Fig 2: Knockout of CD73 reverses the effect of ERCs on intestinal barrier restoration in colitis. (A) Alcian blue staining showing goblet cells (blue) in colon tissue (Scale bars: 400 or 100μm). (B) Quantitative analysis indicates the number of goblet cells in colon tissue (n = 6). (C) Representative TEM images show colonic epithelial cells’ ultrastructure (scale bar:1μm). Red arrows indicate colonic cell microvilli, and blue arrows outline tight junctions and the intercellular space between two adjacent cells. (D) The concentration of serum FITC-dextran suggests the intestinal mucosa’s permeability in each group (n=6). (E-H) Quantitative analysis of intestinal interepithelial tight junction proteins ZO-1, Occludin, and Claudin-1 with β-actin as a reference in colon tissues measured by western blot (n=3). Data are presented as mean ± s.e.m (SEM). ns, no significance; *P < 0.05; **P < 0.01; ***P < 0.001; ****P <0.0001, analyzed by one-way ANOVA with LSD multiple comparison post hoc test.

Fig 3: Knockout of CD73 reverses the effect of ERCs on CD4+T cell differentiation in colitis. MLN cells were collected on day 10 after DSS induction. To identify Th1 and Th17 cells, MLN cells were first incubated with a stimulation cocktail for 5 h, followed by staining with a fluorescent antibody. Flow cytometry plots and graph analysis of CD4+IFN-γ+ Th1 (A), CD4+IL-17+Th17 (B), and CD4+CD25+Foxp3+Tregs (C) in the MLNs from the Control, untreated, ERCs-treated and CD73-/-ERCs-treated groups. (D-F) Percentage of CD4+IFN-γ+Th1, CD4+IL-17+Th17 cells and CD4+CD25+Foxp3+Tregs (n=5). Data are presented as mean ± s.e.m (SEM). ns, no significance; *P < 0.05; **P < 0.01; ***P < 0.001; ****P <0.0001, analyzed by one-way ANOVA with LSD multiple comparison post hoc test.

Fig 4: Knockout of CD73 reverses the therapeutic effect of ERCs on colitis. (A) In vivo, BLI of ERCs were tracked at 6, 24, 48, and 72 hours after intraperitoneal or tail intravenous injection in DSS-induced colitis mice. (B) Daily body weight changes in each group for 10 days (n = 6). (C) Representative pictures showed blood stools in BALB/c mice on day 10 after DSS induction. (D, E) Representative photographs of the colon in each group and the colon’s length in each group were analyzed on day 10 (n = 6). (F) Representative H&E-stained micrographs of the colon; blue arrows indicate epithelial detachment, red arrows indicate epithelial hyperplasia, black arrows indicate infiltrating inflammatory cells, and black boxes indicate the loss of enteric crypts (scale bar: 200 μm) (n=6). (G) Histopathology scores were calculated to assess colonic damage quantitatively. Data are presented as mean ± s.e.m (SEM). ns, no significance; *P < 0.05; **P < 0.01; ***P < 0.001; ****P <0.0001, analyzed by one-way ANOVA with LSD multiple comparison post hoc test.

Fig 5: Knockout of CD73 reverses the effect of ERCs on decreasing the percentage of mature DCs and enhancing the tolerogenic function of the DCs in vitro. ERCs and CD73-/-ERCs were co-cultured with immature DCs obtained from the bone marrow of BALB/c mice with 200ng/ml LPS for 24 hours in a transwell system. The percentage of CD11c+CD86+ DCs and CD11c+MHCII+ DCs was measured by flow cytometry analysis. (A, B) Representative dot plots of CD11c+CD86+ DCs and CD11c+MHCII+ DCs. (C, D) Percentage of CD11c+CD86+ DCs and CD11c+MHCII+ DCs (n=6). (E-H) The IL-6, TNF-α, IL-1β, and IL-10 were examined in the supernatant of co-cultured cells (n=6). (I) Contour plot of Ki-67+ T cells in vitro. (J) Percentage of Ki-67+ cells (n=4). Data are presented as mean ± s.e.m (SEM). ns, no significance; * P < 0.05; **P < 0.01; ***P < 0.001; ****P <0.0001, analyzed by one-way ANOVA with LSD multiple comparison post hoc test.