Early Life Antibiotic Exposures Inhibits Postnatal Enteric Nervous System Maturation and Alters Neonatal Neuroimmune Interactions

Abstract

Abstract The enteric nervous system (ENS) regulates intestinal homeostasis via interactions with smooth muscle, epithelium, and immune cells. To assess the impact of early life antibiotics on postnatal ENS development, we performed neuron specific translating ribosome affinity purification to isolate RNA from control and antibiotic exposed enteric neurons for RNA sequencing. Colonic enteric neurons from antibiotic exposed pre-weaning mice expressed significantly less mature neuron markers, and acquired a more inflammatory cytokine signaling signature (e.g. IL18, MCP-1, TLR4). Immunofluorescence staining and ELISA revealed increased enteric neuron density in antibiotic exposed colons, yet reduced expression of neuronal markers including Tuj1 (neuron specific tubulin), synaptophysin (synapse marker) and acetylcholine. Notably, antibiotics did not affect ENS development in germ free mice. By confocal microscopy, antibiotic exposure appeared to reduce phagocytosis of neuronal cell bodies by macrophages, but phagocytosis of synapses by macrophages was increased. Flow cytometry revealed that antibiotic exposure reduced the proportion of macrophages expressing the “M2” marker CD163 compared to controls. This observation is reminiscent of Hirschsprung disease (HSCR), a congenital absence of the ENS from the distal intestine, which causes intestinal macrophages to adopt a pro-inflammatory phenotype and predisposes patients for HSCR-associated enterocolitis secondary to decreased cholinergic signaling. These data suggest that early life antibiotic exposure disrupts how macrophages participate in postnatal ENS development in a microbiota dependent mechanism. NIH NIAID U01AI131342, NIH NIDDK P30DK052574, T32DK077653, NIH T32HD43010 American Academy of Pediatrics Marshall Klaus 2022