Early life antibiotics influence in vivo and in vitro mouse intestinal epithelium maturation and functioning

Background & Aims The use of antibiotics (AB) is a common practice during the first months of life. AB can perturb the intestinal microbiota, indirectly influencing the intestinal epithelial cells (IECs), but also directly affect IECs, independent of the microbiota. Previous studies in rodents have mostly focused on the impact of AB treatment during adulthood. However, the difference between the adult and neonatal intestine warrants careful investigation of the effects of AB in early life.   Methods Neonatal mice were treated with a combination of amoxicillin, vancomycin, and metronidazole, from postnatal day 10 to 20. Intestinal permeability and whole intestine gene and protein expression were analyzed. IECs were FACS-sorted and their genome-wide gene expression analyzed. Mouse fetal intestinal organoids were treated with the same AB combination and their gene and protein expression, and metabolic capacity determined. Results We found that in vivo treatment of neonatal mice led to decreased intestinal permeability and reduced number of specialized vacuolated cells, characteristic of the neonatal period and necessary for absorption and digestion of milk macromolecules. Additionally, the expression of genes typically present in the neonatal intestinal epithelium was lower, whereas the adult gene expression signature was higher. Moreover, we found altered epithelial defense and transepithelial sensing capacity. In vitro treatment of intestinal fetal organoids with AB showed that part of the consequences observed in vivo is a result of a direct action of the AB on IECs. Lastly, AB reduced the metabolic capacity of intestinal fetal organoids.   Conclusion Our results demonstrate that early life AB treatment induces direct and indirect effects on IECs, influencing their maturation and functioning. Postnatal (P)10 C57BL/6 wild-type mice pups were randomly distributed in 2 groups: treatment group (2 litters, 6 pups per litter) and control group (2 litters, 6 pups per litter). Treatment group received daily via oral gavage antibiotic mix (25mg/kg/day amoxicillin, 50mg/kg/day metronidazole, and 50mg/kg/day vancomycin), while control group received PBS. At P20, proximal and distal small intestine were separated and epithelial cells were FACS-sorted using the EpCAM marker. The epithelial cells of 3 different pups, belonging to the same litter, were combined into one sample, resulting in 4 samples per group. RNA was extracted and microarray analysis was performed using Affymetrix Clariom® S 8-Array HT Plate