Journal: Advanced Science

Article Title: T Cell‐Derived Apoptotic Extracellular Vesicles Hydrolyze cGAMP to Alleviate Radiation Enteritis via Surface Enzyme ENPP1

doi: 10.1002/advs.202401634

Figure Lengend Snippet: ApoEVs hydrolyze cGAMP by ENPP1 to inhibit the activation of the cGAS‐STING pathway. A) Western blot analysis of ENPP1 expression in naive T cells, activated T cells, apoptotic T cells, and ApoEVs. B) Immunoelectron microscopy detection of ENPP1 on ApoEVs. Scale bar, 200 nm. Arrows indicate ENPP1 adhered by gold particles. C) The activity of ApoEVs to hydrolyze cGAMP with or without ENPP1‐IN (ENPP1 inhibitor, 100 µ m ) was measured in vitro ( n = 3). DMSO was used as solvent control. D) Schematic diagram of the experimental procedure. To evaluate the effect of ENPP1 on ApoEVs surface on neutrophils and macrophages, ApoEVs treated with or without ENPP1‐IN were added to cGAMP‐pretreated neutrophils or BMDM. DMSO was used as solvent control. 6 h after ApoEVs were added, the cGAMP concentration, STING protein, and IFNB mRNA expression of cells were detected. E) The detection of extracellular and intracellular cGAMP concentration for neutrophils ( n = 3). F) Western blot analysis of STING protein in neutrophils. G) The mRNA expression of IFNB in neutrophils ( n = 3). H) The detection of extracellular and intracellular cGAMP concentration for BMDM ( n = 3). I) Western blot analysis of STING protein in BMDM. J) The mRNA expression of IFNB in BMDM ( n = 3). The data are represented as mean ± SD. Statistical analyses are performed by one‐way ANOVA with Tukey's post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001; ns, p > 0.05.

Article Snippet: Subsequently, 0, 25, 50, 100 µg mL −1 ApoEVs added with or without 100 µ m ENPP1 inhibitor (ENPP1‐IN‐12, MedChemExpress, HY‐143256) were reacted with 10 µ m cGAMP (MedChemExpress, HY‐100564A) in 100 µL of DMEM (free EVs) for 2 h at room temperature.

Techniques: Activation Assay, Western Blot, Expressing, Immuno-Electron Microscopy, Activity Assay, In Vitro, Solvent, Control, Concentration Assay

Journal: Advanced Science

Article Title: T Cell‐Derived Apoptotic Extracellular Vesicles Hydrolyze cGAMP to Alleviate Radiation Enteritis via Surface Enzyme ENPP1

doi: 10.1002/advs.202401634

Figure Lengend Snippet: ApoEVs hydrolyze intracellular cGAMP. A, B) The presence of ENPP1 on the surface of intracellular ApoEVs was observed after the uptake of ApoEVs by BMDM. To avoid the effects of ENPP1 on the cell itself, ApoEVs were incubated with ENPP1 antibodies before they were added to BMDM. A) Representative images of PKH26‐labeled ApoEVs (red) and ENPP1 (green) detected by laser scanning confocal microscopy. Scale bar, 50 µm in low‐magnification images and 10 µm in high‐magnification images. B) ENPP1 of intracellular ApoEVs detected by immunoelectron microscopy. Scale bar, 2 µm in low‐magnification images and 200 nm in high‐magnification images. C) Schematic diagram of the experimental procedure. To evaluate the ENPP1 enzymatic activity of intracellular ApoEVs in BMDM, ApoEVs treated with or without ENPP1‐IN (ENPP1 inhibitor, 100 µ m ) were added to BMDM. DMSO was used as solvent control. After ApoEVs were added, unengulfed ApoEVs were removed by changing the medium, and cells were stimulated with cGAMP for 5 h. Concurrently, by the addition of purified soluble recombinant mouse ENPP1, it was proved that the secretory ENPP1 could not hydrolyze intracellular cGAMP. D) Concentration of extracellular and intracellular cGAMP in BMDM. E) Western blot analysis of the STING expression. F) The mRNA expression of IFNB . The data are represented as mean ± SD. Statistical analyses are performed by one‐way ANOVA with Tukey's post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001; ns, p > 0.05.

Article Snippet: Subsequently, 0, 25, 50, 100 µg mL −1 ApoEVs added with or without 100 µ m ENPP1 inhibitor (ENPP1‐IN‐12, MedChemExpress, HY‐143256) were reacted with 10 µ m cGAMP (MedChemExpress, HY‐100564A) in 100 µL of DMEM (free EVs) for 2 h at room temperature.

Techniques: Incubation, Labeling, Confocal Microscopy, Immuno-Electron Microscopy, Activity Assay, Solvent, Control, Purification, Recombinant, Concentration Assay, Western Blot, Expressing

Journal: Advanced Science

Article Title: T Cell‐Derived Apoptotic Extracellular Vesicles Hydrolyze cGAMP to Alleviate Radiation Enteritis via Surface Enzyme ENPP1

doi: 10.1002/advs.202401634

Figure Lengend Snippet: ApoEV administration alleviates radiation enteritis by hydrolyzing cGAMP. A) Schematic diagram of the in vivo experimental procedure. Before ApoEVs injection, ApoEVs were incubated with ENPP1‐IN to block ENPP1 enzyme activity. The mice exposed to ionizing radiation (IR) were intravenously injected with PBS (150 µL), ApoEVs (150 µg in 150 µL PBS), or ENPP1‐IN incubated ApoEVs (150 µg in 150 µL PBS) on days 0.5, 2 and 4 after radiation. B) Survival rate of mice ( n = 10), significance tested using Log‐rank test. C) Body weight of mice ( n = 5–6). D) Representative morphology images of the colon and quantitative analysis of the colon length in each group ( n = 6). E) Representative H&E staining of the small intestine and colon tissues, and quantitative analysis of the histological activity index ( n = 6). Scale bar, 500 µm in low‐magnification images and 75 µm in high‐magnification images. F–H) cGAMP concentration ( n = 5, F), STING expression (G), and IFNB mRNA level (H) in the small intestine. I–K) cGAMP concentration ( n = 5) (I), STING expression (J), and IFNB mRNA level (K) in the colon. L) Serum concentrations of IFNβ, IL‐6, and IL‐10 detected by ELISA ( n = 6). The data are represented as mean ± SD. Statistical analyses are performed by one‐way ANOVA with Tukey's post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001; ns, p > 0.05.

Article Snippet: Subsequently, 0, 25, 50, 100 µg mL −1 ApoEVs added with or without 100 µ m ENPP1 inhibitor (ENPP1‐IN‐12, MedChemExpress, HY‐143256) were reacted with 10 µ m cGAMP (MedChemExpress, HY‐100564A) in 100 µL of DMEM (free EVs) for 2 h at room temperature.

Techniques: In Vivo, Injection, Incubation, Blocking Assay, Activity Assay, Staining, Concentration Assay, Expressing, Enzyme-linked Immunosorbent Assay