Journal: Advanced Science

Article Title: IL‐7R‐Enriched Extracellular Vesicles From the Thymus Drive Colitis via Promoting Neutrophil Extracellular Trap Formation

doi: 10.1002/advs.202520331

Figure Lengend Snippet: Plasma‐derived EVs of colitis are enriched with IL‐7R and induce colonic NET formation through IL‐7R. (a) IL‐7R levels in EVs were detected using western blotting. (b) The levels of IL‐7R in plasma, EVs‐free plasma, and EVs were detected by western blotting. (c) Neutrophils were treated with UC‐EVs, UC‐EVs + anti‐IL‐7R antibody, or UC‐EVs + recombinant mouse IL‐7R protein, and NET formation was assessed by SYTOX Green/H3cit co‐staining. (d) Neutrophils were treated with CD‐EVs, CD‐EVs + anti‐IL‐7R antibody, or CD‐EVs + recombinant mouse IL‐7R protein, and NET formation was assessed by SYTOX Green/H3cit co‐staining. (e) Immunofluorescence analyses were performed to evaluate IL‐7 and IL‐7R expression in colon tissues from IBD patients and non‐IBD controls. (f, g) Western blotting and immunofluorescence analyses were used to assess the levels of IL‐7R in the colon tissues of the experimental DSS‐induced colitis model. (h) Intraperitoneal injections of GW4869 were administered on days 1, 3, 5, and 7 to inhibit EVs release in DSS‐induced colitis mice, and immunofluorescence analysis was performed to examine IL‐7R expression in colon tissues. (i) Immunofluorescence analysis demonstrating that IL‐7R promotes the formation of NETs. (j) Immunofluorescence showing that IL‐7R can promote the formation of PMA‐induced NETs. (k) Intraperitoneal injections of anti‐IL‐7R antibody (DSS + IL‐7R (+)) and recombinant mouse IL‐7R protein (DSS + IL‐7R (‐)) were administered on days 1, 3, 5, and 7 to modulate IL‐7R expression in DSS‐induced colitis mice. (l) On Day 9, the mice were sacrificed, and the level of neutrophils in the colon tissues was measured by immunofluorescence. (m) Immunofluorescence was used to analyze the formation of NETs in colon tissues. n = 5–10 mice per group; results are presented as the mean ± SD; *** p < 0.001, **** p < 0.0001.

Article Snippet: The intervention groups (DSS + GW4869, DSS + anti‐Ly‐6G, DSS + DNase I, DSS + IL‐7R (‐), DSS + IL‐7R (+), LPS, LPS + anti‐TLR4 antibody) received intraperitoneal injections of GW4869 (MCE, HY‐19363,2.5 mg/kg), anti‐Ly‐6G antibody (BioLegend, 127680, 200 μg per mouse), DNase I (Sigma–Aldrich, 10104159001, 20 mg/kg), anti‐IL‐7R antibody (Elabscience, E‐AB‐ F10230 , 20 μg per mouse), Recombinant mouse IL‐7R (MCE, HY‐ P72536 , 20 μg per mouse), LPS (intravenous injection via tail vein, Solarbio, L8880, 200 μg per mouse), and anti‐TLR4 antibody (intravenous injection via tail vein, BioLegend, 117617, 200 μg per mouse), respectively, on days 1, 3, 5, and 7.

Techniques: Clinical Proteomics, Derivative Assay, Western Blot, Recombinant, Staining, Immunofluorescence, Expressing

Journal: Advanced Science

Article Title: IL‐7R‐Enriched Extracellular Vesicles From the Thymus Drive Colitis via Promoting Neutrophil Extracellular Trap Formation

doi: 10.1002/advs.202520331

Figure Lengend Snippet: IL‐7R exacerbates the severity of experimental DSS‐induced colitis. (a) Mice with DSS‐induced colitis received either anti‐IL‐7R antibodies or recombinant mouse IL‐7R to modulate IL‐7R expression, with body weight changes monitored over time (Day 1 was set as 0%). (b) Changes in the DAI, which were assessed based on diarrhea, bleeding, and body weight loss. (c) Colon length of mice with different treatments. (d) Macroscopic appearance of the colon, as represented by the colon with the mean colon length and typical injury findings. (e) Mean colon macroscopic scores were evaluated for each group. (f) Histopathological changes in the colon tissue samples were examined using H&E staining. (g) Histopathological scores for the colon tissue samples were determined. (h, i) Goblet cell depletion in the colon was assessed using AB‐PAS staining (h) and quantified by counting goblet cells (i). (j) Relative mRNA expression levels of the inflammatory cytokines in colon tissue were quantified by qRT‐PCR. (k) IL‐17A, TNF‐α, iNOS, TGF‐β1, and IL‐1β protein expression in colon tissue was detected by immunohistochemistry. n = 5–10 mice per group; results are presented as the mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The intervention groups (DSS + GW4869, DSS + anti‐Ly‐6G, DSS + DNase I, DSS + IL‐7R (‐), DSS + IL‐7R (+), LPS, LPS + anti‐TLR4 antibody) received intraperitoneal injections of GW4869 (MCE, HY‐19363,2.5 mg/kg), anti‐Ly‐6G antibody (BioLegend, 127680, 200 μg per mouse), DNase I (Sigma–Aldrich, 10104159001, 20 mg/kg), anti‐IL‐7R antibody (Elabscience, E‐AB‐ F10230 , 20 μg per mouse), Recombinant mouse IL‐7R (MCE, HY‐ P72536 , 20 μg per mouse), LPS (intravenous injection via tail vein, Solarbio, L8880, 200 μg per mouse), and anti‐TLR4 antibody (intravenous injection via tail vein, BioLegend, 117617, 200 μg per mouse), respectively, on days 1, 3, 5, and 7.

Techniques: Recombinant, Expressing, Staining, Quantitative RT-PCR, Immunohistochemistry

Journal: Advanced Science

Article Title: IL‐7R‐Enriched Extracellular Vesicles From the Thymus Drive Colitis via Promoting Neutrophil Extracellular Trap Formation

doi: 10.1002/advs.202520331

Figure Lengend Snippet: IL‐7R aggravates colitis by inducing NETs via the PAD4 pathway. (a) Relative mRNA expression level of PAD4 in neutrophils stimulated with IL‐7R (qRT‐PCR). (b) Schematic of IL‐7R protein administration to WT and PAD4 ‐KO DSS‐induced colitis mice. (c) Immunofluorescence analysis and quantification of NET formation in colon tissues. (d) Percentage change in body weight relative to Day 1. (e) Changes in the DAI. (f) Colon lengths measured on Day 10. (g) Characterization of the macroscopic appearance of the colon. (h) Mean macroscopic colon damage scores. (i) Representative H&E‐stained sections of colon tissue. (j) Histopathological scores. (k, l) Representative AB‐PAS stained sections (k) and quantification of goblet cells (l). n = 5 mice per group; results are presented as the mean ± SD; ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns: non‐significance.

Article Snippet: The intervention groups (DSS + GW4869, DSS + anti‐Ly‐6G, DSS + DNase I, DSS + IL‐7R (‐), DSS + IL‐7R (+), LPS, LPS + anti‐TLR4 antibody) received intraperitoneal injections of GW4869 (MCE, HY‐19363,2.5 mg/kg), anti‐Ly‐6G antibody (BioLegend, 127680, 200 μg per mouse), DNase I (Sigma–Aldrich, 10104159001, 20 mg/kg), anti‐IL‐7R antibody (Elabscience, E‐AB‐ F10230 , 20 μg per mouse), Recombinant mouse IL‐7R (MCE, HY‐ P72536 , 20 μg per mouse), LPS (intravenous injection via tail vein, Solarbio, L8880, 200 μg per mouse), and anti‐TLR4 antibody (intravenous injection via tail vein, BioLegend, 117617, 200 μg per mouse), respectively, on days 1, 3, 5, and 7.

Techniques: Expressing, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: Advanced Science

Article Title: IL‐7R‐Enriched Extracellular Vesicles From the Thymus Drive Colitis via Promoting Neutrophil Extracellular Trap Formation

doi: 10.1002/advs.202520331

Figure Lengend Snippet: The thymus is the primary source of circulating IL‐7R‐enriched EVs. (a) qRT‐PCR analysis of IL‐7R expression in colon, thymus, bone marrow, brain, and liver tissues of mice. (b) Immunofluorescence evaluation of IL‐7R expression in the thymus. (c) Nanoflow cytometry analysis of IL‐7R levels in serum EVs. (d) TD‐EVs‐Normal and TD‐EVs‐Colitis were analyzed using negative‐staining TEM and western blotting detection of TSG101, CD9, and CD81. (e) Nanoflow cytometry analysis of IL‐7R levels in TD‐EVs‐Normal and TD‐EVs‐Colitis. (f) Western blotting detection of IL‐7R in TD‐EVs‐Normal and TD‐EVs‐Colitis. (g) ELISA quantification of LPS levels in serum and thymus. (h) LPS administration via tail vein injection in mice, LPS levels in serum and thymus were quantified using ELISA. (i) qRT‐PCR analysis of IL‐7R expression post‐LPS treatment. (j) Immunofluorescence of thymic IL‐7R post‐LPS treatment. (k) TD‐EVs‐LPS was analyzed using negative‐staining TEM and western blotting detection of TSG101, CD9, CD81, IL‐7R levels. (l) Ex vivo imaging of DiR‐labeled TD‐EVs‐Colitis and TD‐EVs‐LPS in colon, spleen, liver, femur, tibia, brain, thymus, and lung. (m) Neutrophils were incubated with PKH26‐labeled TD‐EVs‐Normal, TD‐EVs‐Colitis, TD‐EVs‐LPS, and EVs internalization was examined using laser scanning confocal microscopy. (n–q) NET formation assay: Neutrophils were treated with PBS, TD‐EVs‐Normal, TD‐EVs‐Colitis, TD‐EVs‐LPS, or PMA, with/without anti‐IL‐7R antibody. NETs were quantified. TD‐EVs‐Normal: EVs from thymus of normal mice; TD‐EVs‐Colitis: EVs from thymus of DSS‐induced colitis mice; TD‐EVs‐LPS: EVs from thymus of LPS‐treated mice; IL‐7R (‐): anti‐IL‐7R antibody. n = 4–5 mice per group; results are presented as the mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns: non‐significance.

Article Snippet: The intervention groups (DSS + GW4869, DSS + anti‐Ly‐6G, DSS + DNase I, DSS + IL‐7R (‐), DSS + IL‐7R (+), LPS, LPS + anti‐TLR4 antibody) received intraperitoneal injections of GW4869 (MCE, HY‐19363,2.5 mg/kg), anti‐Ly‐6G antibody (BioLegend, 127680, 200 μg per mouse), DNase I (Sigma–Aldrich, 10104159001, 20 mg/kg), anti‐IL‐7R antibody (Elabscience, E‐AB‐ F10230 , 20 μg per mouse), Recombinant mouse IL‐7R (MCE, HY‐ P72536 , 20 μg per mouse), LPS (intravenous injection via tail vein, Solarbio, L8880, 200 μg per mouse), and anti‐TLR4 antibody (intravenous injection via tail vein, BioLegend, 117617, 200 μg per mouse), respectively, on days 1, 3, 5, and 7.

Techniques: Quantitative RT-PCR, Expressing, Immunofluorescence, Cytometry, Negative Staining, Western Blot, Enzyme-linked Immunosorbent Assay, Injection, Ex Vivo, Imaging, Labeling, Incubation, Confocal Microscopy, Tube Formation Assay