Colonization of fecal microbiota from patients with neonatal necrotizing enterocolitis exacerbates intestinal injury in germfree mice subjected to necrotizing enterocolitis-induction protocol via alterations in butyrate and regulatory T cells

Abstract

Abstract Background Necrotizing enterocolitis (NEC) remains a life-threatening disease in neonates. Numerous studies have shown a correlation between the intestinal microbiota and NEC, but the causal link remains unclear. This study aimed to demonstrate the causal role of gut microbiota in NEC and explore potential mechanisms involved. Methods Eighty-one fecal samples from patients with NEC and eighty-one matched controls (matched to the NEC infants by gestational age, birth weight, date of birth, mode of delivery and feeding patterns) were collected. To explore if altered gut microbiota contributes to the pathogenesis of NEC, fecal microbiota transplantation (FMT) was carried out in germ-free (GF) mice prior to a NEC-induction protocol that included exposure to hypoxia and cold stress. Butyric acid was also administered to demonstrate its role in NEC. The fecal microbiota from patients and mice were analyzed by 16S rRNA gene sequencing analysis. Short chain fatty acid (SCFA) levels were measured by gas chromatography-mass spectrometry (GC–MS). The ontogeny of T cells and regulatory T cells (Tregs) in lamina propria mononuclear cells (LPMC) from the ileum of patients and mice were isolated and analyzed by flow cytometry.The transcription of inflammatory cytokines was quantified by qRT-PCR. Results NEC patients had increased Proteobacteria and decreased Firmicutes and Bacteroidetes compared to fecal control samples, and the level of butyric acid in the NEC group was lower than the control group. FMT in GF mice with samples from NEC patients achieved a higher histological injury scores when compared to mice that received FMT with control samples. Alterations in microbiota and butyrate levels were maintained in mice following FMT. The ratio of Treg/CD4+T (Thelper) cells was reduced in both NEC patients and mice modeling NEC following FMT. Conclusions The microbiota was found to have NEC and the microbial butyrate-Treg axis was identified as a potential mechanism for the observed effects.

Keywords

• Necrotizing enterocolitis
• Fecal microbiota transplantation
• Gut microbiota
• Germ free
• Butyric acid