Exosomes Derived from Dendritic Cells Treated with Schistosoma japonicum Soluble Egg Antigen Attenuate DSS-Induced Colitis

Abstract

Exosomes are 30-150 nm small membrane vesicles that are released into the extracellular medium via cells that function as a mode of intercellular communication. Dendritic cell (DC)-derived exosomes modulate immune responses and prevent the development of autoimmune diseases. Moreover, Schistosoma japonicum eggs show modulatory effects in a mouse model of colitis. Therefore, we hypothesized that exosomes derived from DCs treated with S. japonicum soluble eggs antigen (SEA; SEA-treated DC exosomes) would be useful for treating inflammatory bowel disease (IBD). Exosomes were purified from the supernatant of DCs treated or untreated with SEA and identified via transmission electron microscopy, western blotting and NanoSight. Acute colitis was induced via the administration of dextran sulfate sodium (DSS) in drinking water (5.0%, wt/vol). Treatment with exosomes was conducted via intraperitoneal injection (i.p.; 50 μg per mouse) from day 0 to day 6. Clinical scores were calculated based on weight loss, stool type, and bleeding. Colon length was measured as an indirect marker of inflammation, and colon macroscopic characteristics were determined. Body weight loss and the disease activity index of DSS-induced colitis mice decreased significantly following treatment with SEA-treated DC exosomes. Moreover, the colon lengths of SEA-treated DC exosomes treated colitis mice improved, and their mean colon macroscopic scores decreased. In addition, histologic examinations and histological scores showed that SEA-treated DC exosomes prevented colon damage in acute DSS-induced colitis mice. These results indicate that SEA-treated DC exosomes attenuate the severity of acute DSS-induced colitis mice more effectively than DC exosomes. The current work suggests that SEA-treated DC exosomes may be useful as a new approach to treat IBD.

Keywords: dendritic cell; dextran sulfate sodium; exosomes; inflammatory bowel disease; soluble egg antigen.

FIGURE 1

Successful isolation of exosomes from the supernatant of DCs treated with SEA (SEA-EXO) or untreated (EXO). (A) Exosome ultrastructure was confirmed via negative-staining TEM. (B) The expressions of the exosome-specific markers CD63, CD9, and CD81 were confirmed via western blotting. (C) The size distribution profile of the exosomes was investigated using NanoSight.

FIGURE 2

Clinical scores of acute DSS-induced colitis mice treated with exosomes. (A) The effect of exosomes on the body weight of mice (50 μg/mouse). Mice were weighed daily. (B) The effect of exosomes on the DAI. The DAI was evaluated using the parameters of weight loss, diarrhea, and bleeding. Statistical analyses were performed using a one-way ANOVA followed by Dunnett’s multiple comparison post hoc test (^∗P < 0.05, DSS+SEA-EXO versus DSS+PBS).

FIGURE 3

Colon length and colon macroscopic scores in acute DSS-induced colitis mice treated with exosomes. (A) On day 6, the mice were sacrificed, and their colons were removed. Colon length was measured as an indirect marker of inflammation. (B) The macroscopic appearance of colons. Mean colon length and typical injury findings are presented. (C) Mean macroscopic scores of colons. Statistical analyses of multiple groups were performed using a one-way ANOVA followed by Dunnett’s multiple comparison post hoc test versus DSS+PBS (^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001). Statistical significance between two experimental groups was assessed using the Independent-Sample t-test (^&P < 0.05, ^&&P < 0.01).

FIGURE 4

Histopathological changes in the colons of acute DSS-induced colitis mice treated with exosomes. (A) Colon tissue samples were examined using H&E staining (5×, 10×, 20×, and 40×). (B) Histopathological scores of colon tissue samples. Statistical analyses of multiple groups were performed using a one-way ANOVA followed by Dunnett’s multiple comparison post hoc test versus DSS+PBS (^∗∗P < 0.01, ^∗∗∗P < 0.001). Statistical significance between two experimental groups was assessed using the Independent-Sample t-test (^&P < 0.05).

FIGURE 5

Pro-inflammatory cytokine and immunoregulatory cytokine expression in the colons of acute DSS-induced colitis were determined by Real-time PCR. The mRNA expression levels of TNF- α, IFN-γ, IL-17A, IL-12, IL-22, and TGF-β were detected by Real-time PCR, the housekeeping gene GAPDH was used as internal reference. Statistical analyses of multiple groups were performed using a one-way ANOVA followed by Dunnett’s multiple comparison post hoc test versus DSS+PBS (^∗P < 0.05, ^∗∗P < 0.01). Statistical significance between two experimental groups was assessed using the Independent-Sample t-test (^&P < 0.05, ^&&P < 0.01).