O2198: Extracellular vesicles derived from intestinal epithelial organoids improve intestinal barrier function during necrotizing enterocolitis

Background: Necrotizing enterocolitis (NEC) is a devastating intestinal disease that mainly affects premature infants. NEC is characterized by impaired intestinal permeability which allows the invasion of bacteria. Important to intestinal barrier integrity are tight junction proteins which are decreased in NEC. It has been demonstrated that transplantation of intestinal epithelial organoids (IEO) and administration of IEO derived extracellular vesicles (IEO-EV) can rescue experimental NEC injury. In this study, we investigated whether IEO-EVs can improve intestinal barrier function during NEC.

Methods: IEOs were derived from postnatal day (p) 9 C57BL/6 mice and the culture media was collected on days 3,5, and 7. IEO-EVs were isolated from the IEO culture media via ultracentrifugation (100000g x 2.5h). Three groups were studied: (i) breast-fed mice as control (n=10); (ii) experimental NEC mice receiving PBS (n=8); and (iii) experimental NEC mice receiving IEO-EVs (n=9). NEC was induced between p5-9 by gavage feeding of hyperosmolar formula and lipopolysaccharide, as well as administration of hypoxia prior to each feed. Enema administration of either PBS or IEOs (2.75 x 108 particles per day) was given from p6 to p9. The terminal ileum was harvested for analysis on p9.

Results: IEO-EVs were successfully isolated and characterized by nanoparticle tracking and protein expression analysis of extracellular vesicle markers (data not shown). Experimental NEC injury was confirmed using a histological scoring system, with IEO-EV treatment reducing intestinal epithelial damage (p = 0.0241). In the NEC+IEO-EV group, there was decreased inflammation compared to NEC+PBS as indicated by gene expression analysis of inflammatory marker Tnfα (Figure A). Furthermore, immunostaining and protein expression analysis for tight junction marker claudin 4 (CLDN4) shows that the NEC+IEO-EV group had an increased expression compared to NEC+PBS, indicating improved intestinal permeability (Figure B & C).

Conclusion: IEO-EVs were successfully isolated as indicated by their size and the presence of specific markers. Administration of IEO-EVs resulted in reduced NEC incidence and inflammation, as well as improved intestinal barrier function. IEO-EVs may represent a novel therapeutic compound for the treatment of NEC.