Neonates are particularly susceptible to invasion by colonizing bacteria and thus mammalian mothers protect their children via antibodies and in particular, Immunoglobulin A (IgA), secreted into maternal milk. Necrotizing Enterocolitis (NEC) is a disease of premature infants characterized by invasion of the neonatal intestine by the microbiota. Studies have shown that the incidence of NEC is significantly reduced in infants fed with breast milk, though the mechanism is unclear. Our preliminary studies show that maternal IgA (mIgA) is an important factor in protection against NEC. Analysis of IgA-binding on fecal samples from premature infants indicated that breast milk was the predominant source of IgA in the first month post-delivery and that a relative drop in the fraction of bacteria bound by IgA is associated with the development of NEC. Sequencing of IgA-bound and unbound bacteria indicated that NEC was associated with a unique increase in IgA negative Enterobacteriaceae. Further, we confirmed that IgA is critical in preventing NEC in the murine model, where we demonstrate that pups reared by mIgA deficient mothers are susceptible to disease, indicating that IgA in breast milk is necessary for protection. Together these data indicate that mIgA binding is critical in preventing the development of NEC by shaping the neonatal microbiota. In order to mediate effects on bacterial communities IgA acts on bacterial sub- populations. Therefore, our central hypothesis is that IgA in maternal milk modifies intestinal bacteria directly, preventing their invasion of the neonatal intestine and the induction of inflammatory immune responses. We will test this hypothesis in three aims. In the first aim we will investigate the mechanisms of how individual sub- populations of Enterobacteriaceae change to elude mIgA. Further we will investigate the mIgA repertoire of different breast milk donors to test the hypothesis that variation in this repertoire is important in binding different intestinal bacteria.
In aim 2 we will investigate the mechanism of IgA binding on the neonatal microbiota at the community level, via 16S rRNA gene sequencing and by transcriptional analysis of a model organism in gnotobiotic mice. Our hope is to associate these data with analysis of the role of IgA on epithelial association of intestinal bacteria and the downstream immune responses that are induced. In the third aim we will investigate whether the production of mammary gland antibodies that protect against NEC are dependent upon maternal T cells.
These aims will lead to a better understanding of how mIgA shapes the nascent microbiome and the neonatal immune response. In addition, our proposal may improve provide strategies to prevent NEC via the targeted provision of breast milk containing protective antibodies to the most at-risk children.

Public Health Relevance

Necrotizing enterocolitis (NEC) is a terrible disease that affects approximately 7% of premature children and is lethal in 25-30% of cases. Maternal milk has been shown to reduce the incidence of NEC and this study proposes to investigate the role of maternal antibodies secreted into the milk in preventing bacterial invasion of the intestine and disease. Our proposal aims to describe the mechanism via which maternal milk protects the intestine via shaping the neonatal bacterial flora via specific binding to individual bacterial strains.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Gastrointestinal Mucosal Pathobiology Study Section (GMPB)
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Perrin, Peter J
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University of Pittsburgh
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