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: Journal of thrombosis and haemostasis : JTH

Article Title: A novel pathway of cellular activation mediated by antiphospholipid antibody-induced extracellular vesicles.

doi: 10.1111/jth.13072

Figure Lengend Snippet: Fig. 1. Extracellular vesicles (EVs) released in response to anti-b2- glycoprotein I (b2GPI) antibodies activate unstimulated endothelial cells (ECs). ECs were cultured for 6 h in the presence of medium alone, b2GPI and control rabbit IgG, or b2GPI and rabbit anti- b2GPI antibodies. EVs were isolated from conditioned medium and incubated with unstimulated ECs at three different ratios of donor cells (from which the EVs were derived) to recipient cells (0.8 : 1, 4 : 1, and 20 : 1; represented by hatched, open and gray bars, respectively), for 6 h. EC activation was measured by the expression of E-selectin on the cell surface following incubation with EVs. OD450 nm values from triplicate points from a single experiment rep- resentative of three experiments performed are depicted. The upper bar represents the endothelial response to purified interleukin (IL)-1b (50 pg mL1). EVs from anti-b2GPI-treated cells caused a concentra- tion-dependent increase in E-selectin expression.

Article Snippet: Recombinant human IL-1R antagonist and mouse and goat polyclonal antibodies against IL-1b were from R&D Systems (Minneapolis, MN, USA).

Techniques: Cell Culture, Control, Isolation, Incubation, Derivative Assay, Activation Assay, Expressing

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: A novel pathway of cellular activation mediated by antiphospholipid antibody-induced extracellular vesicles.

doi: 10.1111/jth.13072

Figure Lengend Snippet: Fig. 2. Anti-b2-glycoprotein I (b2GPI) antibodies stimulate interleukin (IL)-1b mRNA expression in endothelial cells (ECs), accompanied by release of extracellular vesicles (EVs) enriched in active IL-1b. (A) ECs were incubated with medium alone, b2GPI and control human or rabbit IgG, or b2GPI and affinity-purified rabbit or human anti-b2GPI antibodies, as described in Materials and methods. Cells were then harvested, and the content of IL-1b mRNA was quantified by quantitative real-time PCR (qPCR). (B) Analysis of the 337 nucleotide IL-1b qPCR prod- ucts with 1.8% agarose gel electrophoresis. (C, D) Immunoblot analysis of IL-1b in ECs, isolated EVs and EV-free conditioned medium (CM) following incubation of ECs with medium, control IgG, or affinity-purified rabbit or human anti-b2GPI antibodies. Increased amounts of pro- IL-1b were present in treated cells, but only the cleaved, active form of IL-1b was present in EVs. GAPDH, glyceraldehyde-3-phosphate dehy- drogenase; LPS, lipopolysaccharide.

Article Snippet: Recombinant human IL-1R antagonist and mouse and goat polyclonal antibodies against IL-1b were from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Incubation, Control, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Western Blot, Isolation

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: A novel pathway of cellular activation mediated by antiphospholipid antibody-induced extracellular vesicles.

doi: 10.1111/jth.13072

Figure Lengend Snippet: Fig. 4. Activation of endothelial cells (ECs) by anti-b2-glycoprotein I (b2GPI) antibody-derived extracellular vesicles (EVs) is not interleukin (IL)-1b-dependent. EVs were isolated from ECs incubated in medium alone, with b2GPI and control IgG, or with b2GPI and affinity-purified human anti-b2GPI IgG, and then incubated with unstimulated ECs as in Fig. 1. (A–C) Incubations were performed in the absence or presence of a neutralizing IL-1 antibody (IL-1 Ab) (A), in the absence or presence of a peptide IL-1 receptor (IL-1R) antagonist (IL-1RA), and with ECs pretreated with IL-1R or control, scrambled small interfering RNA (siRNA) (C). Cells were also treated with recombinant IL-1b as a positive control. The IL-1b antibody or IL-1R inhibitors (IL-1RIs) blocked activation in response to IL-1b, but not in response to EVs. (D) Immunoblot of IL-1R after siRNA pretreatment of ECs, demonstrating > 90% knockdown.

Article Snippet: Recombinant human IL-1R antagonist and mouse and goat polyclonal antibodies against IL-1b were from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Derivative Assay, Isolation, Incubation, Control, Small Interfering RNA, Recombinant, Positive Control, Western Blot, Knockdown

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: A novel pathway of cellular activation mediated by antiphospholipid antibody-induced extracellular vesicles.

doi: 10.1111/jth.13072

Figure Lengend Snippet: Fig. 5. Extracellular vesicles (EVs) from anti-b2-glycoprotein I (b2GPI) antibody-treated endothelial cells (ECs) stimulate interleukin (IL)-1 receptor-associated kinase 4 (IRAK4) phosphorylation. (A) Unstimulated ECs were incubated with EVs from cells incubated in medium alone, with b2GPI and control IgG, or with b2GPI and affinity-purified human anti-b2GPI antibodies. Cell extracts were then prepared and analyzed for IRAK4 phosphorylation by immunoblotting with antibodies against phospho-IRAK4 (Thr345-Ser346; upper panel) or total IRAK4. (B) IRAK4 expression in ECs treated with IRAK4 small interfering RNA (siRNA). (C) IRAK4 siRNA blocks EC activation induced by EVs from b2GPI and anti-b2GPI antibody-treated cells, and in response to IL-1b, used as a positive control.

Article Snippet: Recombinant human IL-1R antagonist and mouse and goat polyclonal antibodies against IL-1b were from R&D Systems (Minneapolis, MN, USA).

Techniques: Phospho-proteomics, Incubation, Control, Western Blot, Expressing, Small Interfering RNA, Activation Assay, Positive Control