A novel method of high-purity extracellular vesicle enrichment from microliter-scale human serum for proteomic analysis

We have developed a rapid, low-cost, and simple separation strategy to separate extracellular vesicles (EVs) from a small amount of serum (i.e.,<100 μL) with minimal contamination by serum proteins and lipoprotein particles to meet the high purity requirement for EV proteome analysis. EVs were separated by a novel polyester capillary channel polymer (PET C-CP) fiber phase/hydrophobic interaction chromatography (HIC) method which is rapid and can process small size samples. The collected EV fractions were subjected to a post-column cleanup protocol using a centrifugal filter to perform buffer exchange and eliminate potential coeluting non-EV proteins while minimizing EV sample loss. Downstream characterization demonstrated that our current strategy can separate EVs with the anticipated exosome-like particle size distribution and high yield (∼1 × 10¹¹ EV particles per mL of serum) in approximately 15 min. Proteome profiling of the EVs reveals that a group of genuine EV components were identified that have significantly less high-abundance blood proteins and lipoprotein particle contamination in comparison to traditional separation methods. The use of this methodology appears to address the major challenges facing EV separation for proteomics analysis. In addition, the EV post-column cleanup protocol proposed in the current work has the potential to be combined with other separation methods, such as ultracentrifugation (UC), to further purify the separated EV samples.     

Selective In Situ Analysis of Mature microRNAs in Extracellular Vesicles Using a DNA Cage-Based Thermophoretic Assay

Mature microRNAs (miRNAs) in extracellular vesicles (EVs) are involved in different stages of cancer progression, yet it remains challenging to precisely detect mature miRNAs in EVs due to the presence of interfering RNAs (such as longer precursor miRNAs, pre-miRNAs) and the low abundance of tumor-associated miRNAs. By leveraging the size-selective ability of DNA cages and polyethylene glycol (PEG)-enhanced thermophoretic accumulation of EVs, we devised a DNA cage-based thermophoretic assay for highly sensitive, selective, and in situ detection of mature miRNAs in EVs with a low limit of detection (LoD) of 2.05 fM. Our assay can profile EV mature miRNAs directly in serum samples without the interference of pre-miRNAs and the need for ultracentrifugation. A clinical study showed that EV miR-21 or miR-155 had an overall accuracy of 90 % for discrimination between breast cancer patients and healthy donors, which outperformed conventional molecular probes detecting both mature miRNAs and pre-miRNAs. We envision that our assay can advance EV miRNA-based diagnosis of cancer.     

A new horizon of precision medicine: combination of the microbiome and extracellular vesicles

Over several decades, the disease pattern of intractable disease has changed from acute infection to chronic disease accompanied by immune and metabolic dysfunction. In addition, scientific evidence has shown that humans are holobionts; of the DNA in humans, 1% is derived from the human genome, and 99% is derived from microbial genomes (the microbiome). Extracellular vesicles (EVs) are lipid bilayer-delimited nanoparticles and key messengers in cell-to-cell communication. Many publications indicate that microbial EVs are both positively and negatively involved in the pathogenesis of various intractable diseases, including inflammatory diseases, metabolic disorders, and cancers. Microbial EVs in feces, blood, and urine show significant differences in their profiles between patients with a particular disease and healthy subjects, demonstrating the potential of microbial EVs as biomarkers for disease diagnosis, especially for assessing disease risk. Furthermore, microbial EV therapy offers a variety of advantages over live biotherapeutics and human cell EV (or exosome) therapy for the treatment of intractable diseases. In summary, microbial EVs are a new tool in medicine, and microbial EV technology might provide us with innovative diagnostic and therapeutic solutions in precision medicine.Subject terms: Biomarkers, Biological therapy     

Microfluidics-based technologies for the analysis of extracellular vesicles at the single-cell level and single-vesicle level

Extracellular vesicles(EVs) are membrane vesicles secreted by cells, playing critical roles in mediating intercellular communications for various physiological and pathological processes. Most of the EV analysis is currently performed at the bulk level, obscuring the origin of the EVs and diverse characteristics of the individual extracellular vesicle. Technologies to analyze the extracellular vesicles at the single-cell and single-vesicle levels are needed to evaluate EV comprehensively and dec...     

Rapid isolation and quantification of extracellular vesicles from suspension-adapted human embryonic kidney cells using capillary-channeled polymer fiber spin-down tips

Exosomes, a subset of extracellular vesicles (EVs, 30–200-nm diameter), serve as biomolecular snapshots of their cell of origin and vehicles for intercellular communication, playing roles in biological processes, including homeostasis maintenance and immune modulation. The large-scale processing of exosomes for use as therapeutic vectors has been proposed, but these applications are limited by impure, low-yield recoveries from cell culture milieu (CCM). Current isolation methods are also limited by tedious and laborious workflows, especially toward an isolation of EVs from CCM for therapeutic applications. Employed is a rapid (<10 min) EV isolation method on a capillary-channeled polymer fiber spin-down tip format. EVs are isolated from the CCM of suspension-adapted human embryonic kidney cells (HEK293), one of the candidate cell lines for commercial EV production. This batch solid-phase extraction technique allows 10¹² EVs to be obtained from only 100-µl aliquots of milieu, processed using a benchtop centrifuge. The tip-isolated EVs were characterized using transmission electron microscopy, multi-angle light scattering, absorbance quantification, an enzyme-linked immunosorbent assay to tetraspanin marker proteins, and a protein purity assay. It is believed that the demonstrated approach has immediate relevance in research and analytical laboratories, with opportunities for production-level scale-up projected.     

Analysis of the human breast milk microbiome and bacterial extracellular vesicles in healthy mothers

The microbiota of human breast milk (HBM) contribute to infant gut colonization; however, whether bacterial extracellular vesicles (EVs) are present in HBM or might contribute to this process remains unknown. In this study, we characterized the HBM microbiota of healthy Korean mothers and measured the key bacteria likely affecting infant gut colonization by analyzing both the microbiota and bacterial EVs. A total of 22 HBM samples were collected from lactating mothers. The DNA of bacteria and bacteria-derived EVs was extracted from each sample. In alpha-diversity analyses, bacterial samples showed higher richness and evenness than bacterial EV samples, and beta-diversity analyses showed significant differences between bacteria and bacterial EVs within identical individual samples. Firmicutes accounted for the largest proportion among the phyla, followed by Proteobacteria, Bacteroidetes, and Actinobacteria, in both bacteria and bacterial EV samples. At the genus level, Streptococcus (25.1%) and Staphylococcus (10.7%) were predominant in bacterial samples, whereas Bacteroides (9.1%), Acinetobacter (6.9%), and Lactobacillaceae(f) (5.5%) were prevalent in bacterial EV samples. Several genera, including Bifidobacterium, were significantly positively correlated between the two samples. This study revealed the diverse bacterial communities in the HBM of healthy lactating mothers, and found that gut-associated genera accounted for a high proportion in bacterial EV samples. Our findings suggest the existence of key bacteria with metabolic activity that are independent of the major bacterial populations that inhabit HBM, and the possibility that EVs derived from these bacteria are involved in the vertical transfer of gut microbiota.Subject terms: Immunogenetics, Immunization     

Vaccination with Klebsiella pneumoniae-derived extracellular vesicles protects against bacteria-induced lethality via both humoral and cellular immunity

The emergence of multidrug-resistant Klebsiella pneumoniae highlights the need to develop preventive measures to ameliorate Klebsiella infections. Bacteria-derived extracellular vesicles (EVs) are spherical nanometer-sized proteolipids enriched with outer membrane proteins. Gram-negative bacteria-derived EVs have gained interest for use as nonliving complex vaccines. In the present study, we evaluated whether K. pneumoniae-derived EVs confer protection against bacteria-induced lethality. K. pneumoniae-derived EVs isolated from in vitro bacterial culture supernatants induced innate immunity, including the upregulation of co-stimulatory molecule expression and proinflammatory mediator production. EV vaccination via the intraperitoneal route elicited EV-reactive antibodies and interferon-gamma-producing T-cell responses. Three vaccinations with the EVs prevented bacteria-induced lethality. As verified by sera and splenocytes adoptive transfer, the protective effect of EV vaccination was dependent on both humoral and cellular immunity. Taken together, these findings suggest that K. pneumoniae-derived EVs are a novel vaccine candidate against K. pneumoniae infections.     

基于微流控芯片的细胞外囊泡分离技术研究进展

细胞外囊泡（extracellular vesicles，EVs）是脂质双分子层包绕形成的半球状囊泡。研究表明EVs存在重要的生物学功能，同时EVs排放的数量、种类以及内含蛋白质、脂质或RNA等构成变化与疾病密切相关。EVs的研究将有助于理解其生物学功能和作用机制，同时也有望用于疾病的诊断和治疗，因此拥有巨大的临床应用前景。从复杂的体液样品中分离捕获EVs是实现基于EVs开展医学研究以及临床诊断的前提，但是目前绝大多数的EVs分离捕获仍然是采用传统分离手段，纯度低、效率差，迫切需要高效和高选择性的EVs分离手段。先进的微流控芯片技术具有微型化、集成化和自动化的优势，利用微流控芯片的EVs分离技术研究已成热点，本文围绕相关研究的最新进展进行了综述。     

Theragnostic Applications of Mammal and Plant-Derived Extracellular Vesicles: Latest Findings,Current Technologies,and Prospects

The way cells communicate is not fully understood. However, it is well-known that extracellular vesicles (EVs) are involved. Researchers initially thought that EVs were used by cells to remove cellular waste. It is now clear that EVs function as signaling molecules released by cells to communicate with one another, carrying a cargo representing the mother cell. Furthermore, these EVs can be found in all biological fluids, making them the perfect non-invasive diagnostic tool, as their cargo causes functional changes in the cells upon receiving, unlike synthetic drug carriers. EVs last longer in circulation and instigate minor immune responses, making them the perfect drug carrier. This review sheds light on the latest development in EVs isolation, characterization and, application as therapeutic cargo, novel drug loading techniques, and diagnostic tools. We also address the advancement in plant-derived EVs, their characteristics, and applications; since plant-derived EVs only recently gained focus, we listed the latest findings. Although there is much more to learn about, EV is a wide field of research; what scientists have discovered so far is fascinating. This paper is suitable for those new to the field seeking to understand EVs and those already familiar with it but wanting to review the latest findings.     

Identification of extracellular nanoparticle subsets by nuclear magnetic resonance

Exosomes are a subset of secreted lipid envelope-encapsulated extracellular vesicles (EVs) of 50–150 nm diameter that can transfer cargo from donor to acceptor cells. In the current purification protocols of exosomes, many smaller and larger nanoparticles such as lipoproteins, exomers and microvesicles are typically co-isolated as well. Particle size distribution is one important characteristics of EV samples, as it reflects the cellular origin of EVs and the purity of the isolation. However, most of the physicochemical analytical methods today cannot illustrate the smallest exosomes and other small particles like the exomers. Here, we demonstrate that diffusion ordered spectroscopy (DOSY) nuclear magnetic resonance (NMR) method enables the determination of a very broad distribution of extracellular nanoparticles, ranging from 1 to 500 nm. The range covers sizes of all particles included in EV samples after isolation. The method is non-invasive, as it does not require any labelling or other chemical modification. We investigated EVs secreted from milk as well as embryonic kidney and renal carcinoma cells. Western blot analysis and immuno-electron microscopy confirmed expression of exosomal markers such as ALIX, TSG101, CD81, CD9, and CD63 in the EV samples. In addition to the larger particles observed by nanoparticle tracking analysis (NTA) in the range of 70–500 nm, the DOSY distributions include a significant number of smaller particles in the range of 10–70 nm, which are visible also in transmission electron microscopy images but invisible in NTA. Furthermore, we demonstrate that hyperpolarized chemical exchange saturation transfer (Hyper-CEST) with ¹²⁹Xe NMR indicates also the existence of smaller and larger nanoparticles in the EV samples, providing also additional support for DOSY results. The method implies also that the Xe exchange is significantly faster in the EV pool than in the lipoprotein/exomer pool.

Diffusion and xenon NMR based methods to determine a very broad range of sizes and sub-sets of extracellular vesicles.     

Profiling Phosphoproteome Landscape in Circulating Extracellular Vesicles from Microliters of Biofluids through Functionally Tunable Paramagnetic Separation

Many biological processes are regulated through dynamic protein phosphorylation. Monitoring disease-relevant phosphorylation events in circulating biofluids is highly appealing but also technically challenging. We introduce here a functionally tunable material and a strategy, extracellular vesicles to phosphoproteins (EVTOP), which achieves one-pot extracellular vesicles (EVs) isolation, extraction, and digestion of EV proteins, and enrichment of phosphopeptides, with only a trace amount of starting biofluids. EVs are efficiently isolated by magnetic beads functionalized with Ti^IV ions and a membrane-penetrating peptide, octa-arginine R₈⁺, which also provides the hydrophilic surface to retain EV proteins during lysis. Subsequent on-bead digestion concurrently converts EVTOP to Ti^IV ion-only surface for efficient enrichment of phosphopeptides for phosphoproteomic analyses. The streamlined, ultra-sensitive platform enabled us to quantify 500 unique EV phosphopeptides with only a few μL of plasma and over 1200 phosphopeptides with 100 μL of cerebrospinal fluid (CSF). We explored its clinical application of monitoring the outcome of chemotherapy of primary central nervous system lymphoma (PCNSL) patients with a small volume of CSF, presenting a powerful tool for broad clinical applications.     

16S rRNA gene-based metagenomic analysis reveals differences in bacteria-derived extracellular vesicles in the urine of pregnant and non-pregnant women

Recent evidence has indicated that bacteria-derived extracellular vesicles (EVs) are important for host–microbe communication. The aims of the present study were to evaluate whether bacteria-derived EVs are excreted via the urinary tract and to compare the composition of bacteria-derived EVs in the urine of pregnant and non-pregnant women. Seventy-three non-pregnant and seventy-four pregnant women were enrolled from Dankook University and Ewha Womans University hospitals. DNA was extracted from urine EVs after EV isolation using the differential centrifugation method. 16S ribosomal RNA (16S rRNA) gene sequencing was performed using high-throughput 454 pyrosequencing after amplification of the V1–V3 region of the 16S rDNA. The composition of 13 taxa differed significantly between the pregnant and non-pregnant women. At the genus level, Bacillus spp. EVs were more significantly enriched in the urine of the pregnant women than in that of the non-pregnant women (45.61% vs 0.12%, respectively). However, Pseudomonas spp. EVs were more dominant in non-pregnant women than in pregnant women (13.2% vs 4.09%, respectively). Regarding the compositional difference between pregnant women with normal and preterm delivery, EVs derived from Ureaplasma spp. and the family Veillonellaceae (including Megasphaera spp.) were more abundant in the urine of preterm-delivered women than in that of women with normal deliveries. Taken together, these data showed that Bacillus spp. EVs predominate in the urine of pregnant women, whereas Pseudomonas spp. EVs predominate in the urine of non-pregnant women; this suggests that Bacillus spp. EVs might have an important role in the maintenance of pregnancy.     

Size-Selective Harvesting of Extracellular Vesicles for Strategic Analyses Towards Tumor Diagnoses

Extracellular vesicles (EV), typified by exosomes or microvesicles, are expected to be effective diagnostic markers for cancers. The sizes of the vesicles range from 20 to 1000 nm, but the size-dependent variations of the contents of EVs are still poorly understood. We succeeded in the size-selective harvesting of the vesicles by utilizing the molecular weight-dependent characteristics of a variety of polyethylene glycols (PEG) as precipitating reagents and analyzed the antigens displayed on the surfaces of the vesicles and the miRNAs included in the vesicles from each size group. As a result, the relatively larger (<100 nm) particles precipitated by PEG5k clearly exhibited the greatest amount of epithelial cell adhesion molecule (EpCAM), from both breast cancer (MCF-7) and colon cancer (HCT116) cells, and a larger quantity of microRNA (miRNA) specific to breast cancer cells (miRNA155 for MCF-7) seemed to be contained in the PEG-precipitated particles. The results demonstrated that the quantities of both the tumor-specific miRNA and protein were similarly distributed among the several classes of the size-sorted EVs and that the size-selective harvesting of EVs may be informative for strategic analyses towards the diagnoses of cancers.     

The Role of Extracellular Vesicles in the Pathogenesis,Diagnosis, and Treatment of Osteoarthritis

Osteoarthritis (OA) is a degenerative joint disease that affects the entire joint and has been a tremendous burden on the health care system worldwide. Although cell therapy has made significant progress in the treatment of OA and cartilage regeneration, there are still a series of problems. Recently, more and more evidence shows that extracellular vesicles (EVs) play an important role in the progression and treatment of OA. Here, we discuss that EVs from different cell sources not only participate in OA progression, but can also be used as effective tools for the diagnosis and treatment of OA. In addition, cell pretreatment strategies and EV tissue engineering play an increasingly prominent role in the field of OA treatment. This article will systematically review the latest developments in these areas. As stated above, it may provide new insights for improving OA and cartilage regeneration.     

Electrospray ionization mass spectrometry and partial least squares discriminant analysis applied to the quality control of olive oil

Direct infusion electrospray ionization mass spectrometry in the positive ion mode [ESI(+)‐MS] is used to obtain fingerprints of aqueous–methanolic extracts of two types of olive oils, extra virgin (EV) and ordinary (OR), as well as of samples of EV olive oil adulterated by the addition of OR olive oil and other edible oils: corn (CO), sunflower (SF), soybean (SO) and canola (CA). The MS data is treated by the partial least squares discriminant analysis (PLS‐DA) protocol aiming at discriminating the above‐mentioned classes formed by the genuine olive oils, EV (1) and OR (2), as well as the EV adulterated samples, i.e. EV/SO (3), EV/CO (4), EV/SF (5), EV/CA (6) and EV/OR (7). The PLS‐DA model employed is built with 190 and 70 samples for the training and test sets, respectively. For all classes (1–7), EV and OR olive oils as well as the adulterated samples (in a proportion varying from 0.5 to 20.0% w/w) are properly classified. The developed methodology required no ions identification and demonstrated to be fast, as each measurement lasted about 3 min including the extraction step and MS analysis, and reliable, because high sensitivities (rate of true positives) and specificities (rate of true negatives) were achieved. Finally, it can be envisaged that this approach has potential to be applied in quality control of EV olive oils. Copyright © 2013 John Wiley & Sons, Ltd.     

Brain tumor diagnostic model and dietary effect based on extracellular vesicle microbiome data in serum

The human microbiome has been recently associated with human health and disease. Brain tumors (BTs) are a particularly difficult condition to directly link to the microbiome, as microorganisms cannot generally cross the blood–brain barrier (BBB). However, some nanosized extracellular vesicles (EVs) released from microorganisms can cross the BBB and enter the brain. Therefore, we conducted metagenomic analysis of microbial EVs in both serum (152 BT patients and 198 healthy controls (HC)) and brain tissue (5 BT patients and 5 HC) samples based on the V3–V4 regions of 16S rDNA. We then developed diagnostic models through logistic regression and machine learning algorithms using serum EV metagenomic data to assess the ability of various dietary supplements to reduce BT risk in vivo. Models incorporating the stepwise method and the linear discriminant analysis effect size (LEfSe) method yielded 12 and 29 significant genera as potential biomarkers, respectively. Models using the selected biomarkers yielded areas under the curves (AUCs) >0.93, and the model using machine learning resulted in an AUC of 0.99. In addition, Dialister and [Eubacterium] rectale were significantly lower in both blood and tissue samples of BT patients than in those of HCs. In vivo tests showed that BT risk was decreased through the addition of sorghum, brown rice oil, and garlic but conversely increased by the addition of bellflower and pear. In conclusion, serum EV metagenomics shows promise as a rich data source for highly accurate detection of BT risk, and several foods have potential for mitigating BT risk.Subject terms: Diagnostic markers, Machine learning     

The Latest Trends in Electric Vehicles Batteries

Global energy demand is rapidly increasing due to population and economic growth, especially in large emerging countries, which will account for 90% of energy demand growth to 2035. Electric vehicles (EVs) play a paramount role in the electrification revolution towards the reduction of the carbon footprint. Here, we review all the major trends in Li-ion batteries technologies used in EVs. We conclude that only five types of cathodes are used and that most of the EV companies use Nickel Manganese Cobalt oxide (NMC). Most of the Li-ion batteries anodes are graphite-based. Positive and negative electrodes are reviewed in detail as well as future trends such as the effort to reduce the Cobalt content. The electrolyte is a liquid/gel flammable solvent usually containing a LiFeP6 salt. The electrolyte makes the battery and battery pack unsafe, which drives the research and development to replace the flammable liquid by a solid electrolyte.     

Proteomic Signature of Extracellular Vesicles for Lung Cancer Recognition

The proteins of extracellular vesicles (EVs) that originate from tumors reflect the producer cells’ proteomes and can be detected in biological fluids. Thus, EVs provide proteomic signatures that are of great interest for screening and predictive cancer diagnostics. By applying targeted mass spectrometry with stable isotope-labeled peptide standards, we assessed the levels of 28 EV-associated proteins, including the conventional exosome markers CD9, CD63, CD81, CD82, and HSPA8, in vesicles derived from the lung cancer cell lines NCI-H23 and A549. Furthermore, we evaluated the detectability of these proteins and their abundance in plasma samples from 34 lung cancer patients and 23 healthy volunteers. The abundance of TLN1, TUBA4A, HSPA8, ITGB3, TSG101, and PACSIN2 in the plasma of lung cancer patients was measured using targeted mass spectrometry and compared to that in plasma from healthy volunteers. The most diagnostically potent markers were TLN1 (AUC, 0.95), TUBA4A (AUC, 0.91), and HSPA8 (AUC, 0.88). The obtained EV proteomic signature allowed us to distinguish between the lung adenocarcinoma and squamous cell carcinoma histological types. The proteomic cargo of the extracellular vesicles represents a promising source of potential biomarkers.     

Plasma extracellular vesicle delivery of miR-210-3p by targeting ATG7 to promote sepsis-induced acute lung injury by regulating autophagy and activating inflammation

Extracellular vesicles (EVs) can be used for intercellular communication by facilitating the transfer of miRNAs from one cell to a recipient cell. MicroRNA (miR)-210-3p is released into the blood during sepsis, inducing cytokine production and promoting leukocyte migration. Thus, the current study aimed to elucidate the role of plasma EVs in delivering miR-210-3p in sepsis-induced acute lung injury (ALI). Plasma EVs were isolated from septic patients, after which the expression of various inflammatory factors was measured using enzyme-linked immunosorbent assay. Cell viability and apoptosis were measured via cell counting kit-8 and flow cytometry. Transendothelial resistance and fluorescein isothiocyanate fluorescence were used to measure endothelial cell permeability. Matrigel was used to examine the tubulogenesis of endothelial cells. The targeting relationship between miR-210-3p and ATG7 was assessed by dual-luciferase reporter assays. The expression of ATG7 and autophagy-related genes was determined to examine autophagic activation. A sepsis mouse model was established by cecal ligation and puncture (CLP)-induced surgery. The level of miR-210-3p was highly enriched in septic EVs. MiR-210-3p enhanced THP-1 macrophage inflammation, BEAS-2B cell apoptosis, and HLMVEC permeability while inhibiting angiogenesis and cellular activity. MiR-210-3p overexpression reduced ATG7 and LC3II/LC3I expression and increased P62 expression. Improvements in vascular density and autophagosome formation, increased ATG7 expression, and changes in the ratio of LC3II/LC3I were detected, as well as reduced P62 expression, in adenovirus-anti-miR-210-3p treated mice after CLP injury. Taken together, the key findings of the current study demonstrate that plasma EVs carrying miR-210-3p target ATG7 to regulate autophagy and inflammatory activation in a sepsis-induced ALI model.Subject terms: Infection, Immunological disorders     

Phosphorylation of ERK-Dependent NF-κB Triggers NLRP3 Inflammasome Mediated by Vimentin in EV71-Infected Glioblastoma Cells

Enterovirus 71 (EV71) is a dominant pathogenic agent that may cause severe central nervous system (CNS) diseases among infants and young children in the Asia-pacific. The inflammasome is closely implicated in EV71-induced CNS injuries through a series of signaling pathways. However, the activation pathway of NLRP3 inflammasome involved in EV71-mediated CNS injuries remains poorly defined. In the studies, EV71 infection, ERK1/2 phosphorylation, and activation of NLRP3 are abolished in glioblastoma cells with low vimentin expression by CRISPR/Cas9-mediated knockdown. PD098059, an inhibitor of p-ERK, remarkably blocks the vimentin-mediated ERK1/2 phosphorylation in EV71-infected cells. Nuclear translocation of NF-κB p65 is dependent on p-ERK in a time-dependent manner. Moreover, NLRP3 activation and caspase-1 production are limited in EV71-infected cells upon the caffeic acid phenethyl ester (CAPE) administration, an inhibitor of NF-κB, which contributes to the inflammasome regulation. In conclusion, these results suggest that EV71-mediated NLRP3 inflammasome could be activated via the VIM-ERK-NF-κB pathway, and the treatment of the dephosphorylation of ERK and NF-κB inhibitors is beneficial to host defense in EV71-infected CNS.