Milk-derived Exosomes Prevent Activation of Inflammatory Pathways in the Lung During Necrotizing Enterocolitis

Background: Necrotizing enterocolitis (NEC) is an inflammatory disease commonly observed in premature infants. In addition to intestinal damage, lung injury has been observed in neonates with NEC. Breast-milk derived exosomes have been established as a therapeutic modality for NEC, capable of rescuing intestinal injury caused by the disease. However, the extent to which exosome treatment during NEC impacts the lung has not been assessed. Therefore, the aim of this study is to examine the effect of exosome administration on inflammatory pathways in the lungs during experimental NEC.

Methods: Experimental NEC was induced by gavage feeding of hyperosmolar formula, hypoxia, and lipopolysaccharide administration 3 times a day in neonatal C57BL/6 mice from postnatal days (P) 5-9 (approval n.32238). Hyperosmolar formula was supplemented with bovine milk exosomes (1.0 μg/μl), obtained by ultracentrifugation, in each feed during NEC induction. Experimental groups included control, NEC, and NEC+Exosomes. On P9, pups were sacrificed, and lungs and intestine were collected. The lungs were assessed using western blot to examine the presence of TLR4/NF-κB pathway and NLRP3 inflammasome markers. Results are presented as means ± SD, as data was normally distributed. Group comparisons were done using one-way ANOVA with Bonferroni-corrected post-hoc tests. p<0.05 was considered significant.

Results: NEC resulted in intestinal damage that was reduced by exosome administration. Total activation of transcription factor NF-κB, as determined by the ratio of phosphorylated-NF-κB to total NF-κB, was significantly increased in the NEC lungs and reduced upon exosome administration (Fig. A). Activation of TLR4, the receptor involved in activating the pathway, was significantly decreased in the lungs of the NEC+Exosomes and control groups relative to NEC (Fig. B). NLRP3 inflammasome components, NLRP3 and Caspase 1, were significantly increased in the NEC lungs and reduced following exosome administration (Fig. C, D). Mature Il-1β, the active form of Il-1β that is cleaved by the NLRP3 inflammasome, was significantly reduced in the NEC+Exosomes and control groups relative to NEC (Fig. E).

Conclusion: We demonstrated that milk-derived exosomes are capable of reducing inflammatory pathway activation in the lungs during NEC. These novel findings emphasize the beneficial effects of milk-derived exosomes in protecting organs, such as the lung, from injury caused by NEC.