, resulting in apoptotic cell death and astrogliosis. Microglia, the resident immune cells of the CNS, were activated by WNV infection, as exemplified by their amoeboid morphology, the development of filopodia and lamellipodia, and phagocytosis of WNV-infected cells and debris. Microglial cell activation was concomitant with increased expression of proinflammatory cytokines and chemokines, including CXCL10, CXCL1, CCL5, CCL3, CCL2, tumor necrosis factor alpha (TNF-α), TNF-related apoptosis-inducing ligand (TRAIL), and interleukin-6 (IL-6). The application of minocycline, an inhibitor of neuroinflammation, altered the WNV-induced proinflammatory cytokine/chemokine expression profile, with inhibited production of CCL5, CCL2, and IL-6. Our findings establish that CNS-resident cells have the capacity to initiate a robust innate immune response against WNV infection in the absence of infiltrating inflammatory cells and systemic immune responses. IMPORTANCE: There are no specific treatments of proven efficacy available for WNV neuroinvasive disease. A better understanding of the pathogenesis of WNV CNS infection is crucial for the rational development of novel therapies. Development of a spinal cord slice culture (SCSC) model facilitates the study of WNV pathogenesis and allows investigation of the intrinsic immune responses of the CNS. Our studies demonstrate that robust CNS innate immune responses, including microglial activation and proinflammatory cytokine/chemokine production, develop independently of contributions from the peripheral immune system and CNS-infiltrating inflammatory cells.


Assuntos
Microglia/imunologia , Fagocitose , Medula Espinal/imunologia , Medula Espinal/virologia , Vírus do Nilo Ocidental/crescimento & desenvolvimento , Vírus do Nilo Ocidental/imunologia , Animais , Apoptose , Astrócitos/fisiologia , Astrócitos/virologia , Camundongos , Neurônios/fisiologia , Neurônios/virologia , Técnicas de Cultura de Órgãos , Cultura de Vírus
3.
J Virol ; 88(2): 1080-9, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24198425

RESUMO

Apoptosis is an important mechanism of West Nile virus (WNV) pathogenesis within the central nervous system (CNS). The signaling pathways that result in WNV-induced apoptotic neuronal death within the CNS have not been established. In this study, we identified death receptor (DR)-induced apoptosis as a pathway that may be important in WNV pathogenesis, based on the pattern of differential gene expression in WNV-infected, compared to uninfected, brains. Reverse transcription-PCR (RT-PCR) and Western blotting confirmed that genes involved in DR-induced apoptotic signaling are upregulated in the brain following WNV infection. Activity of the DR-associated initiator caspase, caspase 8, was also increased in the brains of WNV-infected mice and occurred in association with cleavage of Bid and activation of caspase 9. These results demonstrate that DR-induced apoptotic signaling is activated in the brain following WNV infection and suggest that the caspase 8-dependent cleavage of Bid promotes intrinsic apoptotic signaling within the brains of infected animals. Utilization of a novel ex vivo brain slice culture (BSC) model of WNV encephalitis revealed that inhibition of caspase 8 decreases virus-induced activation of caspase 3 and tissue injury. The BSC model allows us to examine WNV-induced pathogenesis in the absence of a peripheral immune response. Thus, our results indicate that WNV-induced neuronal injury in the brain is mediated by DR-induced apoptosis signaling and can occur in the absence of infiltrating immune cells. However, astrocytes and microglia were activated in WNV-infected BSC, suggesting that local immune responses influence WNV pathogenesis.


Assuntos
Apoptose , Encéfalo/citologia , Sistema Nervoso Periférico/imunologia , Receptores de Morte Celular/imunologia , Febre do Nilo Ocidental/fisiopatologia , Vírus do Nilo Ocidental/fisiologia , Animais , Encéfalo/enzimologia , Encéfalo/imunologia , Encéfalo/virologia , Caspase 3/genética , Caspase 3/imunologia , Caspase 8/genética , Caspase 8/imunologia , Caspase 9/genética , Caspase 9/imunologia , Feminino , Humanos , Masculino , Camundongos , Sistema Nervoso Periférico/enzimologia , Sistema Nervoso Periférico/virologia , Receptores de Morte Celular/genética , Transdução de Sinais , Febre do Nilo Ocidental/enzimologia , Febre do Nilo Ocidental/genética , Febre do Nilo Ocidental/imunologia , Vírus do Nilo Ocidental/genética
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