Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inhibition of Glycolysis in Pathogenic T H 17 Cells through Targeting a miR -21-Peli1 -c-Rel Pathway Prevents Autoimmunity.

doi: 10.4049/jimmunol.2000060

Figure Lengend Snippet: FIGURE 1. TH17 cells derived in vitro show different metabolic states. (A) RT-PCR analysis of key glycolytic pathway genes in TH17 (b) and TH17 (23) cells differentiated in vitro for 48 h; gene expression was normalized to Actb. (B) ECAR of TH17 (b) and TH17 (23) cells differentiated for 96 h assessed by a glycolytic stress test. ECAR was measured under basal conditions and in response to glucose (10 mM), oligomycin (1.0 mM), and 2-deoxyglucose (2-DG) (50 mM). (C) Principal component analysis (PCA) analysis of identified metabolites of TH17 (b) and TH17 (23) cells (n = 5) differentiated in vitro for 48 h by metabolomics. (D) Metabolomics analysis of TH17 (b) and TH17 (23) cells differentiated in vitro for 48 h; the top differentially observed metabolites are shown in heat map. Data are representative of three independent experiments (A and B). Error bars represent SEM. *p , 0.05, **p , 0.01, ***p , 0.001, determined by two-tailed unpaired t test.

Article Snippet: Naive CD4+ T cells were stimulated with plate-bound anti-CD3 mAb (catalog no. 16-0038-85, 5 mg/ml; Thermo Fisher Scientific) in the presence of antiCD28 mAb (catalog no. 16-0289-85, 2 mg/ml; Thermo Fisher Scientific) in a 48-well plate under neutral condition (catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), TH17 (b) condition (catalog no. 7666-MB-005, 2 ng/ml TGF-b1; R&D Systems; catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), and TH17 (23) condition (catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 401-ML-025, 20 ng/ml IL-1b; R&D Systems; catalog no. 1887-ML-010, 20 ng/ml IL-23; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific).

Techniques: Derivative Assay, In Vitro, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Two Tailed Test

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inhibition of Glycolysis in Pathogenic T H 17 Cells through Targeting a miR -21-Peli1 -c-Rel Pathway Prevents Autoimmunity.

doi: 10.4049/jimmunol.2000060

Figure Lengend Snippet: FIGURE 2. TH17 cells show differential metabolic pathway gene activation in vivo. (A) Scatter plot shows differential expressed genes between CNS TH17 (red) and ileum TH17 (blue) cells with differential TH17 signature genes highlighted (black dot). differentially-expressed genes are filtered as false discovery rate (FDR) , 0.05 and FC $ 1.5 from DESeq2. (B) Scatter plot shows differential expressed genes between CNS TH17 (red) and ileum TH17 (blue) cells with glycolysis pathway genes highlighted (black dot) and differential genes labeled. DEGs are filtered as in (A). (C) Scatter plot shows differential expressed genes between CNS TH17 (red) and ileum TH17 (blue) cells with glutaminolysis pathway genes highlighted (black dot) and differential genes labeled. DEGs are filtered as in (A). (D) CNS TH17 and ileum TH17 cells depend on distinct metabolic pathways. Numbers indicate the differentially expressed genes and total genes for each selected pathway. (E) KEGG pathway enrichment for CNS TH17 cell highly expressed genes. (F) KEGG pathway enrichment for ileum TH17 cell highly expressed genes. Color represents log-transferred FDR, and dot size represents the number of differentially expressed genes observed for this pathway (E and F).

Article Snippet: Naive CD4+ T cells were stimulated with plate-bound anti-CD3 mAb (catalog no. 16-0038-85, 5 mg/ml; Thermo Fisher Scientific) in the presence of antiCD28 mAb (catalog no. 16-0289-85, 2 mg/ml; Thermo Fisher Scientific) in a 48-well plate under neutral condition (catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), TH17 (b) condition (catalog no. 7666-MB-005, 2 ng/ml TGF-b1; R&D Systems; catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), and TH17 (23) condition (catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 401-ML-025, 20 ng/ml IL-1b; R&D Systems; catalog no. 1887-ML-010, 20 ng/ml IL-23; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific).

Techniques: Activation Assay, In Vivo, Labeling

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inhibition of Glycolysis in Pathogenic T H 17 Cells through Targeting a miR -21-Peli1 -c-Rel Pathway Prevents Autoimmunity.

doi: 10.4049/jimmunol.2000060

Figure Lengend Snippet: FIGURE 3. TH17 cells derived in vitro show discrete chromatin states. (A) Scatter plot for differentially opened/closed regions (OCRs) between TH17 (23) (red) and TH17 (b) cells (blue) with differential numbers labeled. (B) ATAC-seq reads density heatmap around the OCRs (shows the peak summit 6 1 kb region) between TH17 (23) and TH17 (b) cells. (C) The enriched transcription factor motifs identified from top 1000 differentially OCRs between TH17 (23) cells and TH17 (b) cells; color bar represents log-transferred p values. Right panel shows the identified motif sequences for key regulators. (D) WashU Epigenome Browser visualization of differentially OCR signals for key glycolytic genes in TH17 (23) and TH17 (b) cells. Arrows represent the NF-kB binding motif sites.

Article Snippet: Naive CD4+ T cells were stimulated with plate-bound anti-CD3 mAb (catalog no. 16-0038-85, 5 mg/ml; Thermo Fisher Scientific) in the presence of antiCD28 mAb (catalog no. 16-0289-85, 2 mg/ml; Thermo Fisher Scientific) in a 48-well plate under neutral condition (catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), TH17 (b) condition (catalog no. 7666-MB-005, 2 ng/ml TGF-b1; R&D Systems; catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), and TH17 (23) condition (catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 401-ML-025, 20 ng/ml IL-1b; R&D Systems; catalog no. 1887-ML-010, 20 ng/ml IL-23; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific).

Techniques: Derivative Assay, In Vitro, Labeling, Binding Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inhibition of Glycolysis in Pathogenic T H 17 Cells through Targeting a miR -21-Peli1 -c-Rel Pathway Prevents Autoimmunity.

doi: 10.4049/jimmunol.2000060

Figure Lengend Snippet: FIGURE 4. TH17 cells show discrete chromatin states in vivo. (A) Scatter plot for differentially opened/closed regions (OCRs) between CNS TH17 (red) and ileum TH17 cells (blue) with differential numbers labeled. (B) ATAC-seq reads density heatmap around the OCRs (shows the peak summit 6 1 kb region) between CNS TH17 and ileum TH17 cells. (C) The enriched transcription factor motifs identified from top 1000 differential OCRs between CNS TH17 cells and ileum TH17 cells; color bar represents log-transferred p values. Right panel shows the identified motif sequences for key regulators. (D) WashU Epigenome Browser visualization of differentially OCR signals with corresponding gene expression levels for key TH17 signature genes and metabolic genes in CNS TH17 and ileum TH17 cells. Arrows represent the NF-kB binding motif sites.

Article Snippet: Naive CD4+ T cells were stimulated with plate-bound anti-CD3 mAb (catalog no. 16-0038-85, 5 mg/ml; Thermo Fisher Scientific) in the presence of antiCD28 mAb (catalog no. 16-0289-85, 2 mg/ml; Thermo Fisher Scientific) in a 48-well plate under neutral condition (catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), TH17 (b) condition (catalog no. 7666-MB-005, 2 ng/ml TGF-b1; R&D Systems; catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), and TH17 (23) condition (catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 401-ML-025, 20 ng/ml IL-1b; R&D Systems; catalog no. 1887-ML-010, 20 ng/ml IL-23; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific).

Techniques: In Vivo, Labeling, Gene Expression, Binding Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inhibition of Glycolysis in Pathogenic T H 17 Cells through Targeting a miR -21-Peli1 -c-Rel Pathway Prevents Autoimmunity.

doi: 10.4049/jimmunol.2000060

Figure Lengend Snippet: FIGURE 5. Identification of a miR-21–Peli1–c-Rel pathway controlling pathogenic TH17 cell glycolysis. (A) WashU Epigenome Browser visualization of ATAC-seq signals across selected microRNA genome loci in ileum homeostatic and CNS-infiltrated pathogenic TH17 cells. (B) Relative expression of microRNA in ileum homeostatic and CNS-infiltrated pathogenic TH17 cells (n = 3). (C) KEGG pathway enrichment for genes downregulated in miR-212/2

Article Snippet: Naive CD4+ T cells were stimulated with plate-bound anti-CD3 mAb (catalog no. 16-0038-85, 5 mg/ml; Thermo Fisher Scientific) in the presence of antiCD28 mAb (catalog no. 16-0289-85, 2 mg/ml; Thermo Fisher Scientific) in a 48-well plate under neutral condition (catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), TH17 (b) condition (catalog no. 7666-MB-005, 2 ng/ml TGF-b1; R&D Systems; catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), and TH17 (23) condition (catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 401-ML-025, 20 ng/ml IL-1b; R&D Systems; catalog no. 1887-ML-010, 20 ng/ml IL-23; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific).

Techniques: Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inhibition of Glycolysis in Pathogenic T H 17 Cells through Targeting a miR -21-Peli1 -c-Rel Pathway Prevents Autoimmunity.

doi: 10.4049/jimmunol.2000060

Figure Lengend Snippet: FIGURE 6. Pharmaceutical inhibition c-Rel–mediated glycolysis in pathogenic TH17 cells prevents autoimmunity. (A) Western blot results for c-Rel and RelA from TH17 cells differentiated under indicated conditions. (B) Western blot results for c-Rel and RelA from TH17 cells differentiated under indicated conditions. (C) Western blot results for c-Rel and RelA from TH17 cells differentiated under indicated conditions. (D) Normalized RT-PCR results for key glycolytic pathway genes in TH17 (23) cells differentiated in vitro for 48 h, treated with H2O or 500 mg/ml PTXF for 16 h. (E) ECAR of TH17 (23) cells differentiated for 48 h and then were treated with H2O or 500 mg/ml PTXF for 16 h, assessed by a glycolysis stress test. (F) Basal and maximal glycolytic capability of TH17 (23) cells differentiated for 48 h and then were treated with H2O or PTXF for 16 h. (G) EAE development in C57BL/6 mice, i.p. PBS or 100 mg/kg PTXF per mouse (n = 6) from day 7 to day 14. (H) EAE development in recipient C57BL/6 mice (sublethal irradiation; n = 6) i.p. with pathogenic TH17 (23) cells differentiated from naive 2D2 CD4+ T cells for 96 h and then treated with H2O or 500 mg/ml PTXF for 16 h. Data shown are one experiment representative of three independent experiments (A–C, G, and H). Data are representative of three independent experiments (D–F). Error bars represent SEM. *p , 0.05, **p , 0.01, ***p , 0.001, determined by two-tailed unpaired t test.

Article Snippet: Naive CD4+ T cells were stimulated with plate-bound anti-CD3 mAb (catalog no. 16-0038-85, 5 mg/ml; Thermo Fisher Scientific) in the presence of antiCD28 mAb (catalog no. 16-0289-85, 2 mg/ml; Thermo Fisher Scientific) in a 48-well plate under neutral condition (catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), TH17 (b) condition (catalog no. 7666-MB-005, 2 ng/ml TGF-b1; R&D Systems; catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), and TH17 (23) condition (catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 401-ML-025, 20 ng/ml IL-1b; R&D Systems; catalog no. 1887-ML-010, 20 ng/ml IL-23; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific).

Techniques: Inhibition, Western Blot, Reverse Transcription Polymerase Chain Reaction, In Vitro, Irradiation, Two Tailed Test

Journal: Frontiers in immunology

Article Title: Bacterial Outer Membrane Vesicle-Mediated Cytosolic Delivery of Flagellin Triggers Host NLRC4 Canonical Inflammasome Signaling.

doi: 10.3389/fimmu.2020.581165

Figure Lengend Snippet: FIGURE 1 | Culture supernatants from Salmonella typhimurium promotes NLRC4-dependent inflammasome activation. (A) Illustration of experimental scheme to determine inflammasome activation in BMDMs by bacteria-secreted molecules using transwell plate. (B) Immunoblots from mouse BMDMs in the lower well (A) incubated with wild-type (WT) or DfliC-fljB S. typhimurium SL1344 in the upper well (A, MOI 30) for 7 h. (C) Immunoblots from mouse BMDMs untreated or treated with culture supernatant (CS, 1/33 or 1/10 volume of culture medium), derived from 6 h culture of WT or DfliC–fljB S. typhimurium, for 6 h. (D) Immunoblots from Nlrc4+/+ or Nlrc4−/−mice BMDMs treated with WT or DfliC–fljB S. typhimurium CS (1/10) for 6 h. (E) Immunoblots from Nlrp3+/+ or Nlrp3−/−mice BMDMs treated with WT or DfliC–fljB S. typhimurium CS (1/10) for 6 h, or primed with LPS (0.25 µg/ml, 3 h), followed by treatment with ATP (2.5 mM, 40 min). (F) Immunoblots from mouse BMDMs incubated with S. typhimurium CS (1/20) with or without heat treatment (97°C, 30 min) or proteinase K treatment (10 µg/ml, 30 min), for 6 h. (G, H) Quantification of IL-1b (G) or IL-6 (H) in the supernatant of mouse BMDMs treated with S. typhimurium CS (1/20) as same as in (F). (n = 3, one-way ANOVA) (I, J) Quantification of IL-1b (I) or IL-6 (J) in the culture supernatants of mouse BMDMs treated with WT, DfliC–fljB, or DfliC–fljB–prgJ S. typhimurium CS (1/100) for 6 h. (n = 4, one-way ANOVA). (K) Immunoblots from mouse BMDMs treated with culture supernatant (CS, 1/100 or 1/20), derived from 6 h culture of WT, DfliC–fljB, DfliC–fljB–prgJ S. typhimurium 14028s, for 6 h. (L) Immunoblots from mouse BMDMs treated with S. typhimurium flagellin (ST-FLA, 250 ng/ml) with or without premixing with DOTAP (DT) liposomal transfection reagent for 6 h or treated with LPS (0.25 µg/ml, 3 h), followed by ATP treatment (2.5 mM, 40 min). Culture supernatants (Sup) or cellular lysates (Lys) were immunoblotted with the indicated antibodies. Data were expressed as the mean ± SEM. Asterisks indicate significant differences compared with S. typhimurium CS-treated group. (*P < 0.05, ***P < 0.001, n.s. not significant).

Article Snippet: The following antibodies were used for detecting caspase-1 (Adipogen), NLRP3 (Adipogen), IL-1b (R&D Systems), ASC (Santa Cruz Biotechnology), flagellin (InvivoGen), OmpA (Biorbyt), and gasdermin D (Novus Biologicals).

Techniques: Activation Assay, Bacteria, Western Blot, Incubation, Derivative Assay, Transfection

Journal: Frontiers in immunology

Article Title: Bacterial Outer Membrane Vesicle-Mediated Cytosolic Delivery of Flagellin Triggers Host NLRC4 Canonical Inflammasome Signaling.

doi: 10.3389/fimmu.2020.581165

Figure Lengend Snippet: FIGURE 2 | Outer membrane vesicles from Salmonella typhimurium activate inflammasome signaling via receptor-mediated endocytosis. (A) Immunoblots from the culture supernatants of mouse BMDMs treated with S. typhimurium CS, or supernatant (CS-Sup) or pellet (CS-Pel) from ultracentrifugation (150,000 × g, 18 h) of S. typhimurium CS. (B) Immunoblots from wild-type (WT) or DfliC–fljB S. typhimurium CS, filtrates from Centricon concentration (Supplementary Figure S3A) and the extract of OMVs. (C) Nanoparticle Tracking Analysis of OMVs isolated from WT or DfliC–fljB S. typhimurium. (D) Transmission electron microscopy of OMVs isolated from wild-type S. typhimurium. Scale bar, 100 nm. (E) Representative confocal microscopy images of BMDMs treated with Vybrant Dil-labeled PBS or S. typhimurium OMVs. Scale bar, 20 µm. (F) Immunoblots from mouse BMDMs treated with OMVs isolated from S. typhimurium SL1344 or 14028s (0.5 or 5 µg/ml) for 8 h or treated with Pam3CSK4 (1 µg/ml, 4 h), followed by the transfection of LPS (2 µg/ml, 5 h) using a DOTAP (DT). (G) Immunoblots from mouse BMDMs treated with WT or DfliC–fljB S. typhimurium (SL1344) OMV (1 or 5 µg/ml, 8 h). (H) Quantification of IL-1b in the culture supernatants of mouse BMDMs treated with WT, DfliC–fljB, or DfliC–fljB–prgJ S. typhimurium (SL1344) OMVs (5 µg/ml, 8 h) (n = 7, one-way ANOVA). (I) Immunoblots from mouse BMDMs treated with S. typhimurium OMV (5 µg/ml, 6 h) in the presence of cytochalasin D (5 µM) or Pitstop 2 (PS2, 10 µM), or treated with S. typhimurium CS (1/20) in the presence of PS2 (10 µM) pretreatment (10 min before Salmonella CS treatment). (J) Representative confocal microscopy images of BMDMs treated with Vybrant Dil-labeled S. typhimurium OMVs (5 µg/ml, 6 h) in the presence of Pitstop 2 treatment (10 µM, 30 min pretreat). Scale bar, 20 µm. (K) Relative Dil fluorescence intensity per DAPI signals of BMDMs as treated in (J). (n = 6, one-way ANOVA). (L) Quantification of IL-1b in the culture supernatants of mouse BMDMs treated as in (I, left panel). (n = 3, one-way ANOVA). Culture supernatants (Sup) or cellular lysates (Lys) were immunoblotted with the indicated antibodies. Data were expressed as the mean ± SEM. Asterisks indicate significant differences compared with S. typhimurium CS-treated group. (*P < 0.05, ***P < 0.001).

Article Snippet: The following antibodies were used for detecting caspase-1 (Adipogen), NLRP3 (Adipogen), IL-1b (R&D Systems), ASC (Santa Cruz Biotechnology), flagellin (InvivoGen), OmpA (Biorbyt), and gasdermin D (Novus Biologicals).

Techniques: Membrane, Western Blot, Concentration Assay, Isolation, Transmission Assay, Electron Microscopy, Confocal Microscopy, Labeling, Transfection

Journal: Frontiers in immunology

Article Title: Bacterial Outer Membrane Vesicle-Mediated Cytosolic Delivery of Flagellin Triggers Host NLRC4 Canonical Inflammasome Signaling.

doi: 10.3389/fimmu.2020.581165

Figure Lengend Snippet: FIGURE 4 | Flagellated bacteria-released outer membrane vesicles trigger NLRC4-dependent inflammasome activation. (A) Immunoblots from mouse BMDMs treated with OMV (0.5 or 3 µg/ml) isolated from Pseudomonas aeruginosa PAO1 for 8 h. (B) Immunoblots from Nlrc4+/+ or Nlrc4−/−mice BMDMs treated with P. aeruginosa OMV (1 or 7 µg/ml) for 8 h. (C) Immunoblots from Nlrp3 +/+ or Nlrp3−/−mice BMDMs treated with P. aeruginosa OMV (5 µg/ml) for 8 h. (D, E) Quantification of IL-1b in the culture supernatants of Nlrc4+/+ or Nlrc4−/−(D) or Nlrp3+/+ or Nlrp3−/−(E) mice BMDMs treated with P. aeruginosa OMV (1 or 7 µg/ml, D; 5 µg/ml, E) for 8 h, or treated with Pam3CSK4 (1 µg/ml, 3 h), followed by the transfection of LPS (2 µg/ml, D; 1 µg/ml, E) for 6 h. (n = 3) (F) Immunoblots from Nlrp3+/+ or Nlrp3−/−mice BMDMs treated with E. coli BL21 or DH5a OMVs (5 µg/ml) for 8 h. (G) Immunoblots from Nlrc4+/+ or Nlrc4−/−mice BMDMs treated with E. coli BL21 OMVs (1 or 5 µg/ml) for 8 h. (H, I) Quantification of IL-1b in the culture supernatants of Nlrp3+/+ or Nlrp3−/−(H) or Nlrc4+/+ or Nlrc4−/−(I) mice BMDMs treated with E. coli BL21-derived OMVs (5 µg/ml, H; 1 or 5 µg/ml, I) or DH5a-derived OMVs (5 µg/ml, H) for 8 h, or treated with Pam3CSK4 (1 µg/ml, 3 h), followed by the transfection of LPS (1 µg/ml, H; 2 µg/ml, I) for 6 h. (n = 3) Culture supernatants (Sup) or cellular lysates (Lys) were immunoblotted with the indicated antibodies. Data were expressed as the mean ± SEM. Asterisks indicate significant differences compared with the group in the Nlrp3+/+ cells. (*P < 0.05, ***P < 0.001, n.s. not significant).

Article Snippet: The following antibodies were used for detecting caspase-1 (Adipogen), NLRP3 (Adipogen), IL-1b (R&D Systems), ASC (Santa Cruz Biotechnology), flagellin (InvivoGen), OmpA (Biorbyt), and gasdermin D (Novus Biologicals).

Techniques: Bacteria, Membrane, Activation Assay, Western Blot, Isolation, Transfection, Derivative Assay

Journal: Frontiers in immunology

Article Title: Bacterial Outer Membrane Vesicle-Mediated Cytosolic Delivery of Flagellin Triggers Host NLRC4 Canonical Inflammasome Signaling.

doi: 10.3389/fimmu.2020.581165

Figure Lengend Snippet: FIGURE 5 | Salmonella-released outer membrane vesicles exhibit heat-resistant and GBP2-independent inflammasome activation potential. (A) Immunoblots of OptiPrep density gradient fractions of S. typhimurium OMVs. (B) Immunoblots of the extracts of OMVs in the presence of heat treatment (97°C, 30 min) or proteinase K treatment (10 µg/ml, 30 min) by anti-ST-flagellin antibody. (C) Immunoblots from mouse BMDMs primed with LPS (0.25 µg/ml, 3 h), followed by the treatment with intact or heat-treated S. typhimurium flagellin (250 ng/ml) for 6 h. (D) Immunoblots of mouse BMDMs treated with intact or heat-treated S. typhimurium OMVs (5 µg/ml) for 6 h. (E) Quantification of IL-1b in the culture supernatants of mouse BMDMs treated with intact or heat-treated S. typhimurium OMVs for 6 h. (n = 5). (F) Immunoblots from Gbp2+/+ or Gbp2−/−mice BMDMs treated with E. coli BL21 OMVs (5 µg/ml) or S. typhimurium (14028s) OMVs (5 µg/ml) or for 8 h. (G, H) Quantification of IL-1b in the culture supernatants of Gbp2+/+ or Gbp2−/−mice BMDMs treated as same as (F) (n = 3). (I, J) Immunoblots of mouse BMDMs treated with intact or proteinase-treated S. typhimurium OMVs (10 µg/ml) in the presence of MCC 950 (100 nM) as indicated for 8 h. After proteinase treatment, PMSF was added to eliminate proteinase K activity. Culture supernatants (Sup) or cellular lysates (Lys) were immunoblotted with the indicated antibodies. Data were expressed as the mean ± SEM. Asterisks indicate significant differences. (**P < 0.01, ***P < 0.001, n.s. not significant).

Article Snippet: The following antibodies were used for detecting caspase-1 (Adipogen), NLRP3 (Adipogen), IL-1b (R&D Systems), ASC (Santa Cruz Biotechnology), flagellin (InvivoGen), OmpA (Biorbyt), and gasdermin D (Novus Biologicals).

Techniques: Membrane, Activation Assay, Western Blot, Activity Assay

Journal: Frontiers in immunology

Article Title: Bacterial Outer Membrane Vesicle-Mediated Cytosolic Delivery of Flagellin Triggers Host NLRC4 Canonical Inflammasome Signaling.

doi: 10.3389/fimmu.2020.581165

Figure Lengend Snippet: FIGURE 6 | Salmonella-released outer membrane vesicles trigger in vivo NLRC4-dependent interleukin-1b secretion. (A) Immunoblots from mouse BMDMs treated with wild-type (WT) or DfliC–fljB S. typhimurium (SL1344) OMV (5 µg/ml) for 2, 4, 8 h. (B) Quantification of IL-1b in the culture supernatants of mouse BMDMs treated with WT or DfliC–fljB S. typhimurium (SL1344) OMVs (5 µg/ml) for 2, 8, 16 h. (n = 3) (C) Quantification of LDH release into culture supernatants of mouse BMDMs treated with WT or DfliC–fljB S. typhimurium (SL1344) OMVs (5 µg/ml) for 2, 8, 16 h. (n = 3) (D) Immunoblots from mouse BMDMs treated with S. typhimurium (SL1344) or E. coli (BL21) OMVs (5 µg/ml) for 2, 4, 8 h as indicated. GSDMD, gasdermin D (E) Quantification of LDH release into culture supernatants of mouse BMDMs treated with S. typhimurium (SL1344) or E. coli (BL21) OMVs (5 µg/ml) for 2, 4, 8 h as indicated. (n = 3). (F, G) Quantification of IL-1b in the peritoneal lavage fluid of Nlrc4+/+ or Nlrc4−/−(F), or Nlrp3+/+ or Nlrp3−/−(G) mice 6 h after intraperitoneal injection of PBS or S. typhimurium OMVs (50 µg/mice). (n = 2, PBS; n = 3, OMV) (H) Quantification of IL-1b in the peritoneal lavage fluid of Nlrp3+/+ or Nlrp3−/−mice 6 h after intraperitoneal injection of DfliC–fljB S. typhimurium OMVs (50 µg/mice). (n = 5, Nlrp3+/+; n = 6, Nlrp3−/−) (I, J) Quantification of IL-6 in the peritoneal lavage fluid of Nlrc4+/+ or Nlrc4−/−(I), or Nlrp3+/+ or Nlrp3−/−(J) mice 6 h after intraperitoneal injection of PBS or S. typhimurium OMVs (50 µg/mice). (n = 2, PBS; n = 3, OMV) Culture supernatants (Sup) or cellular lysates (Lys) were immunoblotted with the indicated antibodies. Data were expressed as the mean ± SEM. Asterisks indicate significant differences. (*P < 0.05, ***P < 0.001, n.s. not significant).

Article Snippet: The following antibodies were used for detecting caspase-1 (Adipogen), NLRP3 (Adipogen), IL-1b (R&D Systems), ASC (Santa Cruz Biotechnology), flagellin (InvivoGen), OmpA (Biorbyt), and gasdermin D (Novus Biologicals).

Techniques: Membrane, In Vivo, Western Blot, Injection

Journal: Frontiers in immunology

Article Title: Bacterial Outer Membrane Vesicle-Mediated Cytosolic Delivery of Flagellin Triggers Host NLRC4 Canonical Inflammasome Signaling.

doi: 10.3389/fimmu.2020.581165

Figure Lengend Snippet: FIGURE 7 | Proposed mechanism of host inflammasome activation by flagellated or non-flagellated bacteria-released outer membrane vesicles. Flagellated bacteria such as S. typhimurium or P. aeruginosa facilitate the delivery of bacterial flagellin and LPS into the cytoplasm of host cells via OMV cargo. In the initial phase of OMV exposure, OMV-delivered flagellin causes the assembly of NLRC4 inflammasome leading to caspase-1 activation and the subsequent IL-1b secretion. Besides flagellin, intracellular OMV-associated LPS can activate caspase-11 non-canonical inflammasome signaling, which includes GSDMD-dependent pyroptosis and NLRP3-mediated caspase-1 activation and IL-1b secretion. On the contrary, OMVs derived from non-flagellated bacteria such as E. coli mediates the cytosolic delivery of bacterial LPS. Non-flagellated bacterial -delivered LPS mainly causes the activation of caspase-11 and GSDMD-mediated pyroptosis, contributing to potential endotoxic shock. NAIP5, NLR family, apoptosis inhibitory protein 5; NLRC4, NLR family, CARD domain-containing protein 4; NLRP3, NLR family, pyrin domain-containing protein 3; GSDMD, gasdermin D.

Article Snippet: The following antibodies were used for detecting caspase-1 (Adipogen), NLRP3 (Adipogen), IL-1b (R&D Systems), ASC (Santa Cruz Biotechnology), flagellin (InvivoGen), OmpA (Biorbyt), and gasdermin D (Novus Biologicals).

Techniques: Activation Assay, Bacteria, Membrane, Derivative Assay