GAS6 as a potential target to alleviate neuroinflammation during Japanese encephalitis in mouse models.

Viral encephalitis is characterized by inflammation of the brain parenchyma caused by a variety of viruses, among which the Japanese encephalitis (JE) virus (JEV) is a typical representative arbovirus. Neuronal death, neuroinflammation, and breakdown of the blood brain barrier (BBB) constitute vicious circles of JE progression. Currently, there is no effective therapy to prevent this damage. Growth arrest specific gene 6 (GAS6) is a secreted growth factor that binds to the TYRO3, AXL, and MERTK (TAM) family of receptor tyrosine kinases and has been demonstrated to participate in neuroprotection and suppression of inflammation in many central nervous system (CNS) diseases which has great potential for JE intervention. In this study, we found that GAS6 expression in the brain was decreased and was reversely correlated with viral load and neuronal loss. Mice with GAS6/TAM signalling deficiency showed higher mortality and accelerated neuroinflammation during peripheral JEV infection, accompanied by BBB breakdown. GAS6 directly promoted the expression of tight junction proteins in bEnd.3 cells and strengthened BBB integrity, partly via AXL. Mice administered GAS6 were more resistant to JEV infection due to increased BBB integrity, as well as decreased viral load and neuroinflammation. Thus, targeted GAS6 delivery may represent a strategy for the prevention and treatment of JE especially in patients with impaired BBB.

The protective role of Mertk in JEV-induced encephalitis by maintaining the integrity of blood-brain barrier.

Japanese encephalitis is an acute infectious disease of the central nervous system caused by neurotropic Japanese encephalitis virus (JEV). As a member of TAM (Tyro3, Axl and Mertk) family, Mertk has involved in multiple biological processes by engaging with its bridging ligands Gas6 and Protein S, including invasion of pathogens, phagocytosis of apoptotic cells, inflammatory response regulation, and the maintenance of blood brain barrier (BBB) integrity. However, its role in encephalitis caused by JEV infection has not been studied in detail. Here, we found that Mertk-/- mice exhibited higher mortality and more rapid disease progression than wild-type mice after JEV challenge. There were no significant differences in viral load and cytokines expression level in peripheral tissues between Wild type and Mertk-/- mice. Furthermore, the absence of Mertk had little effect on the inflammatory response and immunopathological damage while it can cause an increased viral load in the brain. For the in vitro model of BBB, Mertk was shown to maintain the integrity of the BBB. In vivo, Mertk-/- mice exhibited higher BBB permeability and lower BBB integrity. Taken together, our findings demonstrate that Mertk acts as a protective factor in the development of encephalitis induced by JEV infection, which is mainly associated with its beneficial effect on BBB integrity, rather than its regulation of inflammatory response.

Structure-based discovery of antiviral inhibitors targeting the E dimer interface of Japanese encephalitis virus.

Flaviviruses are emerging arthropod-borne viruses posing a great threat to human beings worldwide. The E dimer configuration of the flavivirus was prominent during viral assembly, maturation and entry. Neutralization antibodies targeting E dimer played the important role in controlling the flavivirus infection. Previously, the ideal drug target of small molecular inhibitors of JEV was viral proteases and polymerases. The crystal structure of JEV E protein showed a conserved pocket in it is important at membrane fusion step. Recently, a set of anti-virus drugs has been found by virtual screening. Here, we show that the fusion-loop pocket of JEV E protein was a conservative region and an ideal drug target. ChemDiv-3 from virtual screening as the lead compound was found to show a relatively modest inhibition effect for JEV in vitro and in vivo test and could interfere with the formation of JEV sE dimer. ChemDiv-3 interacts with the amino acid residues ASN 313, PRO 314, ALA 315, and VAL 323 in E protein via hydrogen bonds for occupation of the fusion-loop pocket. The key binding sites LYS 312, ALA 513 and THR 317 forming the fusion-loop pocket are the same and other auxiliary sites are similar among the flavivirus. Taken together, the fusion-loop pocket of the flavivirus could be one promising target for drug discovery.

Exploring the shared genetic basis of major depressive disorder and frailty.

BACKGROUND: Major depressive disorder (MDD) and frailty impose substantial health and economic burdens. MDD is recognized as a significant risk factor for frailty, but the genetic associations between these conditions remain unclear. This study investigates the genetic correlation, shared pleiotropic loci, causal relationships, and comorbid genes between MDD and frailty. METHODS: The genetic correlation between MDD and frailty was assessed using linkage disequilibrium score regression (LDSC) based on data from genome-wide association studies (GWAS). A detailed analysis was performed to identify shared pleiotropic loci and causal relationships through cross-phenotype association tests and Mendelian randomization. Additionally, tissue enrichment analysis was conducted using stratified LDSC, gene-based associations with both conditions were assessed using Multimarker Analysis of Genomic Annotation (MAGMA), and pathway analysis of comorbid genes was performed using the g: GOSt tool. RESULTS: Our findings revealed a significant positive genetic correlation between MDD and frailty (rg = 0.65, P = 1.49E-219). We identified 57 shared risk SNPs between the two conditions, including 6 novel SNPs. Mendelian randomization analyses indicated robust causal effects of MDD on frailty and vice versa. Furthermore, we observed tissue-specific heritability enrichment in 9 brain tissues. By combining MAGMA and CPASSOC analyses, we identified 10 comorbid genes associated with both MDD and frailty, primarily involved in synapse formation, modulation, plasticity, and desaturase activity. CONCLUSION: This study provides strong evidence for a shared genetic basis between MDD and frailty. The identification of comorbid genes offers new insights into the mechanisms underlying the relationship between these conditions.

QKI deficiency in macrophages protects mice against JEV infection by regulating cell migration and antiviral response.

Japanese encephalitis (JE) is a major reason to cause viral encephalitis, with 50% patients suffering from severe neuro-inflammation and permanent neural injury. Effective anti-viral treatment is urgently needed. Here, we found RNA binding protein quaking (QKI) was involved in the progression of JE by regulating migration and anti-viral response of macrophages. After JE virus (JEV) infection, QKI-deficient mice had lower viral loads in the brain and fewer neurological symptoms. In comparison with control mice, proinflammatory cytokines in the brain of QKI-deficient animals revealed distinct patterns, with lower levels of IL-6 (interleukin-6) and IFN-ß (interferon-ß) at the early stage but higher levels at the end of JE. Then we found infiltration of CCR2 positive ((C-C motif) receptor 2) peripheral macrophages and CCR2 expression on macrophages were inhibited in QKI-deficient mice, while the expression of CCR2 ligands was not changed. Bioinformatical analysis showed that a QRE (quaking response element) located on 3'UTR (untranslated region) of Ccr2. We further verified that QKI was able to interact with Ccr2 mRNA and regulate its degradation in vitro. Additionally, since the IFN-ß production was increased in QKI-ablation mice after JEV infection, the anti-viral response was analyzed. Results in QKI-silenced N9 cells showed that the expression of RIG-I (retinoic acid-inducible gene-I) and TBK1 (TANK binding kinase 1) was increased, thus further inducing IRF3 (interferon regulatory factor 3) phosphorylation and interferon activation. Overall, these results revealed QKI mediated the anti-viral process via interfering migration of macrophages to CNS (central nervous system) and enhancing RIG-I/IRF3/IFN-ß pathway to restrict virus dissemination.

The Comparison of Inflammatory Cytokines (IL-6 and IL-18) and Immune Cells in Japanese Encephalitis Patients With Different Progression.

Background: Japanese encephalitis virus (JEV) is the main cause of viral encephalitis in Asia. Nowadays, no effective and specific therapy for JE patients is available except supportive treatment. The fatality rate of JE patients is still about 30%, and more than half of survivors suffered from various neuropsychiatric sequelae. Thus, more attention should be paid to JE. Methods: In this study, a retrospective cohort of JE patients was collected and the general features of JE patients admitted into the Department of Infectious Diseases were analyzed. Meanwhile, the dynamic change of plasma cytokines and immune cells in JE patients with divergent prognosis was detected and analyzed. Results: We found a mounted proportion of adult/old patients in JE cases. The level of IL-6 and IL-18 increased in JE patients especially in fatal individuals. There was a continuous decreased percentage of CD4+ T and B cells in severe JE patients with fatal outcome compared with the surviving JE patients. Conclusions: The consistent high level of IL-6 and IL-18 in the plasma and low proportion of CD4+ T and B cells in the PBMCs might be the indicators of poor prognosis.

Axl-/- neurons promote JEV infection by dampening the innate immunity.

Japanese encephalitis virus (JEV) causes the most commonly diagnosed viral encephalitis in Asia. JEV is a highly neurotropic flavivirus that can replicate efficiently in the brain. Axl belongs to the TAM (Tyro3, Axl, Mer) family, a group of tyrosine kinase receptors involved in the viral entry, micked as apoptotic bodies and regulation of innate immunity. However, the underlying mechanisms on its regulation in the neurons for JEV are unclear. Here, we found that Axl was upregulated in neurons after JEV infection. Unexpectedly, Axl deficient (Axl-/-) mice were more susceptible to JEV infection with increased viral loads in neurons. The RNA-sequencing analysis between the wild type neurons and Axl-/- neurons infected with JEV showed that many interferon-stimulated genes were downregulated in the Axl-/- neurons which innate immunity was attenuated largely. The rescue experiment in Axl-/- neurons indicated that Axl may be positively involved in the regulation of antiviral immunity. Taken together, our data demonstrated that Axl may play an antiviral role in JEV replication within neurons by modulating neuronal innate immunity.

miR-155 regulates pro- and anti-inflammatory cytokine expression in human monocytes during chronic hepatitis C virus infection.

BACKGROUND: Hepatitis C virus (HCV) dysregulates innate and adaptive immune responses while monocytes (M) play a crucial role in linking innate and adaptive immunity to control viral infection. A transcription factor T-bet is upregulated to dampen M functions via the c-Jun N-terminal kinase (JNK) pathway, followed by enhanced Tim-3 expression in chronic HCV infection. However, the molecular mechanisms that control the expression in M are yet unknown. miR-155 has been implicated as a key regulator controlling diverse biological processes through posttranscriptional repression, but the influences of miR-155 on these regulators and effectors still need to be studied. METHODS: Forty HCV-infected patients and 40 healthy subjects (HS) were recruited, THP-1 cells (human acute monocyte leukemia cell line) were cultured with HCV-infected Huh 7.5 cells. The expression levels of miR-155 and JNK1/JNK2/JNK3 were measured by real-time RT-PCR. IL-10/IL-12 was detected by flow cytometry. THP-1 cells were transfected with mimics-155 and negative control, SOCS1, p-STAT1, p65, p-smad, p-p38, and p-JNK were measured by Western blot. TNF-α levels were measured by ELISA. Student's t-test was used in statistics. RESULTS: The study showed that miR-155 was upregulated in CD14+ M in HCV-infected patients compared to healthy subjects (P<0.05). Moreover, the upregulation of miR-155 in CD14+ M from HCV-infected patients induced TNF-α production and JNK gene expression, which, in turn, led to T-bet upregulation. Also, miR-155 upregulation in CD14+ M of HCV-infected patients increased the IL-12 and decreased the IL-10 production. CONCLUSIONS: The obtained results indicated that miR-155 upregulation in M during HCV infection enhances the activation of TNF-α and JNK pathways, promotes the expression of transcription factor T-bet, and triggers pro- and anti-inflammatory mediators. Together, these data reveal new information regarding the mechanisms of chronic HCV infection.

Integrated Metabolomics and Transcriptomics Analyses Reveal Metabolic Landscape in Neuronal Cells during JEV Infection.

Japanese encephalitis virus (JEV) is a leading cause of viral encephalitis in endemic regions of Asia. The neurotropism of JEV and its high-efficiency replication in neurons are the key events for pathogenesis. Revealing the interplay between virus and host cells in metabolic facet is of great importance both for unraveling the pathogenesis mechanisms and providing novel antiviral targets. This study took advantage of the integration analysis of metabolomics and transcriptomics to depict the metabolic profiles of neurons during the early stage of JEV infection. Increased glycolysis and its branched pentose phosphate pathway (PPP) flux and impaired oxidative phosphorylation (OXPHOS) in glucose utilization, and the catabolic patterns of lipid metabolism were created to facilitate the biosynthesis of precursors needed for JEV replication in neurons. Pharmacological inhibitions of both glycolysis pathway and PPP in neurons suggested its indispensable role in maintaining the optimal propagation of JEV. In addition, analysis of metabolomic-transcriptomic regulatory network showed the pivotal biological function of lipid metabolism during JEV infection. Several pro-inflammatory lipid metabolites were significantly up-regulated and might partially be responsible for the progression of encephalitis. These unique metabolic reprogramming features might give deeper insight into JEV infected neurons and provide promising antiviral approaches targeting metabolism.

RIPK3 Promotes JEV Replication in Neurons via Downregulation of IFI44L.

Japanese encephalitis virus (JEV), the leading cause of viral encephalitis in Asia, is neurovirulent and neuroinvasive. Neurons are the main target of JEV infection and propagation. Receptor interacting serine/threonine-protein kinase 3 (RIPK3) has been reported to contribute to neuroinflammation and neuronal death in many central nervous system diseases. In this study, we found that the progression of JE was alleviated in RIPK3-knockout (RIPK3-/-) mice in both peripheral and intracerebral infection. RIPK3-knockdown (RIPK3-RNAi) neuro2a cells showed higher cell viability during JEV infection. Moreover, the JEV load was significantly decreased in RIPK3-/- mouse-derived primary neurons and RIPK3-RNAi neuro2a cells compared with wild-type neurons, but this was not observed in microglia. Furthermore, RNA sequencing of brain tissues showed that the level of the interferon (IFN)-induced protein 44-like gene (IFI44L) was significantly increased in JEV-infected RIPK3-/- mouse brains, RIPK3-/- neurons, and RIPK3-RNAi-neuro2a cells. Then, it was demonstrated that the propagation of JEV was inhibited in IFI44L-overexpressing neuro2a cells and enhanced in IFI44L and RIPK3 double knockdown neuro2a cells. Taken together, our results showed that the increased expression of RIPK3 following JEV infection played complicated roles. On the one hand, RIPK3 participated in neuroinflammation and neuronal death during JEV infection. On the other hand, RIPK3 inhibited the expression of IFI44L to some extent, leading to the propagation of JEV in neurons, which might be a strategy for JEV to evade the cellular innate immune response.

Mesenchymal stem cells alleviate Japanese encephalitis virus-induced neuroinflammation and mortality.

BACKGROUND: Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis in Asia. Japanese encephalitis (JE) caused by JEV is characterized by extensive inflammatory cytokine secretion, microglia activation, blood-brain barrier (BBB) breakdown, and neuronal death, all of which contribute to the vicious cycle of inflammatory damage. There are currently no effective treatments for JE. Mesenchymal stem cells (MSCs) have been demonstrated to have a therapeutic effect on many central nervous system (CNS) diseases by regulating inflammation and other mechanisms. METHODS: In vivo, 8- to 10-week-old mice were infected intraperitoneally with JEV and syngeneic bone marrow MSCs were administered through the caudal vein at 1 and 3 days post-infection. The mortality, body weight, and behavior were monitored daily. Brains from each group were harvested at the indicated times for hematoxylin and eosin staining, immunohistochemical observation, flow cytometric analysis, TUNEL staining, Western blot, quantitative real-time polymerase chain reaction, and BBB permeability assays. In vitro, co-culture and mixed culture experiments of MSCs with either microglia or neurons were performed, and then the activation state of microglia and survival rate of neurons were tested 48 h post-infection. RESULTS: MSC treatment reduced JEV-induced mortality and improved the recovery from JE in our mouse model. The inflammatory response, microglia activation, neuronal damage, BBB destruction, and viral load (VL) were significantly decreased in the MSC-treated group. In co-culture experiments, MSCs reprogrammed M1-to-M2 switching in microglia and improved neuron survival. Additionally, the VL was decreased in Neuro2a cells in the presence of MSCs accompanied by increased expression of interferon-α/ß. CONCLUSION: MSC treatment alleviated JEV-induced inflammation and mortality in mice.

MLKL Mediated Necroptosis Accelerates JEV-Induced Neuroinflammation in Mice.

Japanese encephalitis virus (JEV) is the most prevalent cause of viral encephalitis in Asia and the western Pacific. Neuronal death caused by JEV infection and inflammation induced cytotoxicity leads to progression and deterioration of Japanese encephalitis (JE). Mixed-lineage kinase domain-like protein (MLKL) mediated necroptosis is a newly discovered pathway of programmed cell death and participates in many inflammatory diseases. In this study, we demonstrated for the first time that necroptosis was involved in the neuronal loss during JE via immune-electron microscopy and immunochemistry. The expression of MLKL in neurons was upregulated in presence of JEV infection in vitro and in vivo. Deletion of MLKL alleviated the progression of JE and decreased the level of inflammatory cytokines in mice model. Taken together, this study provides evidence for the participation of necroptosis in the pathogenesis of JEV infection.

[Alterations and clinical signifecance of exosome-containing innate immunity related lncRNAs in patients of hemorrhagic fever with renal syndrome].

Objective To observe the alterations of innate immunity related long non-coding RNAs (lncRNAs) in exosomes extracted from the plasma of hemorrhagic fever with renal syndrome (HFRS) patients, and analyze their relationship with the disease stage and severity. Methods Exosomes were extracted from the plasma samples of HFRS patients, healthy controls and recovered HFRS patients. Transmission electronic microscopy and Western blotting were performed to confirm the efficiency of the extraction. lncRNA profiles in the different groups were determined by high-throughput sequencing. The contents of several innate immunity related lncRNAs were detected by quantitative real-time PCR, and their relationship with the disease stage and severity was analyzed. Results Exosomes from the plasma were accurately extracted. Innate immunity related lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1), negative regulator of interferon response (NRIR), negative regulator of antiviral response (NRAV) were found in exosomes. NEAT1 content was significantly reduced in the exosomes from HFRS patients compared with healthy controls and it was significantly restored in recovered HFRS patients. The exosome NEAT1 content was correlated with the epidemic of HFRS but had no relationship with the stage and severity of the disease. Conclusion Several innate immunity related lncRNAs exist in the exosome from HFRS patients, among which NEAT1 content significantly decreases in HFRS patients compared with healthy controls and recovered HFRS patients. The reduced NEAT1 level is correlated with the epidemic of HFRS.

[Eukaryotic expression and application of HCV Hebei strain E2 extracellular core region].

Objective To express core region of HCV1b (Hebei strain) E2 protein (E2c) by eukaryotic system, and establish the detection method of specific anti-HCV E2 antibody in the sera from hepatitis C patients. Methods Based on the literature, the E2c gene was modified from the HCV1b gene and synthesized via overlapping PCR. Thereafter, the E2c gene including tissue-type plasminogen activator (tPA) signal peptide was cloned into the pCI-neo eukaryotic expression vector, and the product was named pCI-tpa-1bE2c. After HEK293T cells were transfected with pCI-tpa-1bE2c, the supernatant was collected, condensed and purified. Its specificity was identified by Western blotting. Galanthus nivalis agglutinin (GNA)-based ELISA was used to detect the antibody against HCVE2 in the sera from hepatitis C patients. Results Modified HCV E2c protein was successfully expressed in HEK293T cells and the GNA-based ELISA was developed for detecting the antibody against HCV E2 in the sera from hepatitis C patients. Conclusion HCV-1bE2c protein can be effectively expressed in HEK293T cells and applied clinically.