RESUMEN
Japanese encephalitis virus (JEV), a member of the Flaviviridae family, is a leading cause of viral encephalitis in humans. Survivors of this infection often develop lifelong neurological sequelae. Short-chain fatty acids (SCFAs) produced in the gut are vital mediators of the gut-brain axis. We aimed to study microRNA-based mechanisms of SCFAs in an in vitro model of JEV infection. N9 microglial cells were pretreated with SCFA cocktail before JEV infection. Cytokine bead analysis, immunoblotting, and PCR were performed to analyze relevant inflammatory markers. microRNA sequencing was performed using Illumina Hiseq, and bioinformatics tools were used for differentially expressed (DE) miRNAs and weighted gene co-expression network analysis (WGCNA). microRNA mimic/inhibitor experiments and luciferase assay were performed to study miRNA-target interaction. A significant reduction in monocyte chemoattractant protein (MCP1) and tumor necrosis factor alpha (TNFα) along with reduced expression of phospho-nuclear factor kappa B (phospho-NF-κB) was observed in SCFA conditions. Significant attenuation of histone deacetylase activity and protein expression was recorded. miRNA sequencing revealed 160 DE miRNAs in SCFA + JEV-treated cells at 6 h post-infection. WGCNA revealed miR-200a-3p, a hub miRNA significantly upregulated in SCFA conditions. Transcription factor ZBTB20 was bioinformatically predicted and validated as a gene target for miR-200a-3p. Further miRNA mimic/inhibitor assay demonstrated that miR-200-3p regulated ZBTB20 along with Iκßα that possibly dampened NF-κB signal activation downstream. IMPORTANCE: The gut-brain axis plays a pivotal role in the physiological state of an organism. Gut microbiota-derived metabolites are known to play a role in brain disorders including neuroviral infections. Short-chain fatty acids (SCFAs) appear to quench inflammatory markers in Japanese encephalitis virus-infected microglial cells in vitro. Mechanistically, we demonstrate the interaction between miR-200a-3p and ZBTB20 in regulating the canonical nuclear factor kappa B (NF-κB) signaling pathway via transcriptional regulation of Iκßα. Findings of this study pave the way to a better understanding of SCFA mechanisms that can be used to develop strategies against viral neuroinflammation.
Asunto(s)
Virus de la Encefalitis Japonesa (Especie) , Ácidos Grasos Volátiles , Inflamación , MicroARNs , Microglía , MicroARNs/genética , MicroARNs/metabolismo , Virus de la Encefalitis Japonesa (Especie)/fisiología , Virus de la Encefalitis Japonesa (Especie)/genética , Microglía/virología , Microglía/metabolismo , Ácidos Grasos Volátiles/metabolismo , Ácidos Grasos Volátiles/farmacología , Ratones , Animales , Inflamación/genética , Inflamación/virología , Encefalitis Japonesa/virología , Encefalitis Japonesa/genética , Encefalitis Japonesa/metabolismo , Línea Celular , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Inhibidor NF-kappaB alfa/metabolismo , Inhibidor NF-kappaB alfa/genética , FN-kappa B/metabolismo , FN-kappa B/genética , Transducción de Señal , HumanosRESUMEN
Short-chain fatty acids (SCFAs) are gut microbial metabolic derivatives produced during the fermentation of ingested complex carbohydrates. SCFAs have been widely regarded to have a potent anti-inflammatory and neuro-protective role and have implications in several disease conditions, such as, inflammatory bowel disease, type-2 diabetes, and neurodegenerative disorders. Japanese encephalitis virus (JEV), a neurotropic flavivirus, is associated with life threatening neuro-inflammation and neurological sequelae in infected hosts. In this study, we hypothesize that SCFAs have potential in mitigating JEV pathogenesis. Postnatal day 10 BALB/c mice were intraperitoneally injected with either a SCFA mixture (acetate, propionate, and butyrate) or PBS for a period of 7 days, followed by JEV infection. All mice were observed for onset and progression of symptoms. The brain tissue was collected upon reaching terminal illness for further analysis. SCFA-supplemented JEV-infected mice (SCFA + JEV) showed a delayed onset of symptoms, lower hindlimb clasping score, and decreased weight loss and increased survival by 3 days (p < 0.0001) upon infection as opposed to the PBS-treated JEV-infected animals (JEV). Significant downregulation of inflammatory cytokines TNF-α, MCP-1, IL-6, and IFN-Υ in the SCFA + JEV group relative to the JEV-infected control group was observed. Inflammatory mediators, phospho-NF-kB (P-NF-kB) and iba1, showed 2.08 ± 0.1 and 3.132 ± 0.43-fold upregulation in JEV versus 1.19 ± 0.11 and 1.31 ± 0.11-fold in the SCFA + JEV group, respectively. Tissue section analysis exhibited reduced glial activation (JEV groupâ42 ± 2.15 microglia/ROI; SCFA + JEV groupâ27.07 ± 1.8 microglia/ROI) in animals that received SCFA supplementation prior to infection as seen from the astrocytic and microglial morphometric analysis. Caspase-3 immunoblotting showed 4.08 ± 1.3-fold upregulation in JEV as compared to 1.03 ± 0.14-fold in the SCFA + JEV group and TUNEL assay showed a reduced cellular death post-JEV infection (JEV-6.4 ± 1.5 cells/ROI and SCFA + JEV-3.7 ± 0.73 cells/ROI). Our study critically contributes to the increasing evidence in support of SCFAs as an anti-inflammatory and neuro-protective agent, we further expand its scope as a potential supplementary intervention in JEV-mediated neuroinflammation.
Asunto(s)
Encefalitis Japonesa , Ácidos Grasos Volátiles , Microbioma Gastrointestinal , Enfermedades Neuroinflamatorias , Microbioma Gastrointestinal/fisiología , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Enfermedades Neuroinflamatorias/inmunología , Enfermedades Neuroinflamatorias/metabolismo , Enfermedades Neuroinflamatorias/microbiología , Microglía/efectos de los fármacos , Microglía/inmunología , Encefalitis Japonesa/tratamiento farmacológico , Encefalitis Japonesa/inmunología , Encefalitis Japonesa/microbiología , Encefalitis Japonesa/prevención & control , Encefalitis Japonesa/virología , Ácidos Grasos Volátiles/farmacología , Ácidos Grasos Volátiles/uso terapéutico , Virus de la Encefalitis Japonesa (Subgrupo)/efectos de los fármacos , Virus de la Encefalitis Japonesa (Subgrupo)/inmunología , Virus de la Encefalitis Japonesa (Subgrupo)/patogenicidad , Análisis de Supervivencia , Quimiocinas/inmunología , Quimiocinas/metabolismo , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Síndrome de Liberación de Citoquinas/inmunología , Síndrome de Liberación de Citoquinas/metabolismo , Síndrome de Liberación de Citoquinas/prevención & control , Humanos , Femenino , Animales , Ratones , Apoptosis/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/virología , Carga Viral/efectos de los fármacos , Factores de TiempoRESUMEN
The gut-brain axis (GBA) is the umbrella term to include all bidirectional communication between the brain and gastrointestinal (GI) tract in the mammalian body. Evidence from over two centuries describes a significant role of GI microbiome in health and disease states of the host organism. Short-chain fatty acids (SCFAs), mainly acetate, butyrate, and propionate that are the physiological forms of acetic acid, butyric acid, and propionic acid respectively, are GI bacteria derived metabolites. SCFAs have been reported to influence cellular function in multiple neurodegenerative diseases (NDDs). In addition, the inflammation modulating properties of SCFAs make them suitable therapeutic candidates in neuroinflammatory conditions. This review provides a historical background of the GBA and current knowledge of the GI microbiome and role of individual SCFAs in central nervous system (CNS) disorders. Recently, a few reports have also identified the effects of GI metabolites in the case of viral infections. Among these viruses, the flaviviridae family is associated with neuroinflammation and deterioration of CNS functions. In this context, we additionally introduce SCFA based mechanisms in different viral pathogenesis to understand the former's potential as agents against flaviviral disease.
Asunto(s)
Eje Cerebro-Intestino , Ácidos Grasos Volátiles , Microbioma Gastrointestinal , Enfermedades Neurodegenerativas , Enfermedades Neuroinflamatorias , Virosis , Animales , Humanos , Eje Cerebro-Intestino/fisiología , Ácido Butírico/metabolismo , Ácido Butírico/uso terapéutico , Ácidos Grasos Volátiles/metabolismo , Ácidos Grasos Volátiles/uso terapéutico , Mamíferos/metabolismo , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/metabolismo , Virosis/tratamiento farmacológico , Virosis/metabolismo , Microbioma Gastrointestinal/fisiología , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Enfermedades Neuroinflamatorias/metabolismoRESUMEN
Japanese Encephalitis Virus (JEV) is a neurotropic virus that invades Central Nervous System (CNS) and causes severe neuroinflammation. Given the abundance and the position of astrocytes in the CNS, we speculate that they might play a critical role in the process of neuroinflammation. Unfortunately, the role of astrocytes in JEV-mediated neuroinflammation has long been understated. In this study, we have attempted to assess the role of astrocyte-mediated neuroinflammation upon JEV infection. Mouse model of JEV infection, generated by intraperitoneal injection, showed severe reactive astrogliosis. To further address our hypothesis, we employed immortalized astrocytic cell line (in vitro) and primary astrocyte-enriched culture (ex vivo) as experimental models. JEV infection in the astrocytes induces proinflammatory cytokines like MCP1/CCL2 and IL6 in both ex vivo and in vitro cultures as observed from the cytometric bead array analysis. A significantly altered cytokine profile was observed using PCR analysis in in vitro and ex vivo models upon infection, with respect to control, validating our previous results. We also show that there exists a major inconsistency in the viral replication kinetics, wherein the cell line showed a robust rate of replication whereas the primary astrocyte-enriched culture showed negligibly low number of plaques, underlining the importance of the selection of appropriate experimental model system. In conclusion, we claim that astrocytes significantly contribute to JEV-mediated neuroinflammation, despite not being a CNS immune cell.