PC1109: Sex Differences in the Preterm Gut Microbiome Underlie Enhanced Gastrointestinal Immunity in Female Neonates

Background: Male sex is an established risk factor for common morbidities and mortality in the preterm neonatal population. However, the pathophysiology underlying these sex differences is unclear. The gut microbiome plays an important role in shaping early life immunity, and previous in vitro studies in our lab suggest that preterm female stool elicits an enhanced immune response in macrophages. Our objective was to investigate how differences between preterm male and female gut microbiomes may shape the immune response.

Methods: Neonates born between 33 and 41 weeks and admitted to the level IV Neonatal ICU or Nursery at our academic hospital were enrolled. Neonates who received antibiotics were excluded. Fresh stool was collected and frozen at -80C. The bacterial compositions of the neonatal stool samples were determined by 16S rRNA sequencing. To investigate the effect of sex on the immune response in preterm neonates, germ-free (GF) wild-type C57BL/6 mice were orally gavaged with fecal slurries from 3 preterm males, 3 preterm females of comparable gestational and chronological age, or left as controls (GF group), followed by oral infection with Citrobacter rodentium, a murine model of enteropathogenic/ enterohemorrhagic Escherichia coli (EPEC/EHEC). C. rodentium colony-forming units (CFUs) in the colons, livers, and spleens were quantified 72 hours later. To investigate whether female stool predisposes to increased colonic inflammation, the same treatment groups were provided with 1% dextran sodium sulfate (DSS) water to induce colitis, then sacrificed 8 days later. The local inflammatory response was characterized using flow cytometry, immunohistochemistry, and quantitative PCR (qPCR).

Results: Preliminary 16S rRNA analysis revealed sex differences in the abundance of bacterial genera, including Bacteroides and Staphylococcus, in both preterm and term neonates. In the model of enteric infection, the F-exGF group had significantly reduced dissemination of C. rodentium to the spleens and livers compared to the M-exGF group. The mice in the F-exGF group exposed to DSS had significantly (p < 0.05) less weight loss, lower bleeding scores, and reduced colon shortening at day 8 compared to mice in the M-exGF group, suggesting less severe colitis. Flow cytometry revealed significantly decreased colonic neutrophils in DSS-treated F-exGF mice. qPCR revealed increased colonic expression of Mucin2 and antimicrobial peptide Reg3g in F-exGF mice.

Conclusion: Mice enterally colonized with preterm female stool had reduced bacterial dissemination in a murine model of EPEC/EHEC and less severe disease in a murine model of colitis. Gut bacteria enriched in female preterm infants may confer protection against intestinal insults by promoting the gut barrier through induction of Mucin2 and Reg3g. Future studies will focus on identifying bacteria enriched in female preterm neonates that are inducers of Mucin2 and Reg3g and their potential therapeutic use in protecting preterm neonates against gut inflammation and infection.