Changes in MMΦ Phenotype Influence Early Life Development of the Enteric Nervous System

Abstract

Abstract Vancomycin is a common medication given to premature infants. While protective, this treatment is associated with increased risk of developing disease later in life, including late-onset sepsis and necrotizing enterocolitis (NEC). Previous studies demonstrated that neonatal mice exposed to vancomycin in the early life period exhibit disruptions in the structure and function of the enteric nervous system (ENS). It remains unclear whether these disruptions are a direct consequence of dysbiosis or an indirect result achieved via crosstalk with immune cells. Muscularis macrophages (MMΦs) are a subset of intestinal macrophages identified to interact closely with enteric neurons. We hypothesized that vancomycin impacts ENS structure and function in early life by altering the phenotype of MMΦs. Neonatal mice were fed vancomycin (83mg/kg/day) or water daily from day of life (DOL) 1–10 and sacrificed for analysis on DOL 10. Single cell RNA sequencing performed on isolated MMΦs (CD45+ CXCR1+CD11c−/lo) demonstrated that vancomycin-treated mice had macrophages with a more inflammatory phenotype. Consistent with our RNA seq, MMΦs in mice treated with vancomycin had changes in their morphology consistent with activated macrophages/microglia. Flow cytometry revealed that vancomycin treatment significantly altered the proportion of MMΦs that were yolk sac derived compared to the bone marrow. To investigate how early life bone marrow derived MMΦs contribute to postnatal ENS development, we utilized CCR2 minus;/minus; mice, which cannot recruit macrophages to the colon. These mice exhibited significantly fewer neural cell bodies, suggesting that antibiotic-driven changes in MMΦ origin may influence early postnatal development of the ENS.