Effect Of Intestinal Microbiota On Intestinal Epithelial Fucosylation In Neonatal Necrotizing Enterocolitis

PART I ANALYSIS OF INTESTINAL MICROBIOTA IN NEONATAL NECROTIZING ENTEROCOLITISObjective: Neonatal Necrotizing enterocolitis(NEC)is an acute abdomen that seriously threatens the life and health of newborns,especially very low birth weight or extremely low birth weight premature infants.Its pathogenesis is not yet fully understood.Growing evidence suggests that gut microbiota dysbiosis performs a critical function in NEC pathogenesis.However,the mechanism underlying is not clear.Therefore,this study aims to investigate the characteristics of microbiota composition in NEC and provide a theoretical basis for further revealing its possible role in the pathogenesis of NEC.Methods: Clinical experiment: NEC and non-NEC newborns were recruited from the Neonatology Department of the Chongqing Medical University Affiliated Children’s Hospital.Newborns having Bell stage II or higher NEC were enrolled into the NEC group,while the matched non-NEC infants who were hospitalized during the same period were enrolled into the Control group.We obtained fecal samples from participants and performed16 S r DNA high-throughput sequencing to analyze the characteristics of microbiota composition.Animal experiment: The 10-day-old C57BL/6 neonatal mice were randomly assigned to two groups(Control group and NEC group).The Control group was breastfed.NEC animal model was established by hypoxia,hyperosmolar formula feeding and cold stress for 4 days.The characteristics of microbiota composition was analyzed by 16 S r DNA high-throughput sequencing.Results: Clinical experiment: The prevalence of Proteobacteria was higher in the NEC patients than in the Control patients(P<0.01),while the relative abundance of Bacteroidetes(Bacteroides)in the NEC patients was lower than that in the Control patients(P<0.01).Animal experiment: Compared with the Control group,the abundance of Proteobacteria was higher in the NEC group(P < 0.05),while the proportion of Bacteroidetes(Bacteroides)was lower(P<0.05).Conclusions: Intestinal microbial composition is changed in both mice and humans with NEC,and gut microbiota dysbiosis is closely related to NEC.PART II THE MECHANISM OF CHANGES IN INTESTINAL EPITHELIAL FUCOSYLATION CAUSED BY DYSBIOSIS IN NEONATAL NECROTIZING ENTEROCOLITISObjective: Fucosylated glycans expressed on intestinal epithelial cells(IECs)are important for maintaining intestinal homeostasis.Intestinal epithelial fucosylation is regulated by intestinal microbiota.This study aims to investigate the expression characteristics of intestinal epithelial fucosylation in NEC and reveal its possible role in the pathogenesis of NEC.Methods: The 10-day-old C57BL/6 neonatal mice were randomly divided into six groups: the Control group,the NEC group,the NEC+r IL-22 group,the NEC+PBS group,the NEC+a IL-22 group and the NEC+c Ig G group.The Control group was breastfed.NEC animal model was established by hypoxia,hyperosmolar formula feeding and cold stress for 4 days.The NEC+r IL-22 group,NEC+PBS group,NEC+a IL-22 group and NEC+c Ig G group were anaesthetized and given a single r IL-22,PBS,anti-IL-22 monoclonal antibody or rat Ig G as isotype control intraperitoneally 1 hour before the initiation of NEC,respectively.The weights of all mice were closely monitored and recorded during modeling.Severity of intestinal injury was compared using the macroscopic and histological injury grading.The intestinal permeability was determined using FITC-dextran.The fucosylation status of intestinal epithelial cells(IECs)and proportion of type 3 innate lymphoid cells(ILC3s)in lamina propria lymphocytes(LPLs)were analyzed by flow cytometry.IL-22,fucosyltransferase 2(Fut2),TNF-α and IL-6 gene expression,and IL-22,TNF-α and IL-6 protein expression were detected by q RT-PCR and ELISA,respectively.The 3-day-old germ-free(GF)BALB/C neonatal mice were gavaged with fecal flora from NEC(GNN)or Control infants(GCN)for 7 days and then performed NEC model in the recipient mice.We first performed 16 S r DNA gene sequencing on recipient fecal DNA samples collected before NEC modelling to evaluate whether the recipient mice replicated the microbial signatures of patients.After modeling,the severity of intestinal injury was compared using the HE staining and histopathologic evaluation.The fucosylation status of IECs and proportion of ILC3 s in LPLs were analyzed by flow cytometry.Results: C57BL/6 neonatal mice: NEC mice were generally in poor condition.A significant decrease in body weight and a significant increase in intestinal permeability were observed in the NEC mice(P<0.0001).NEC mice developed more serious intestinal injury than Control mice,as indicated by intestinal morphological and histological changes and scores(both P<0.01).NEC mice had severer intestinal inflammation as evidenced by increased IL-6 and TNF-α transcript and protein levels(both P < 0.01).Decreased epithelial Fut2 expression was found in the NEC mice,as compared with the Control mice(P<0.0001).Lower IL-22 gene and protein expression in the intestine were found in the NEC mice,as compared with the Control mice(both P<0.001).The NEC mice had fewer F-ECs and RORγt+ ILC3 s than did the Control mice(P<0.001 and P<0.05,respectively).NEC+r IL-22 group had increased F-ECs(P < 0.05)and Fut2 expression(P < 0.05). NEC+r IL-22 group exhibited less severe intestinal injury indicated by decreased gut macroscopic and histological scores(both P<0.05),much milder body weight loss and significantly decreased intestinal permeability(P<0.01)when compared with the NEC+PBS group.NEC+r IL-22 group also had milder intestinal inflammation as evidenced by decreased IL-6 and TNF-α transcript and protein levels(P<0.01 for IL-6 and P<0.05 for TNF-α at transcript level;P<0.05 for IL-6 and P<0.05 for TNF-α at protein level).Compared with the NEC+c Ig G group,the NEC+a IL-22 group decreased F-ECs(P<0.01)and Fut2 expression(P<0.0001),significantly exacerbated intestinal injury(P<0.05),exhibited a significant loss in body weight,increased intestinal permeability(P < 0.0001),and aggravated intestinal inflammation(P<0.01 for IL-6 and P<0.001 for TNF-α at transcript level;P<0.001 for IL-6 and P<0.01 for TNF-α at protein level).GF BALB/C neonatal mice: Compared with the GCN group,the abundance of Proteobacteria was higher in the GNN group(P<0.05),while the proportion of Bacteroidetes(Bacteroides)was lower(P<0.05)after fecal microbiota transplantation.The GNN group showed increasingly aggravated intestinal damage,as indicated by gut histological scores(P<0.05).The frequencies of F-ECs and ILC3 s were significantly lower in the GNN group than in the GCN group(both P<0.01).Conclusions: Intestinal dysbiosis may play an important role in the pathogenesis of NEC.NEC-associated dysbiosis,especially the decrease of Bacteroidetes(Bacteroides),decreases expression of epithelial fucosylation accompanied by reduced ILC3 and IL-22 in the intestine,leading to the destruction of the integrity of the intestinal barrier and the pathogens invaded into the intestinal mucosa causing excessive inflammation.