Vancomycin Induced Gut Microbial Dysbiosis Alters Enteric Neuron-Macrophage Interactions During a Critical Period of Postnatal Development
Vancomycin is a broad-spectrum antibiotic widely used in cases of suspected sepsis in premature neonates. While appropriate and potentially lifesaving in this setting, early life antibiotic exposure alters the developing microbiome and is associated with increased risk of deadly complications, including late-onset sepsis (LOS) and necrotizing enterocolitis (NEC). Recent studies show that neonatal vancomycin treatment disrupts postnatal enteric nervous system (ENS) development in mouse pups, which is in part dependent upon neuro-immune interactions. This suggests that early life antibiotic exposure could disrupt these interactions in the neonatal gut. Notably, a subset of tissue-resident intestinal macrophages, muscularis macrophages, have been identified as important contributors to the development of the postnatal ENS. We hypothesized that vancomycin-induced neonatal dysbiosis impacts postnatal ENS development through effects on macrophages. Using a mouse model, we found that exposure to vancomycin in the first ten days of life, but not in adult mice, resulted in an expansion of pro-inflammatory colonic macrophages by increasing the recruitment of bone-marrow derived macrophages. Single cell RNA sequencing of neonatal colonic macrophages revealed that early-life vancomycin exposure was associated with an increase in immature and inflammatory macrophages, consistent with an influx of circulating monocytes differentiating into macrophages. Lineage tracing confirmed that vancomycin significantly increased non-yolk sac derived macrophage population. Consistent with these results, early life vancomycin exposure did not expand the colonic macrophage population nor decrease enteric neuron density in CCR2 deficient mice. Collectively, these findings demonstrate that early life vancomycin exposure alters macrophage number and phenotypes in distinct ways compared to vancomycin exposure in adult mice and results in altered ENS development. Overall design: Mouse pups were treated with vancomycin daily from birth until DOL 11. Cx3CR1+ macrophages were isolated via FACS (selected cells were Live, CD45+ CX3CR1+ CD11c lo/negative). Macropahges from pups in the same litter were pooled.  Control vs. vanomycin treated macropahges were analyzed via RNA seqeuncing.