RESUMO
Cerebral malaria is the deadliest complication that can arise from Plasmodium infection. CD8 T-cell engagement of brain vasculature is a putative mechanism of neuropathology in cerebral malaria. To define contributions of brain endothelial cell major histocompatibility complex (MHC) class I antigen-presentation to CD8 T cells in establishing cerebral malaria pathology, we developed novel H-2Kb LoxP and H-2Db LoxP mice crossed with Cdh5-Cre mice to achieve targeted deletion of discrete class I molecules, specifically from brain endothelium. This strategy allowed us to avoid off-target effects on iron homeostasis and class I-like molecules, which are known to perturb Plasmodium infection. This is the first endothelial-specific ablation of individual class-I molecules enabling us to interrogate these molecular interactions. In these studies, we interrogated human and mouse transcriptomics data to compare antigen presentation capacity during cerebral malaria. Using the Plasmodium berghei ANKA model of experimental cerebral malaria (ECM), we observed that H-2Kb and H-2Db class I molecules regulate distinct patterns of disease onset, CD8 T-cell infiltration, targeted cell death and regional blood-brain barrier disruption. Strikingly, ablation of either molecule from brain endothelial cells resulted in reduced CD8 T-cell activation, attenuated T-cell interaction with brain vasculature, lessened targeted cell death, preserved blood-brain barrier integrity and prevention of ECM and the death of the animal. We were able to show that these events were brain-specific through the use of parabiosis and created the novel technique of dual small animal MRI to simultaneously scan conjoined parabionts during infection. These data demonstrate that interactions of CD8 T cells with discrete MHC class I molecules on brain endothelium differentially regulate development of ECM neuropathology. Therefore, targeting MHC class I interactions therapeutically may hold potential for treatment of cases of severe malaria.
Assuntos
Malária Cerebral , Camundongos , Humanos , Animais , Malária Cerebral/patologia , Malária Cerebral/prevenção & controle , Células Endoteliais/patologia , Encéfalo/patologia , Barreira Hematoencefálica/patologia , Linfócitos T CD8-Positivos , Endotélio/patologia , Camundongos Endogâmicos C57BL , Modelos Animais de DoençasRESUMO
Microglia are key players in maintaining brain homeostasis and exhibit phenotypic alterations in response to epileptic stimuli. However, it is still relatively unknown if these alterations are pro- or anti-epileptic. To unravel this dilemma, we employed chemogenetic manipulation of microglia using the artificial Gi-Dreadd receptor within a kainic acid (KA) induced murine seizure model. Our results indicate that acute Gi-Dreadd activation with Clozapine-N-Oxide can reduce seizure severity. Additionally, we observed increased interaction between microglia and neuronal soma, which correlated with reduced neuronal hyperactivity. Interestingly, prolonged activation of microglial Gi-Dreadds by repeated doses of CNO over 3 days, arrested microglia in a less active, homeostatic-like state, which associated with increased neuronal loss after KA induced seizures. RNAseq analysis revealed that prolonged activation of Gi-Dreadd interferes with interferon ß signaling and microglia proliferation. Thus, our findings highlight the importance of microglial Gi signaling not only during status epilepticus (SE) but also within later seizure induced pathology.
Assuntos
Microglia , Estado Epiléptico , Camundongos , Animais , Microglia/patologia , Convulsões/induzido quimicamente , Estado Epiléptico/induzido quimicamente , Anticonvulsivantes , Encéfalo/patologia , Ácido Caínico/farmacologiaRESUMO
Chemogenetic approaches using Designer Receptors Exclusively Activated by Designer Drugs (DREADD, a family of engineered GPCRs) were recently employed in microglia. Here, we used Cx3cr1CreER/+:R26hM4Di/+ mice to express Gi-DREADD (hM4Di) on CX3CR1+ cells, comprising microglia and some peripheral immune cells, and found that activation of hM4Di on long-lived CX3CR1+ cells induced hypolocomotion. Unexpectedly, Gi-DREADD-induced hypolocomotion was preserved when microglia were depleted. Consistently, specific activation of microglial hM4Di cannot induce hypolocomotion in Tmem119CreER/+:R26hM4Di/+ mice. Flow cytometric and histological analysis showed hM4Di expression in peripheral immune cells, which may be responsible for the hypolocomotion. Nevertheless, depletion of splenic macrophages, hepatic macrophages, or CD4+ T cells did not affect Gi-DREADD-induced hypolocomotion. Our study demonstrates that rigorous data analysis and interpretation are needed when using Cx3cr1CreER/+ mouse line to manipulate microglia.
Assuntos
Microglia , Neurônios , Camundongos , Animais , Neurônios/metabolismo , MacrófagosRESUMO
Activation of innate immunity in the brain is a prominent feature of Alzheimer's disease (AD). The present study investigated the regulation of innate immunity by wild-type serum injection in a transgenic AD mouse model. We found that treatment with wild-type mouse serum significantly reduced the number of neutrophils and microglial reactivity in the brains of APP/PS1 mice. Mimicking this effect, neutrophil depletion via Ly6G neutralizing antibodies resulted in improvements in AD brain functions. Serum proteomic analysis identified vascular endothelial growth factor-A (VEGF-A) and chemokine (C-X-C motif) ligand 1 (CXCL1) as factors enriched in serum samples, which are crucial for neutrophil migration and chemotaxis, leukocyte migration, and cell chemotaxis. Exogenous VEGF-A reversed amyloid ß (Aß)-induced decreases in cyclin-dependent kinase 5 (Cdk5) and increases in CXCL1 in vitro and blocked neutrophil infiltration into the AD brain. Endothelial Cdk5 overexpression conferred an inhibitory effect on CXCL1 and neutrophil infiltration, thereby restoring memory abilities in APP/PS1 mice. Our findings uncover a previously unknown link between blood-derived VEGF signaling and neutrophil infiltration and support targeting endothelial Cdk5 signaling as a potential therapeutic strategy for AD.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Camundongos , Animais , Camundongos Transgênicos , Fator A de Crescimento do Endotélio Vascular , Infiltração de Neutrófilos , Proteômica , Doença de Alzheimer/terapia , Transtornos da Memória , Modelos Animais de Doenças , Precursor de Proteína beta-Amiloide/genética , Presenilina-1/genéticaRESUMO
BACKGROUND: In the aftermath of the COVID-19 pandemic, there has been a surge in human metapneumovirus (HMPV) transmission, surpassing pre-epidemic levels. We aim to elucidate the clinical and epidemiological characteristics of HMPV infections in the post-COVID-19 pandemic era. METHODS: In this retrospective single-center study, participants diagnosed with laboratory confirmed HMPV infection through Targeted Next Generation Sequencing were included. The study encompassed individuals admitted to Henan Children's Hospital between April 29 and June 5, 2023. Demographic information, clinical records, and laboratory indicators were analyzed. RESULTS: Between April 29 and June 5, 2023, 96 pediatric patients were identified as infected with HMPV with a median age of 33.5 months (interquartile range, 12 ~ 48 months). The majority (87.5%) of infected children were under 5 years old. Notably, severe cases were statistically younger. Predominant symptoms included fever (81.3%) and cough (92.7%), with wheezing more prevalent in the severe group (56% vs 21.1%). Coinfection with other viruses was observed in 43 patients, with Epstein-Barr virus (EBV) (15.6%) or human rhinovirus A (HRV type A) (12.5%) being the most common. Human respiratory syncytial virus (HRSV) coinfection rate was significantly higher in the severe group (20% vs 1.4%). Bacterial coinfection occurred in 74 patients, with Haemophilus influenzae (Hin) and Streptococcus pneumoniae (SNP) being the most prevalent (52.1% and 41.7%, respectively). Severe patients demonstrated evidence of multi-organ damage. Noteworthy alterations included lower concentration of IL-12p70, decreased lymphocytes percentages, and elevated B lymphocyte percentages in severe cases, with statistical significance. Moreover, most laboratory indicators exhibited significant changes approximately 4 to 5 days after onset. CONCLUSIONS: Our data systemically elucidated the clinical and epidemiological characteristics of pediatric patients with HMPV infection, which might be instructive to policy development for the prevention and control of HMPV infection and might provide important clues for future HMPV research endeavors.
Assuntos
COVID-19 , Metapneumovirus , Infecções por Paramyxoviridae , Humanos , China/epidemiologia , Pré-Escolar , Metapneumovirus/genética , Metapneumovirus/isolamento & purificação , Estudos Retrospectivos , Feminino , Masculino , Lactente , Infecções por Paramyxoviridae/epidemiologia , Infecções por Paramyxoviridae/virologia , COVID-19/epidemiologia , Criança , Coinfecção/epidemiologia , Coinfecção/virologia , SARS-CoV-2/genéticaRESUMO
Spinal microglia are highly responsive to peripheral nerve injury and are known to be a key player in pain. However, there has not been direct evidence showing that selective microglial activation in vivo is sufficient to induce chronic pain. Here, we used optogenetic approaches in microglia to address this question employing CX3CR1creER/+: R26LSL-ReaChR/+ transgenic mice, in which red-activated channelrhodopsin (ReaChR) is inducibly and specifically expressed in microglia. We found that activation of ReaChR by red light in spinal microglia evoked reliable inward currents and membrane depolarization. In vivo optogenetic activation of microglial ReaChR in the spinal cord triggered chronic pain hypersensitivity in both male and female mice. In addition, activation of microglial ReaChR up-regulated neuronal c-Fos expression and enhanced C-fiber responses. Mechanistically, ReaChR activation led to a reactive microglial phenotype with increased interleukin (IL)-1ß production, which is likely mediated by inflammasome activation and calcium elevation. IL-1 receptor antagonist (IL-1ra) was able to reverse the pain hypersensitivity and neuronal hyperactivity induced by microglial ReaChR activation. Therefore, our work demonstrates that optogenetic activation of spinal microglia is sufficient to trigger chronic pain phenotypes by increasing neuronal activity via IL-1 signaling.
Assuntos
Dor Crônica/etiologia , Microglia/fisiologia , Nervos Espinhais/metabolismo , Animais , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/metabolismo , Channelrhodopsins/metabolismo , Dor Crônica/fisiopatologia , Feminino , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Microglia/metabolismo , Optogenética/métodos , Transdução de Sinais/fisiologia , Medula Espinal/metabolismo , Nervos Espinhais/fisiologiaRESUMO
The contribution of circulating verses tissue resident memory T cells (TRMs) to clinical neuropathology is an enduring question due to a lack of mechanistic insights. The prevailing view is TRMs are protective against pathogens in the brain. However, the extent to which antigen-specific TRMs induce neuropathology upon reactivation is understudied. Using the described phenotype of TRMs, we found that brains of naïve mice harbor populations of CD69+ CD103- T cells. Notably, numbers of CD69+ CD103- TRMs rapidly increase following neurological insults of various origins. This TRM expansion precedes infiltration of virus antigen-specific CD8 T cells and is due to proliferation of T cells within the brain. We next evaluated the capacity of antigen-specific TRMs in the brain to induce significant neuroinflammation post virus clearance, including infiltration of inflammatory myeloid cells, activation of T cells in the brain, microglial activation, and significant blood brain barrier disruption. These neuroinflammatory events were induced by TRMs, as depletion of peripheral T cells or blocking T cell trafficking using FTY720 did not change the neuroinflammatory course. Depletion of all CD8 T cells, however, completely abrogated the neuroinflammatory response. Reactivation of antigen-specific TRMs in the brain also induced profound lymphopenia within the blood compartment. We have therefore determined that antigen-specific TRMs can induce significant neuroinflammation, neuropathology, and peripheral immunosuppression. The use of cognate antigen to reactivate CD8 TRMs enables us to isolate the neuropathologic effects induced by this cell type independently of other branches of immunological memory, differentiating this work from studies employing whole pathogen re-challenge. This study also demonstrates the capacity for CD8 TRMs to contribute to pathology associated with neurodegenerative disorders and long-term complications associated with viral infections. Understanding functions of brain TRMs is crucial in investigating their role in neurodegenerative disorders including MS, CNS cancers, and long-term complications associated with viral infections including COVID-19.
Assuntos
COVID-19 , Viroses , Camundongos , Animais , Células T de Memória , Doenças Neuroinflamatórias , Linfócitos T CD8-Positivos , Encéfalo , Memória ImunológicaRESUMO
Nervous necrosis virus (NNV) is one of the most important fish viral pathogens infecting more than 120 fish species worldwide. Due to the mass mortality rates often seen among larvae and juveniles, few effective vaccines against NNV were developed up to now. Here, the protective effect of recombinant coat protein (CP) from red-spotted grouper nervous necrosis virus (RGNNV) fused with grouper ß-defensin (DEFB) as an oral vaccine was evaluated using Artemia as a biocarrier delivery system in pearl gentian grouper (Epinephelus lanceolatusâ × Epinephelus fuscoguttatusâ). Feeding with Artemia encapsulated with E. coli expressing control vector (control group), CP, or CP-DEFB showed no obvious side effects on the growth of groupers. ELISA and antibody neutralization assay showed that CP-DEFB oral vaccination group induced higher anti-RGNNV CP specific antibodies and exhibited higher neutralization potency than the CP and control group. Meanwhile, the expression levels of several immune and inflammatory factors in the spleen and kidney after feeding with CP-DEFB were also significantly increased compared with the CP group. Consistently, after challenge with RGNNV, groupers fed CP-DEFB and CP exhibited 100% and 88.23% relative percentage survival (RPS), respectively. Moreover, the lower transcription levels of viral genes and milder pathological changes in CP-DEFB group were detected compared with the CP and control group. Thus, we proposed that grouper ß-defensin functioned as an efficient molecular adjuvant for an improved oral vaccine against nervous necrosis virus infection.
Assuntos
Bass , Doenças dos Peixes , Nodaviridae , Infecções por Vírus de RNA , Vacinas Virais , beta-Defensinas , Animais , beta-Defensinas/genética , Infecções por Vírus de RNA/prevenção & controle , Infecções por Vírus de RNA/veterinária , Escherichia coli , Adjuvantes Imunológicos/farmacologia , Proteínas Recombinantes , Nodaviridae/fisiologia , Necrose , Proteínas de Peixes/genéticaRESUMO
Disease caused by Singapore grouper iridovirus (SGIV) results in major economic losses in the global grouper aquaculture industry. Vaccination is considered to be the most effective way to protect grouper from SGIV. In this study, the spores of Bacillus subtilis (B.subtilis) WB600 were utilized as the vehicle that the VP19 protein was displayed on the spores surface. To further investigate the effect of oral vaccination, the grouper were orally immunized with B.s-CotC-19 spores. After challenged, the survival rate of grouper orally vaccinated with B.s-CotC-19 spores was 34.5% and the relative percent survival (RPS) was 28.7% compared to the PBS group. Moreover, the viral load in the tissues of the B.s-CotC-19 group was significantly lower than that of the PBS group. The histopathological sections of head kidney and liver tissue from the B.s-CotC-19 group showed significantly less histopathology compared to the PBS group. In addition, the specific IgM levels in serum in the B.s-CotC-19 group was higher than those in the PBS group. In the hindgut tissue, the immune-related gene expression detected by quantitative real-time PCR (qRT-PCR) exhibited an increasing trend in different degrees in the B.s-CotC-19 group, suggesting that the innate and adaptive immune responses were activated. These results indicated that the oral administration of recombinant B.subtilis spores was effective for preventing SGIV infection. This study provided a feasible strategy for the controlling of fish virus diseases.
Assuntos
Bass , Infecções por Vírus de DNA , Doenças dos Peixes , Iridovirus , Ranavirus , Animais , Iridovirus/fisiologia , Bacillus subtilis/genética , Singapura , Esporos Bacterianos/genética , Ranavirus/fisiologia , Vacinação , Infecções por Vírus de DNA/prevenção & controle , Infecções por Vírus de DNA/veterináriaRESUMO
Hepatocellular carcinoma is a life-threatening malignant tumor found around the world for its high morbidity and mortality. Therefore, it is of great importance for sensitive analysis of liver cancer cells (HepG2 cells) in clinical diagnosis and biomedical research. To fulfill this demand, hollow CdIn2S4/In2S3 heterostructured microspheres (termed CdIn2S4/In2S3 for clarity) were prepared by a two-step hydrothermal strategy and applied for building a novel photoelectrochemical (PEC) cytosensor for ultrasensitive and accurate detection of HepG2 cells through specific recognition of CD133 protein on the cell surface with the respective aptamer. The optical properties of CdIn2S4/In2S3 were investigated by UV-vis diffuse reflectance spectroscopy (DRS) and PEC technology. By virtue of their appealing PEC characteristics, the resultant PEC sensor exhibited a wider dynamic linear range from 1 × 102 to 2 × 105 cells mL-1 with a lower limit of detection (LOD, 23 cells mL-1), combined by evaluating the expression level of CD133 protein stimulated by metformin as a benchmarked inhibitor. This work opens a valuable and feasible avenue for sensitive detection of diverse tumor cells, holding great potential in early clinical diagnosis and treatment coupled by screening inhibitors.
Assuntos
Técnicas Biossensoriais , Técnicas Eletroquímicas , Técnicas Eletroquímicas/métodos , Células Hep G2 , Humanos , MicroesferasRESUMO
The detection and therapy of cancers in the early stage significantly alleviate the associated dangers. Optical devices offer new opportunities for these early measures. However, the clinical translation of the existing methods is severely hindered by their relatively low sensitivity or unclear physiological metabolism. Here, an optical microfiber sensor with a drug loading gold nanorod-black phosphorous nanointerface, as an ultrasensitive biosensor and nanotherapy platform, is developed to meet the early-stage requirement. With interface sensitization and functionalization of the hybrid nanointerface, the microfiber sensor presents an ultrahigh sensing performance, achieving the selective detection of the HER2 biomarker with limits of detection of 0.66 aM in buffer solution and 0.77 aM in 10% serum. It can also distinguish breast cancer cells from other cells in the early stage. Additionally, enabled by the interface, the optical microfiber is able to realize cellular nanotherapy, including photothermal/chemotherapy with pump laser coupling after diagnosis, and evaluate therapy results in real time. The immobilization of the interface on the optical microfiber surface prevents the damage to normal cells induced by nanomaterial enrichment, making the device more efficient and intelligent. This study opens up a new avenue for the development of smart optical platforms for sensitive biosensing and precision therapy.
Assuntos
Técnicas Biossensoriais , Nanotubos , Dispositivos Ópticos , Ouro , FósforoRESUMO
Interferon (IFN)-induced protein 35 (IFI35, also known as IFP35), a member of IFN induced genes (ISGs), participates in virus infection, cancer progression and the chronic inflammatory diseases. However, its roles during fish nodavirus infection still remained largely unknown. In the present study, a homolog of IFI35 from orange spotted grouper (Epinephelus coioides) (EcIFI35) was cloned and characterized. The open reading frame of EcIFI35 was composed of 1,128 bp, and encoded a 375 amino acid polypeptide, which contained two conserved N-myc-interactor (Nmi)/IFP35 domains (NIDs). Homology analysis indicated that EcIFI35 shared 95.73% and 31.96% identity with homologs of giant grouper (E. lanceolatus) and human (Homo sapiens), respectively. The transcription of EcIFI35 was significantly up-regulated in grouper spleen (GS) cells after challenged with red-spotted grouper nervous necrosis virus (RGNNV), polyinosinic:polycytidylic acid [poly(I:C)] or lipopolysaccharide (LPS). The subcellular localization analysis showed that EcIFI35 encoded a cytoplasmic protein. The ectopic expression of EcIFI35 inhibited RGNNV replication by reducing viral genes transcription and protein synthesis. Co-immunoprecipitation (Co-IP) assay demonstrated that EcIFI35 interacted with RGNNV coat protein (CP), and partly co-localized with CP. EcIFI35 overexpression promoted the expression of IFN-related molecules and pro-inflammatory factors, including IFN regulatory factor 7 (IRF7), mitochondrial antiviral signaling protein (MAVS) and myxovirus resistance gene I (MxI), nuclear factor κB (NF-κB), interleukin 6 (IL-6) and IL-8. Together, our results revealed that EcIFI35 interacted with CP and inhibited fish nodavirus replication through positively regulated host innate immune response.
Assuntos
Bass , Infecções por Vírus de DNA , Doenças dos Peixes , Nodaviridae , Sequência de Aminoácidos , Aminoácidos/metabolismo , Animais , Antivirais , Fator VII/genética , Fator VII/metabolismo , Proteínas de Peixes/química , Regulação da Expressão Gênica , Humanos , Imunidade Inata/genética , Interferons/metabolismo , Interleucina-6/genética , Interleucina-8/genética , Lipopolissacarídeos , NF-kappa B/metabolismo , Nodaviridae/fisiologia , Poli I-C/farmacologia , Alinhamento de SequênciaRESUMO
Growing evidences have demonstrated that multiple TRIM (tripartite motif) proteins exert critical roles in host defense against different microbial pathogens. Although mammalian TRIM21 has been reported to function as an important regulatory factor in antiviral immune and inflammatory response, the role of fish TRIM21 against virus infection still remains largely unknown. In the present study, we investigated the characteristics of TRIM21 gene (EcTRIM21) from orange spotted grouper (Epinephelus coioides). The full-length EcTRIM21 cDNA encoded a 557 amino acid peptide with 92.1% and 31.14% identity with giant grouper (Epinephelus lanceolatus) and human (Homo sapiens), respectively. EcTRIM21 contained four conserved domains, including RING, B-Box, PRY and SPRY domain. EcTRIM21 expression was significantly up-regulated in response to Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) infection, suggesting that EcTRIM21 might be involved in host defense against fish virus infections. Subcellular localization showed that EcTRIM21 were distributed in the cytoplasm in a punctate manner. Overexpression of EcTRIM21 in vitro significantly inhibited RGNNV and SGIV replication, as evidenced by the decreased severity of cytopathic effect (CPE) and the reduced expression levels of viral core genes. Consistently, knockdown of EcTRIM21 by small interfering RNA (siRNA) promoted the replication of RGNNV and SGIV in vitro. Furthermore, EcTRIM21 overexpression increased both interferon (IFN) and interferon stimulated response element (ISRE) promoter activities. In addition, the transcription levels of IFN signaling related molecules were positively regulated by EcTRIM21 overexpression. Together, our data demonstrated that fish TRIM21 exerted antiviral activity against fish viruses through positive regulation of host interferon response.
Assuntos
Bass , Infecções por Vírus de DNA , Doenças dos Peixes , Iridovirus , Nodaviridae , Ranavirus , Sequência de Aminoácidos , Animais , Antivirais/farmacologia , Proteínas de Peixes/química , Humanos , Interferons/genética , Mamíferos/genética , Mamíferos/metabolismo , Nodaviridae/fisiologia , Filogenia , Ranavirus/fisiologia , Alinhamento de Sequência , Proteínas com Motivo Tripartido/químicaRESUMO
OBJECTIVES: This pilot study was intended to evaluate the effects of active game play on cognition, quality of life, and depression for older people with dementia. METHODS: Thirty-eight older people with dementia were recruited. Eighteen people received eight-week active game play using Xbox 360 Kinect. Twenty people received their usual care. The Mini Mental State Examination, Quality of Life-Alzheimer's Disease and Cornell Scale for Depression in Dementia were used to measure the outcomes. RESULTS: The results showed that there was no significant improvement on the mean scores of Mini Mental State Examination (P = .252), however, the active game play increased the mean score of Quality of Life-Alzheimer's Disease (P = .005), and reduced the mean score of Cornell Scale for Depression in Dementia (P = .001) in comparison with the usual care group. CONCLUSIONS: The study demonstrated that the active game play was effective in improving quality of life and alleviating depression in older people with dementia. CLINICAL IMPLICATIONS: Findings highlight the potential for gaming as a non-pharmacological interventions for older people with dementia.
Assuntos
Doença de Alzheimer , Demência , Idoso , Cognição , Demência/complicações , Demência/terapia , Depressão/terapia , Humanos , Projetos Piloto , Qualidade de VidaRESUMO
Gliosis is a histopathological characteristic of epilepsy that comprises activated microglia and astrocytes. It is unclear whether or how crosstalk occurs between microglia and astrocytes in the evolution of epilepsy. Here, we report in a mouse model of status epilepticus, induced by intracerebroventricular injection of kainic acid (KA), sequential activation of microglia and astrocytes and their close spatial interaction in the hippocampal CA3 region. Microglial ablation reduced astrocyte activation and their upregulation of complement C3. When compared to wild-type mice, both C3-/- and C3aR-/- mice had significantly less microglia-astrocyte interaction in response to KA-induced status epilepticus. Additionally, KA-injected C3-/- mice had significantly less histochemical evidence of neurodegeneration. The results suggest that the C3-C3aR pathway contributes to KA-induced neurodegeneration by mediating microglia-astrocyte communication. The C3-C3aR pathway may prove to be a potential therapeutic target for epilepsy treatment.
Assuntos
Epilepsia , Estado Epiléptico , Animais , Astrócitos , Complemento C3/genética , Ácido Caínico/toxicidade , Camundongos , Microglia , Estado Epiléptico/induzido quimicamenteRESUMO
Microglia play an important role in the central sensitization and chronic pain. However, a direct connection between microglial function and pain development in vivo remains incompletely understood. To address this issue, we applied chemogenetic approach by using CX3CR1creER/+:R26LSL-hM4Di/+ transgenic mice to enable expression of inhibitory Designer Receptors Exclusively Activated by Designer Drugs (Gi DREADD) in microglia. We found that microglial Gi DREADD activation inhibited spinal nerve transection (SNT)-induced microglial reactivity as well as chronic pain in both male and female mice. Gi DREADD activation downregulated the transcription factor interferon regulatory factor 8 (IRF8) and its downstream target pro-inflammatory cytokine interleukin 1 beta (IL-1ß). Using in vivo spinal cord recording, we found that activation of microglial Gi DREADD attenuated synaptic transmission following SNT. Our results demonstrate that microglial Gi DREADD reduces neuroinflammation, synaptic function and neuropathic pain after SNT. Thus, chemogenetic approaches provide a potential opportunity for interrogating microglial function and neuropathic pain treatment.
Assuntos
Dor Crônica , Neuralgia , Animais , Feminino , Masculino , Camundongos , Microglia , Medula Espinal , Nervos EspinhaisRESUMO
Red-spotted grouper nervous necrosis virus (RGNNV), the causative agent of viral nervous necrosis disease, has caused high mortality and heavy economic losses in marine aquaculture worldwide. However, changes in host cell metabolism during RGNNV infection remain largely unknown. Here, the global metabolic profiling during RGNNV infection and the roles of cellular fatty acid synthesis in RGNNV infection were investigated. As the infection progressed, 71 intracellular metabolites were significantly altered in RGNNV-infected cells compared with mock-infected cells. The levels of metabolites involved in amino acid biosynthesis and metabolism were significantly decreased, whereas those that correlated with fatty acid synthesis were significantly up-regulated during RGNNV infection. Among them, tryptophan and oleic acid were assessed as the most crucial biomarkers for RGNNV infection. In addition, RGNNV infection induced the formation of lipid droplets and re-localization of fatty acid synthase (FASN), indicating that RGNNV induced and required lipogenesis for viral infection. The exogenous addition of palmitic acid (PA) enhanced RGNNV infection, and the inhibition of FASN and acetyl-CoA carboxylase (ACC) significantly decreased RGNNV replication. Additionally, not only inhibition of palmitoylation and phospholipid synthesis, but also destruction of fatty acid ß-oxidation significantly decreased viral replication. These data suggest that cellular fatty acid synthesis and mitochondrial ß-oxidation are essential for RGNNV to complete the viral life cycle. Thus, it has been demonstrated for the first time that RGNNV infection in vitro overtook host cell metabolism and, in that process, cellular fatty acid synthesis was an essential component for RGNNV replication.
Assuntos
Peixes/metabolismo , Peixes/virologia , Metaboloma , Nodaviridae/metabolismo , Aminoácidos/biossíntese , Animais , Células Cultivadas , Doenças dos Peixes/virologia , Peixes/anatomia & histologia , Peixes/genética , Lipogênese , Redes e Vias Metabólicas , Nodaviridae/genética , Baço/citologia , Replicação ViralRESUMO
Immunosuppression of unknown aetiology is a hallmark feature of glioblastoma and is characterized by decreased CD4 T-cell counts and downregulation of major histocompatibility complex class II expression on peripheral blood monocytes in patients. This immunosuppression is a critical barrier to the successful development of immunotherapies for glioblastoma. We recapitulated the immunosuppression observed in glioblastoma patients in the C57BL/6 mouse and investigated the aetiology of low CD4 T-cell counts. We determined that thymic involution was a hallmark feature of immunosuppression in three distinct models of brain cancer, including mice harbouring GL261 glioma, B16 melanoma, and in a spontaneous model of diffuse intrinsic pontine glioma. In addition to thymic involution, we determined that tumour growth in the brain induced significant splenic involution, reductions in peripheral T cells, reduced MHC II expression on blood leucocytes, and a modest increase in bone marrow resident CD4 T cells. Using parabiosis we report that thymic involution, declines in peripheral T-cell counts, and reduced major histocompatibility complex class II expression levels were mediated through circulating blood-derived factors. Conversely, T-cell sequestration in the bone marrow was not governed through circulating factors. Serum isolated from glioma-bearing mice potently inhibited proliferation and functions of T cells both in vitro and in vivo. Interestingly, the factor responsible for immunosuppression in serum is non-steroidal and of high molecular weight. Through further analysis of neurological disease models, we determined that the immunosuppression was not unique to cancer itself, but rather occurs in response to brain injury. Non-cancerous acute neurological insults also induced significant thymic involution and rendered serum immunosuppressive. Both thymic involution and serum-derived immunosuppression were reversible upon clearance of brain insults. These findings demonstrate that brain cancers cause multifaceted immunosuppression and pinpoint circulating factors as a target of intervention to restore immunity.
Assuntos
Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/metabolismo , Tolerância Imunológica , Mediadores da Inflamação/metabolismo , Animais , Células da Medula Óssea/imunologia , Linfócitos T CD4-Positivos/imunologia , Proliferação de Células , Progressão da Doença , Feminino , Genes MHC da Classe II/genética , Glioblastoma/imunologia , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioma/imunologia , Glioma/metabolismo , Glioma/patologia , Masculino , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Parabiose , Convulsões/induzido quimicamente , Baço/imunologia , Baço/patologia , Theilovirus , Timo/patologiaRESUMO
Singapore grouper iridovirus (SGIV), belonging to genus Ranavirus, family Iridoviridae, causes great economic losses in the aquaculture industry. Previous studies demonstrated the lipid composition of intracellular unenveloped viruses, but the changes in host-cell glyceophospholipids components and the roles of key enzymes during SGIV infection still remain largely unknown. Here, the whole cell lipidomic profiling during SGIV infection was analyzed using UPLC-Q-TOF-MS/MS. The lipidomic data showed that glycerophospholipids (GPs), including phosphatidylcholine (PC), phosphatidylserine (PS), glycerophosphoinositols (PI) and fatty acids (FAs) were significantly elevated in SGIV-infected cells, indicating that SGIV infection disturbed GPs homeostasis, and then affected the metabolism of FAs, especially arachidonic acid (AA). The roles of key enzymes, such as cytosolic phospholipase A2 (cPLA2), 5-Lipoxygenase (5-LOX), and cyclooxygenase (COX) in SGIV infection were further investigated using the corresponding specific inhibitors. The inhibition of cPLA2 by AACOCF3 decreased SGIV replication, suggesting that cPLA2 might play important roles in the process of SGIV infection. Consistent with this result, the ectopic expression of EccPLA2α or knockdown significantly enhanced or suppressed viral replication in vitro, respectively. In addition, the inhibition of both 5-LOX and COX significantly suppressed SGIV replication, indicating that AA metabolism was essential for SGIV infection. Taken together, our results demonstrated for the first time that SGIV infection in vitro disturbed GPs homeostasis and cPLA2 exerted crucial roles in SGIV replication.
Assuntos
Peixes/virologia , Iridovirus/genética , Fosfolipases A2 Citosólicas/genética , Replicação Viral/genética , Animais , Aquicultura , Araquidonato 5-Lipoxigenase/genética , Peixes/genética , Glicerofosfolipídeos/genética , Iridovirus/patogenicidade , Fosfatidilcolinas/genética , Fosfatidilserinas/genética , SingapuraRESUMO
Interferon-induced transmembrane proteins (IFITMs) have been identified as important host restriction factors in mammals for the control of infection by multiple viruses. However, the antiviral functions of IFITMs against fish viruses remain largely uncertain. In this study, the IFITM3 homolog from orange spotted grouper (EcIFITM3) was cloned and its roles in grouper virus infection were investigated. The full-length cDNA of EcIFITM3 was 737 bp, which was composed of a 16 bp 5'-UTR, a 274 bp 3'-UTR, and a 447 bp ORF. EcIFITM3 encodes a 148-amino-acid polypeptide, which contains five domains, i.e., the N-terminal domain (aa 1-65), TM1 (aa 66-90), the cytoplasmic domain (aa 91-110), TM2 (aa 111-140), and the C-terminal domain (aa 141-148), and shares 78% and 47% identity with IFITM3 of gilthead seabream (Sparus aurata) and human (Homo sapiens), respectively. EcIFITM3 mRNA was detected in 12 tissues of healthy groupers, with the highest expression levels in the head kidney. Additionally, the in vitro mRNA levels of EcIFITM3 were significantly upregulated by infection with Singapore grouper iridovirus (SGIV) or red spotted grouper nervous necrosis virus (RGNNV), or treatment with polyinosinic-polycytidylic acid (poly I:C) or lipopolysaccharide (LPS). Subcellular localization analysis showed that EcIFITM3 was mainly distributed in the cell membrane of grouper cells. In vitro, the ectopic expression of EcIFITM3 inhibited SGIV and RGNNV infection, as demonstrated by the reduced severity of the cytopathic effect, decreased virus production, and low levels of viral mRNA and proteins. Consistently, knockdown of EcIFITM3 by small interfering RNAs (siRNAs) enhanced SGIV and RGNNV replication. EcIFITM3 overexpression and knockdown experiments both suggested that EcIFITM3 inhibits the infection of SGIV and RGNNV by restricting virus entry.