Vancomycin Treatment in Neonatal Mice V4 16S

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 knockout (KO) 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.

万古霉素（Vancomycin）是一种广谱抗生素，广泛用于早产新生儿疑似脓毒症的临床治疗场景。尽管在此情境下合理用药或可挽救生命，但生命早期的抗生素暴露会改变正在发育的微生物组，且与致命并发症风险升高相关，包括晚发性脓毒症（late-onset sepsis, LOS）与坏死性小肠结肠炎（necrotizing enterocolitis, NEC）。近期研究显示，新生儿万古霉素治疗会破坏幼鼠出生后的肠神经系统（enteric nervous system, ENS）发育，该过程一定程度上依赖于神经-免疫相互作用。这提示生命早期抗生素暴露可能会干扰新生儿肠道内的此类相互作用。值得关注的是，一类组织驻留肠道巨噬细胞——肌层巨噬细胞（muscularis macrophages），已被证实是出生后ENS发育的关键调控因子。我们提出假说：万古霉素诱导的新生儿菌群失调，可通过影响巨噬细胞来干扰出生后ENS的发育。借助小鼠模型开展实验后，我们发现仅在生命最初10天暴露于万古霉素（而非成年小鼠），会通过增加骨髓源性巨噬细胞的招募，扩张促炎性结肠巨噬细胞群体。对新生儿结肠巨噬细胞进行的单细胞RNA测序（single cell RNA sequencing）结果表明，生命早期万古霉素暴露与未成熟及炎性巨噬细胞的增多显著相关，这与循环单核细胞分化为巨噬细胞的浸润现象相符。谱系追踪实验证实，万古霉素会显著增加非卵黄囊来源的巨噬细胞群体。与上述结果一致的是，在CCR2基因敲除（CCR2 knockout, KO）小鼠中，生命早期万古霉素暴露既未扩张结肠巨噬细胞群体，也未降低肠神经元密度。综上，这些研究结果表明，与成年小鼠的万古霉素暴露相比，生命早期万古霉素暴露会以独特的方式改变巨噬细胞的数量与表型，并最终导致ENS发育异常。