Youhui Si’s research while affiliated with Huazhong Agricultural University and other places


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Publications (24)


Isolation and transcriptional profiling of antigen-presenting cells from mouse lung and MLNs following intranasal immunization
  • Article

October 2024

STAR Protocols

Xiuyu Wang

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Yihan Wang

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Qiang Wang

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Youhui Si


Fig. 1. Screening for antiviral compounds against JEV in a library of inhibitors of neurotransmitter-receptor-related molecules. (A) JEV-positive cells were quantified with a high-content screening instrument. Inhibitors with significant anti-JEV effects were selected. (B) Effects of potential anti-JEV compounds on JEV replication. Viral titers were measured with plaque assays. (C) Effects of clomipramine HCl at the indicated concentrations on the viability of primary neurons treated for 48 h. (D-G) Effect of clomipramine HCl on JEV replication. Primary neurons were infected with JEV (MOI = 1) and incubated with clomipramine HCl (10 μM). Viral titers, RNA copies, and E protein expression were measured with plaque assays (D), RT-qPCR (E), western blotting (F), and IFA (G) at indicated time points. Scale bar, 200 μm. (H) Primary neurons were infected with JEV (MOI = 1), and incubated with various concentrations of clomipramine HCl (0, 0.01, 0.1, 1, 5, or 10 μM). At 24 h postinfection, the supernatants and cells were collected. Viral titers and mRNA levels were measured with plaque assay and RT-qPCR, respectively, and IC 50 was calculated. * p < 0.05; * * p < 0.01; * * * p < 0.001; * * * * p < 0.0001; or ns, no significance.
Fig. 2. Clomipramine HCl inhibits JEV replication in vitro. (A) Viability of SH-SY 5Y (upper panel) and N2a cells (lower panel) treated with clomipramine HCl at the indicated concentrations for 48 h. (B and C) SH-SY 5Y and N2a cells were infected with JEV at MOI = 1, and incubated with 10 μM clomipramine HCl. At the indicated times post-infection, the supernatants and cells were collected, and the viral titers and RNA copies were measured with plaque assays (B) and RT-qPCR, respectively (C). (D and E) SH-SY 5Y cells and N2a cells were infected with JEV (MOI = 1) and incubated with various concentrations of clomipramine HCl (0, 0.01, 0.1, 1, 5, or 10 μM). At 24 h post-infection, the supernatants and cells were collected. Viral titers and RNA copies were measured with plaque assays (D) and RT-qPCR, respectively (E), and IC 50 was calculated. * p < 0.05; * * p < 0.01; * * * p < 0.001; * * * * p < 0.0001; or ns, no significance.
Fig. 3. Clomipramine HCl inhibits JEV assembly. (A) Brief schematic diagram of time-of-addition experiment. (B and C) Viral titers and RNA copies in each group were determined with plaque assays (B) and RT-qPCR, respectively (C). (D and E) JEV adsorption and invasion assay in SH-SY 5Y cells. SH-SY 5Y cells were incubated with JEV (MOI = 1) or JEV (MOI = 1) + clomipramine HCl at 4 °C for 1 h. The viral titers were determined with a plaque assay, and the RNA levels were determined with RT-qPCR. (D) SH-SY 5Y cells were incubated with JEV (MOI = 1) or JEV (MOI = 1) + clomipramine HCl at 4 °C for 1 h. The cells were then washed three times with cold alkaline high-salt solution and treated with H 2 O or clomipramine HCl (10 μM) at 37 °C for another 10 min. The cells were collected for a plaque assay to determine the viral titer (E). (F and G) Assembly and release assays in SH-SY 5Y cells. SH-SY 5Y cells were incubated with JEV (MOI = 1) at 4 °C for 1 h. Unattached virus was removed by washing three times with PBS. The cells were then incubated with H 2 O or clomipramine HCl (10 μM) at 37 °C. The supernatants and cells were harvested at the indicated time points. Intracellular and extracellular viral titers were determined with plaque assays. Viral RNA was measured with RT-qPCR. * p < 0.05; * * p < 0.01; * * * p < 0.001; * * * * p < 0.0001; or ns, no significance.
Fig. 4. Clomipramine HCl inhibits JEV replication through SERT. (A) SH-SY 5Y cells were transfected with 3 × Flag-SERT-expressing plasmid or empty vector. Expression levels of SERT were determined with an immunoblotting assay at 48 h post-transfection. (B and C) At 24 h post-transfection, SH-SY 5Y cells were infected with JEV at MOI = 1 and incubated with H 2 O or clomipramine HCl (10 μM). At 24 h post-infection, the viral titers and viral RNA copies were measured with plaque assay (B) and RT-qPCR, respectively (C). * p < 0.05; * * p < 0.01; * * * p < 0.001; * * * * p < 0.0001; or ns, no significance.
Fig. 5. Clomipramine HCl activates ER stress and downstream PERK-mediated UPR. (A) Hierarchical clustering of differentially expressed genes. Red indicates upregulated genes and green indicates downregulated genes. (B) Volcano plot represents modified genes. Blue dots denote non-significantly expressed genes. Upregulated genes are shown as red dots and downregulated genes as green dots. (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the differentially expressed genes (D) Heatmap of genes related to ER stress. (E) RT-qPCR was used to analyze the expression of ER-stress-related genes. (F) SH-SY 5Y cells were treated with clomipramine HCl for 24 h. Levels of BIP protein were determined with an immunoblotting assay (left panel). Quantitative analysis of optical band densitometry (right panel). (G) Whole-cell mRNA was isolated from clomipramine-HCl-treated cells and analyzed with RT-qPCR, which amplified XBP1u and XBP1s. XBP1u, inactive unspliced form; XBP1s, active spliced form. (H) Expression levels of p-PERK, PERK, and ATF6 were determined with immunoblotting assays (left panel). Quantitative analysis of optical band densitometry (right panel). (I) RT-qPCR was used to analyze the expression of EDEM, GADD34, ER57, and calnexin. * p < 0.05; * * p < 0.01; * * * p < 0.001; * * * * p < 0.0001; or ns, no significance.

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Screening a neurotransmitter-receptor-related inhibitor library identifies clomipramine HCl as a potential antiviral compound against Japanese encephalitis virus
  • Article
  • Full-text available

August 2024

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13 Reads

Infectious Medicine

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Japanese encephalitis virus NS1 and NS1′ protein disrupts the blood-brain barrier through macrophage migration inhibitory factor-mediated autophagy

April 2024

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86 Reads

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4 Citations

Journal of Virology

Flaviviruses in the Japanese encephalitis virus (JEV) serogroup, such as JEV, West Nile virus, and St. Louis encephalitis virus, can cause severe neurological diseases. The nonstructural protein 1 (NS1) is a multifunctional protein of flavivirus that can be secreted by infected cells and circulate in the host bloodstream. NS1′ is an additional form of NS1 protein with 52 amino acids extension at its carboxy-terminal and is produced exclusively by flaviviruses in the JEV serogroup. In this study, we demonstrated that the secreted form of both NS1 and NS1′ can disrupt the blood-brain barrier (BBB) of mice, with NS1′ exhibiting a stronger effect. Using the in vitro BBB model, we found that treatment of soluble recombinant JEV NS1 or NS1′ protein increases the permeability of human brain microvascular endothelial cells (hBMECs) and leads to the degradation of tight junction proteins through the autophagy-lysosomal pathway. Consistently, NS1′ protein exhibited a more pronounced effect compared to NS1 in these cellular processes. Further research revealed that the increased expression of macrophage migration inhibitory factor (MIF) is responsible for triggering autophagy after NS1 or NS1′ treatment in hBMECs. In addition, TLR4 and NF-κB signaling was found to be involved in the activation of MIF transcription. Moreover, administering the MIF inhibitor has been shown to decrease viral loads and mitigate inflammation in the brains of mice infected with JEV. This research offers a novel perspective on the pathogenesis of JEV. In addition, the stronger effect of NS1′ on disrupting the BBB compared to NS1 enhances our understanding of the mechanism by which flaviviruses in the JEV serogroup exhibit neurotropism. IMPORTANCE Japanese encephalitis (JE) is a significant viral encephalitis worldwide, caused by the JE virus (JEV). In some patients, the virus cannot be cleared in time, leading to the breach of the blood-brain barrier (BBB) and invasion of the central nervous system. This invasion may result in cognitive impairment, behavioral disturbances, and even death in both humans and animals. However, the mechanism by which JEV crosses the BBB remains unclear. Previous studies have shown that the flavivirus NS1 protein plays an important role in causing endothelial dysfunction. The NS1′ protein is an elongated form of NS1 protein that is particularly produced by flaviviruses in the JEV serogroup. This study revealed that both the secreted NS1 and NS1′ of JEV can disrupt the BBB by breaking down tight junction proteins through the autophagy-lysosomal pathway, and NS1′ is found to have a stronger effect compared to NS1 in this process. In addition, JEV NS1 and NS1′ can stimulate the expression of MIF, which triggers autophagy via the ERK signaling pathway, leading to damage to BBB. Our findings reveal a new function of JEV NS1 and NS1′ in the disruption of BBB, thereby providing the potential therapeutic target for JE.


Single-cell RNA sequencing reveals the immune features and viral tropism in the central nervous system of mice infected with Japanese encephalitis virus

March 2024

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53 Reads

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2 Citations

Journal of Neuroinflammation

Japanese encephalitis virus (JEV) is a neurotropic pathogen that causes lethal encephalitis. The high susceptibility and massive proliferation of JEV in neurons lead to extensive neuronal damage and inflammation within the central nervous system. Despite extensive research on JEV pathogenesis, the effect of JEV on the cellular composition and viral tropism towards distinct neuronal subtypes in the brain is still not well comprehended. To address these issues, we performed single-cell RNA sequencing (scRNA-seq) on cells isolated from the JEV-highly infected regions of mouse brain. We obtained 88,000 single cells and identified 34 clusters representing 10 major cell types. The scRNA-seq results revealed an increasing amount of activated microglia cells and infiltrating immune cells, including monocytes & macrophages, T cells, and natural killer cells, which were associated with the severity of symptoms. Additionally, we observed enhanced communication between individual cells and significant ligand-receptor pairs related to tight junctions, chemokines and antigen-presenting molecules upon JEV infection, suggesting an upregulation of endothelial permeability, inflammation and antiviral response. Moreover, we identified that Baiap2-positive neurons were highly susceptible to JEV. Our findings provide valuable clues for understanding the mechanism of JEV induced neuro-damage and inflammation as well as developing therapies for Japanese encephalitis.


Ferroptosis contributes to JEV-induced neuronal damage and neuroinflammation

December 2023

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32 Reads

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3 Citations

Virologica Sinica

Ferroptosis is a newly discovered prototype of programmed cell death (PCD) driven by iron-dependent phospholipid peroxidation accumulation, and it has been linked to numerous organ injuries and degenerative pathologies. Although studies have shown that a variety of cell death processes contribute to JEV-induced neuroinflammation and neuronal injury, there is currently limited research on the specific involvement of ferroptosis. In this study, we explored the neuronal ferroptosis induced by JEV infection in vitro and in vivo. Our results indicated that JEV infection induces neuronal ferroptosis through inhibiting the function of the antioxidant system mediated by glutathione (GSH)/glutathione peroxidase 4 (GPX4), as well as by promoting lipid peroxidation mediated by yes-associated protein 1 (YAP1)/long-chain acyl-CoA synthetase 4 (ACSL4). Further analyses revealed that JEV E and prM proteins function as agonists, inducing ferroptosis. Moreover, we found that treatment with a ferroptosis inhibitor in JEV-infected mice reduces the viral titers and inflammation in the mouse brains, ultimately improving the survival rate of infected mice. In conclusion, our study unveils a critical role of ferroptosis in the pathogenesis of JEV, providing new ideas for the prevention and treatment of viral encephalitis.


A novel triptolide analog downregulates NF-kB and induces mitochondrial apoptosis pathways in human pancreatic cancer

October 2023

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105 Reads

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6 Citations

eLife

Pancreatic cancer is the seventh leading cause of cancer-related death worldwide, and despite advancements in disease management, the 5-year survival rate stands at only 12%. Triptolides have potent anti-tumor activity against different types of cancers, including pancreatic cancer, however poor solubility and toxicity limit their translation into clinical use. We synthesized a novel pro-drug of triptolide, ( E )-19-[(1'-benzoyloxy-1'-phenyl)-methylidene]-Triptolide (CK21), which was formulated into an emulsion for in vitro and in vivo testing in rats and mice, and using human pancreatic cancer cell lines and patient-derived pancreatic tumor organoids. A time-course transcriptomic profiling of tumor organoids treated with CK21 in vitro was conducted to define its mechanism of action, as well as transcriptomic profiling at a single time point post-CK21 administration in vivo. Intravenous administration of emulsified CK21 resulted in the stable release of triptolide, and potent anti-proliferative effects on human pancreatic cancer cell lines and patient-derived pancreatic tumor organoids in vitro, and with minimal toxicity in vivo . Time course transcriptomic profiling of tumor organoids treated with CK21 in vitro revealed <10 differentially expressed genes (DEGs) at 3 h and ~8,000 DEGs at 12 h. Overall inhibition of general RNA transcription was observed, and Ingenuity pathway analysis together with functional cellular assays confirmed inhibition of the NF-κB pathway, increased oxidative phosphorylation and mitochondrial dysfunction, leading ultimately to increased reactive oxygen species (ROS) production, reduced B-cell-lymphoma protein 2 (BCL2) expression, and mitochondrial-mediated tumor cell apoptosis. CK21 is a novel pro-drug of triptolide that exerts potent anti-proliferative effects on human pancreatic tumors by inhibiting the NF-κB pathway, leading ultimately to mitochondrial-mediated tumor cell apoptosis.


Lung cDC1 and cDC2 dendritic cells priming naive CD8+ T cells in situ prior to migration to draining lymph nodes

October 2023

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72 Reads

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3 Citations

Cell Reports

The current paradigm indicates that naive T cells are primed in secondary lymphoid organs. Here, we present evidence that intranasal administration of peptide antigens appended to nanofibers primes naive CD8⁺ T cells in the lung independently and prior to priming in the draining mediastinal lymph node (MLN). Notably, comparable accumulation and transcriptomic responses of CD8⁺ T cells in lung and MLN are observed in both Batf3KO and wild-type (WT) mice, indicating that, while cDC1 dendritic cells (DCs) are the major subset for cross-presentation, cDC2 DCs alone are capable of cross-priming CD8⁺ T cells both in the lung and draining MLN. Transcription analyses reveal distinct transcriptional responses in lung cDC1 and cDC2 to intranasal nanofiber immunization. However, both DC subsets acquire shared transcriptional responses upon migration into the lymph node, thus uncovering a stepwise activation process of cDC1 and cDC2 toward their ability to cross-prime effector and functional memory CD8⁺ T cell responses.


JEV infection elevates H3K27me3 modification in microglial cells. A, B BV2 cells (A) and primary mouse microglial cells (B) were infected with JEV at an MOI of 5. At 12, 24 and 36 hpi, cells were lysed for protein extraction, and the H3K27me3 modification was detected by immunoblotting (upper panel). Levels of H3K27me3 were quantified by immunoblotting scanning using Image J software and normalized to the amount of histone H3 (lower panel). C, D 6-week-old BALB/c mice (n = 9 /group) were intracerebrally injected with 200 PFU of JEV P3 strain in 20 μL DMEM or equal amount of DMEM (mock infected). The mouse brains were collected at day 1, 3 and 5 post-infection. C H3K27me3 levels and the expression of JEV E protein were detected by immunoblotting (upper panel). H3K27me3 levels in different mice brain samples were quantified by immunoblotting scanning using Image J software and normalized to the amount of histone H3 (lower panel). D Coronal sections of brain tissues were subjected to immunofluorescence assay by using antibodies against microglia marker Iba1 (purple), JEV E protein (green) and H3K27me3 (red). Scale bar, 10 μm. Data are expressed as means ± SEM from three independent experiments. *p < 0.05, **p < 0.01, NS represents no significant difference
H3K27me3 promotes JEV-induced inflammatory response in microglial cells. A, B EZH2 knockdown (EZH2 KD) BV2 cell line or negative control (NC) cells were infected with JEV at an MOI of 5, and cells were harvested at 36 hpi. The protein expression of JEV E, EZH2 and H3K27me3 was detected by immunoblotting (A). RNA was extracted and analyzed by qRT-PCR to measure mRNA levels of inflammatory cytokines (B). C, D BV2 cells were treated with GSK343 (10 μM) or equal volume of DMSO following JEV infection, and cells were harvested at 36 hpi. The levels of H3K27me3 were measured by immunoblotting (C) and mRNA abundance of inflammatory cytokines was examined by qRT-PCR (D). E, F Primary microglial cells were treated with GSK343 (10 μM) or equal volume of DMSO following JEV infection, and cells were harvested at 36 hpi. The levels of H3K27me3 were measured by immunoblotting (E) and mRNA abundance of inflammatory cytokines was determined by qRT-PCR (F). Data are expressed as means ± SEM from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, NS represents no significant difference
ChIP-sequencing analysis of the H3K27me3 target gene profile in BV2 cells infected with JEV and validation of ChIP-sequencing results. A The hierarchical clustering of differently expressed genes. The red color indicates upregulated genes and the green color indicates downregulated genes. B The volcano plot represents the modified genes. The grey dots denote non-significantly expressed genes. The upregulated genes are presented as red dots and the downregulated genes are presented as blue dots. C Distribution of peak binding sites to the TSS of the nearest gene. D The position of the genome annotation peak by barplot in terms of genome features. E GO term enrichment analysis for the differentially regulated genes. F KEGG pathway analysis for the differentially regulated genes. G The recruitment of H3K27me3 to the binding region of Rnf19a, Ipo11 and Cd2ap in BV2 cells. The binding region of Rnf19a, Ipo11 and Cd2ap were ChIP-ed with anti-H3K27me3 antibodies or IgG control. Data are expressed as means ± SEM from three independent experiments. *p < 0.05
H3K27me3 modification of Rnf19a contributes to the JEV-induced inflammatory response in BV2 cells. A, B BV2 cells were infected with JEV at an MOI of 5, mRNA (A) and protein (B) levels of Rnf19a were measured at 12, 24 and 36 hpi. The protein level of Rnf19a was quantified by immunoblotting scanning using Image J software and normalized to the amount of GAPDH (right panel). C, D EZH2 knockdown (EZH2 KD) BV2 cells or negative control (NC) cells were infected with JEV at 5 MOI and cells were harvested at 36 hpi. RNA was extracted and analyzed by qRT-PCR to measure mRNA level of Rnf19a (C). The expression of Rnf19a was detected by immunoblotting (D), and quantified by immunoblotting scanning using Image J software and normalized to the amount of GAPDH (right panel). E, F Rnf19a knockdown (Rnf19a KD) BV2 cells or negative control (NC) cells were infected with JEV at an MOI of 5, and cells were harvested at 36 hpi. The expression of Rnf19a was detected by immunoblotting (E) and mRNA levels of inflammatory cytokines were examined by qRT-PCR (F). G, H BV2 cells were transfected with pCAGGS-Rnf19a plasmid or pCAGGS empty vector following JEV infection (MOI = 5). Cells were harvested at 36 hpi, and the expression of Rnf19a (G) and mRNA abundance inflammatory cytokines (H) were detected by immunoblotting and qRT-PCR, respectively. I Rnf19a knockdown (Rnf19a KD) or negative control (NC) BV2 cells were treated with GSK343 (10 μM) or equal volume of DMSO following JEV infection at MOI of 5. At 36 hpi, the mRNA abundance of inflammatory cytokines was determined by qRT-PCR. Data are expressed as means ± SEM from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, NS represents no significant difference
Rnf19a regulates JEV-induced inflammatory response in BV2 cells by mediating RIG-I degradation. A HEK-293T cells were co-transfected with luc-NF-κB reporter, pRL-TK construct together with pCAGGS-Flag-TRAF6 plasmid, pCAGGS-Rnf19a plasmid or pCAGGS empty vector. The luciferase activity was determined, and the expression of indicated proteins was detected by immunoblot assay at 36 h post-transfection. B HEK-293T cells were co-transfected with the luc-NF-κB reporter, pRL-TK construct and pCAGGS-Flag-RIG-I N plasmid, pCAGGS-Rnf19a plasmid or pCAGGS empty vector. The luciferase activity was determined and the expression of indicated proteins was detected by immunoblot assay at 36 h post-transfection. C BV2 cells were infected with JEV at an MOI of 5. At 12, 24 and 36 hpi, the expression of RIG-I and Rnf19a were determined by immunoblotting (left panel), and quantified by immunoblotting scanning using Image J software and normalized to the amount of GAPDH (right panel). D BV2 cells were transfected with pCAGGS-Rnf19a plasmid or pCAGGS empty vector. The protein level of RIG-I was determined by immunoblot assay at 36 h post-transfection. E HEK-293T cells were co-transfected with pCAGGS-Flag-RIG-I plasmid and pCAGGS-Rnf19a plasmid or pCAGGS empty vector. At 24 h post-transfection, cells were treated with DMSO, the proteasome inhibitor MG132 (10 μM) or the lysosome inhibitor NH4Cl (10 mM). After incubation for 4 h, the protein levels of RIG-I and Rnf19a were determined by immunoblot assay. F HEK-293T cells were transfected with pCAGGS-Flag-RIG-I plasmid along with pCAGGS empty vector or pCAGGS-Rnf19a. The cell lysates were subjected to denaturing immunoprecipitation with anti-Flag beads and the products were analyzed by immunoblotting with the indicated antibodies. G HEK-293T cells were co-transfected with pCAGGS-Flag-RIG-I plasmid, pCAGGS empty vector or a pCAGGS-Rnf19a construct and plasmid encoding HA-WT-ubiquitin (Ub). Followed by treatment with MG132, cells lysates were subjected to denaturing immunoprecipitation with anti-Flag beads and products were analyzed by immunoblotting with indicated antibodies. H BV2 cells were co-transfected with pCAGGS-Rnf19a plasmid and pCAGGS-Flag-RIG-I N plasmid or pCAGGS empty vector following JEV infection at an MOI of 5. At 36 hpi, the mRNA abundance of inflammatory cytokines was measured by qRT-PCR. I Rnf19a knockdown (Rnf19a KD) or negative control (NC) BV2 cells were treated with GSK343 (10 μM) or equal volume of DMSO following JEV infection at MOI of 5. Data are expressed as means ± SEM from three independent experiments. *p < 0.05, ***p < 0.001, ****p < 0.0001, NS represents no significant difference
H3K27me3 of Rnf19a promotes neuroinflammatory response during Japanese encephalitis virus infection

July 2023

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62 Reads

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1 Citation

Journal of Neuroinflammation

Histone methylation is an important epigenetic modification that affects various biological processes, including the inflammatory response. In this study, we found that infection with Japanese encephalitis virus (JEV) leads to an increase in H3K27me3 in BV2 microglial cell line, primary mouse microglia and mouse brain. Inhibition of H3K27me3 modification through EZH2 knockdown and treatment with EZH2 inhibitor significantly reduces the production of pro-inflammatory cytokines during JEV infection, which suggests that H3K27me3 modification plays a crucial role in the neuroinflammatory response caused by JEV infection. The chromatin immunoprecipitation-sequencing (ChIP-sequencing) assay revealed an increase in H3K27me3 modification of E3 ubiquitin ligases Rnf19a following JEV infection, which leads to downregulation of Rnf19a expression. Furthermore, the results showed that Rnf19a negatively regulates the neuroinflammatory response induced by JEV. This is achieved through the degradation of RIG-I by mediating its ubiquitination. In conclusion, our findings reveal a novel mechanism by which JEV triggers extensive neuroinflammation from an epigenetic perspective.


Figures and Figure Legends
Fig.s14 Male mice with AsPC-1 tumors responded to CK21. (a) Subcutaneous AsPC-1 tumor in male mice after CK21 treatment at 1.5 mg/kg. (b) Male mice weight during CK21 treatment. (N=5 for each experimental group)
Fig.s15 Key regulators in NF-kB canonical signaling pathway are significantly downregulated in (a) U049MAI and (b) U123m15-T after treatment with CK21 (50 nM) for 12 hours. Green represent downregulation and red represent upregulation by IPA analysis.
A novel triptolide analog downregulates NF-κB and induces mitochondrial apoptosis pathways in human pancreatic cancer

February 2023

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83 Reads

Background Pancreatic cancer is the seventh leading cause of cancer-related death worldwide, and despite advancements in disease management, the 5-year survival rates stands at only 9%. Triptolides have potent anti-tumor activity against different types of cancers, including pancreatic cancer, however poor solubility and toxicity limit their translation into clinical use. Methods We synthesized a novel pro-drug of triptolide, ( E )-19-[(1’-benzoyloxy-1’-phenyl)-methylidene]-Triptolide (CK21), and formulated into an emulsion for in vitro and in vivo testing in rats and mice, and using human pancreatic cancer cell lines and patient-derived pancreatic tumor organoids. A time-course transcriptomic profiling of tumor organoids treated with CK21 in vitro was conducted to define its mechanism of action, as well as transcriptomic profiling at a single time point post-CK21 administration in vivo. Findings Intravenous administration of emulsified CK21 resulted in the stable release of triptolide, and potent anti-proliferative effects on human pancreatic cancer cell lines and patient-derived pancreatic tumor organoids in vitro, and with minimal toxicity in vivo . Time course transcriptomic profiling of tumor organoids treated with CK21 in vitro revealed <10 differentially expressed genes (DEGs) at 3 h and ∼8,000 DEGs at 12 h. Overall inhibition of general RNA transcription was observed, and Ingenuity pathway analysis together with functional cellular assays confirmed inhibition of the NF-κB pathway, increased oxidative phosphorylation and mitochondrial dysfunction, leading ultimately to increased reactive oxygen species (ROS) production, reduced B-cell-lymphoma protein 2 (BCL2) expression, and mitochondrial-mediated tumor cell apoptosis. Interpretation CK21 is a novel pro-drug of triptolide that exerts potent anti-proliferative effects on human pancreatic tumors by inhibiting the NF-κB pathway, leading ultimately to mitochondrial-mediated tumor cell apoptosis. Funding The study of the anti-tumor efficacy of CK21 supported in part by a research grant from Cinkate Pharmaceutical Corp; the funders had no role in the study design, interpretation or decision to publish. Patient-derived pancreatic tumor organoids were a generous gift from the Organoid and Primary Culture Research Core at University of Chicago.


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Citations (16)


... Viral brain infections are characterized by strong expression of type I interferon (IFN) genes and inflammatory cytokines [4][5][6] . This is accompanied by influx of immune cells from the periphery with an early recruitment of monocytes/macrophages and natural killer cells, and later T cells 7,8 . Both brain resident and infiltrating cell types have been described to contribute to the innate immune response to viral CNS infections. ...

Reference:

Temporally resolved single-cell RNA sequencing reveals protective and pathological responses during herpes simplex virus 1 CNS infection
Single-cell RNA sequencing reveals the immune features and viral tropism in the central nervous system of mice infected with Japanese encephalitis virus

Journal of Neuroinflammation

... In addition, several neurodegenerative diseases have also shown a link to HSV infection (Zhu and Viejo-Borbolla, 2021). Two neuroinvasive viruses, JEV and HSV, both cause severe encephalitis, and the current studies found a strong correlation between symptoms after JEV and HSV infection and ferroptosis (Xu et al., 2023;Zhu et al., 2024). Ferroptosis is a new non-apoptotic form of programmed cell death discovered in recent years, which is usually accompanied by a large amount of iron accumulation and lipid peroxidation (LPO) during cell death. ...

Ferroptosis contributes to JEV-induced neuronal damage and neuroinflammation

Virologica Sinica

... These findings reveal that whereas cDC1 may be primary mediators of tumor antigen presentation in WT mice (Gardner et al., 2020), cDC2 are capable of presenting tumor antigens as well and become more effective than cDC1 in this function in the absence of MHC-II. This is consistent with previous work showing that cDC2 are capable of efficiently cross-presenting antigens to CD8 T lymphocytes in other experimental settings (Ballesteros-Tato et al., 2010;Ji et al., 2013;Sheng et al., 2017;Si et al., 2023;Theisen et al., 2018). ...

Lung cDC1 and cDC2 dendritic cells priming naive CD8+ T cells in situ prior to migration to draining lymph nodes

Cell Reports

... A total of 75% of antitumor compounds currently used to treat human cancers are natural products [24]. With advancements in natural compound extraction and separation technologies, a growing number of compounds with anti-pancreatic cancer activities have been identified in plants [25][26][27][28][29]. For instance, CK21 is a novel pro-drug of triptolide that exerts potent anti-proliferative effects on human pancreatic tumors by inhibiting the NF-κB pathway, ultimately leading to mitochondrial-mediated tumor cell apoptosis [25]. ...

A novel triptolide analog downregulates NF-kB and induces mitochondrial apoptosis pathways in human pancreatic cancer

eLife

... Some results support antiviral efficacy of favipiravir against ZIKV in nonhuman primates [51]. Previous research has also proven that testosterone can reduce mortality and attenuate testicular damage in ZIKVinfected A129 mice [52]. However, our models only indicate that the testosterone could relieve ZIKV-induced testicular lesion. ...

Testosterone protects mice against Zika virus infection and suppresses the inflammatory response in brain

iScience

... (B and C) Data are representative of two placentas for each condition from two dams. of ZIKV infection during pregnancy has been described in animal models Yockey et al., 2016Yockey et al., , 2018, it has not been clear which innate immune responses are most relevant in the fetal compartment in a setting of maternal immunocompetence. While viral sensing via RIG-I/MDA5-MAVS has been shown to restrict ZIKV infection in human placental cell lines Zhao et al., 2022), this pathway has not been studied in vivo; however, deletion of the downstream transcription factors Irf3 and Irf7 facilitated placental and fetal infection in a mouse model of intravaginal ZIKV infection during pregnancy (Yockey et al., 2016). Our breeding schemes with immunocompetent (heterozygous) dams and immunocompetent (heterozygous) or immunodeficient (homozygous) sires enabled the generation of innate immune deficiencies specifically in the fetus. ...

Zika virus causes placental pyroptosis and associated adverse fetal outcomes by activating GSDME

eLife

... Namely, the local tissue milieu can elicit different immune profiles, increasing the complexity of successfully eliciting protective antibacterial responses across the myriad of tissues that S. aureus can cause invasive disease. This has been demonstrated in recent studies where unique immune responses have been observed when S. aureus invades the epidermis vs. dermis in mouse models of cutaneous infection [99][100][101][102][103][104] . ...

Tissue specificity drives protective immunity against Staphylococcus aureus infection

... Due to their rapid processing speed, as well as their heightened sensitivity and specificity, molecular techniques have become the predominant diagnostic method for most viral CNS infections [10,17]. As an accurate and rapid biosensor, the CRISPR/Cas system has expanded to a new use of diagnostics for infectious diseases, including the detection of various viral nucleic acids [18][19][20][21]. For ZIKV detection, the Cas13-based SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) platform can probe and distinguish ZIKV and four dengue virus (DENV) serotypes in infected human-patient bodily fluid samples at concentrations as low as one copy per microliter [22]. ...

Development of efficient, sensitive, and specific detection method for Encephalomyocarditis virus based on CRISPR/Cas13a
  • Citing Article
  • July 2022

Journal of Virological Methods

... A distinctive m6A site is present in the prM (495-695nt) region of the JEV genome, a feature that is not observed in other Flaviviridae viral genomes. The prM protein is largely responsible for the assembly of viruses and their virulence, suggesting that m6A modification of prM may be involved in this process [29,39,41]. ...

Increased Cleavage of Japanese Encephalitis Virus prM Protein Promotes Viral Replication but Attenuates Virulence

Microbiology Spectrum

... 目前, JEV主要在中国、日 本、韩国、菲律宾、印度和俄罗斯东部地区等亚洲地 区流行 [96] . Chen等人 [97] 研发了一种编码经修饰的JEV P3株 prM/E蛋白的mRNA疫苗, 在小鼠中可产生中和抗体, 其抗体的PRNT 50 (50% Plaque Reduction Neutralization Test)滴度达到1/100; 同时引起CD8 + T淋巴细胞介导的 免疫应答. 该疫苗也可保护免疫小鼠免受致死剂量病 毒感染, 并减少病毒引起的神经炎症反应 [97] . ...

Protective Immune Responses Induced by an mRNA-LNP Vaccine Encoding prM-E Proteins against Japanese Encephalitis Virus Infection