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. 2019 Jul;25(7):1110-1115.
doi: 10.1038/s41591-019-0480-9. Epub 2019 Jun 17.

Maternal IgA protects against the development of necrotizing enterocolitis in preterm infants

Kathyayini P Gopalakrishna  1   2 , Benjamin R Macadangdang  1   3 , Matthew B Rogers  4 , Justin T Tometich  1 , Brian A Firek  4 , Robyn Baker  5 , Junyi Ji  1   6 , Ansen H P Burr  1   7 , Congrong Ma  1 , Misty Good  8 , Michael J Morowitz  4 , Timothy W Hand  9   10
Affiliations

Maternal IgA protects against the development of necrotizing enterocolitis in preterm infants

Kathyayini P Gopalakrishna et al. Nat Med. 2019 Jul.

Abstract

Neonates are protected from colonizing bacteria by antibodies secreted into maternal milk. Necrotizing enterocolitis (NEC) is a disease of neonatal preterm infants with high morbidity and mortality that is associated with intestinal inflammation driven by the microbiota1-3. The incidence of NEC is substantially lower in infants fed with maternal milk, although the mechanisms that underlie this benefit are not clear4-6. Here we show that maternal immunoglobulin A (IgA) is an important factor for protection against NEC. Analysis of IgA binding to fecal bacteria from preterm infants indicated that maternal milk was the predominant source of IgA in the first month of life and that a relative decrease in IgA-bound bacteria is associated with the development of NEC. Sequencing of IgA-bound and unbound bacteria revealed that before the onset of disease, NEC was associated with increasing domination by Enterobacteriaceae in the IgA-unbound fraction of the microbiota. Furthermore, we confirmed that IgA is critical for preventing NEC in a mouse model, in which pups that are reared by IgA-deficient mothers are susceptible to disease despite exposure to maternal milk. Our findings show that maternal IgA shapes the host-microbiota relationship of preterm neonates and that IgA in maternal milk is a critical and necessary factor for the prevention of NEC.

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Figures

Extended Data Fig. 1.
Extended Data Fig. 1.
Maternal milk-derived antibodies binding to intestinal bacteria from preterm infants.
Extended Data Fig. 2.
Extended Data Fig. 2.
Fraction of intestinal bacteria bound by IgA in preterm infants.
Extended Data Fig. 3.
Extended Data Fig. 3.
Linear discriminant analysis of the microbiota of infants that will develop NEC and controls.
Extended Data Fig. 4.
Extended Data Fig. 4.
Deconvolution method to decrease the effect of contamination in IgSeq.
Extended Data Fig. 5.
Extended Data Fig. 5.
Longitudinal analysis of the intestinal microbiota of preterm infants.
Extended Data Fig. 6.
Extended Data Fig. 6.
Ratio of IgA− to IgA+ reads for low-abundance taxa.
Extended Data Fig. 7.
Extended Data Fig. 7.
Absolute number of bacteria and number of bacteria associated with the dominant taxa in preterm infants.
Extended Data Fig. 8.
Extended Data Fig. 8.
Enterobacter spp. is enriched in the IgA+ fraction of breast-fed mouse pups.
Figure 1
Figure 1. IgA binding to the intestinal bacteria of preterm infants is positively correlated to maternal milk feeding and negatively correlated to the development of NEC.
Flow cytometric analysis of bacterial IgA binding on fecal samples from preterm infants. a) Example of IgA staining on preterm infants. (b-d) Maternal milk fed infants are indicated by black circles, formula fed infants by open red squares. b) Percent IgA-bound bacteria from maternal milk fed (n=50) vs. formula fed (n=19) infants. The box represents the number of samples with <1% IgA binding of intestinal bacteria, two-sided Mann-Whitney test, Mean±SEM. c) Percent IgA binding was correlated by linear regression with time post-delivery in maternal milk fed (n=50) and formula fed (n=19) infants; Pearson’s correlation coefficient. d) Percent IgA bound bacteria from controls (n=28) or infants diagnosed with NEC (n=23), from samples collected <DOL40; red squares indicate formula fed infants; two-sided Mann-Whitney test, Mean±SEM. e) Percent IgA-bound intestinal bacteria from prospectively collected longitudinal samples of NEC patients (Patient number=10, sample size=39) and Controls (Patient number=13, sample size=59), graphed against DOL, each patient is graphed in a different color; linear regression and Pearson’s correlation coefficient.
Figure 2
Figure 2. Reduced intestinal bacterial diversity driven by increased IgA unbound Enterobacteriaceae precedes the development of NEC.
Longitudinal fecal samples from preterm infants prior to the onset of NEC were selected from NEC infants (n=10; 39 samples combined) and Controls (n=13, 59 samples combined) a) Samples were analyzed for the relative abundance of different bacterial OTUs by targeted sequencing of 16S rRNA genes. Shown is the mean relative abundance of different taxa between NEC patients and controls (pooled from all timepoints and infants). b) Relative abundance of Enterobacteriaceae in preterm fecal samples compared to percent IgA-bound bacteria from controls and prospective NEC patients (c-f) Fecal samples were separated into IgA positive and negative pools prior to 16S rRNA sequencing and deconvolution based on post-sort analysis. c) Shannon diversity scores of IgA positive and IgA negative samples from prospective NEC patients and controls, graphed against DOL. d) Relative abundance of Enterobacteriaceae from IgA positive and IgA negative samples from prospective NEC patients and controls, graphed against DOL. e) Relative abundance of combined anaerobic bacterial OTUs from IgA positive and IgA negative samples from prospective NEC patients and controls, graphed against DOL. f) Ratio of reads (IgA negative/IgA positive; Log2 transformed) from paired IgA positive and negative samples, graphed against DOL. Shown are the ratio of total reads, Enterobacteriaceae reads and combined anaerobes. For b-f, each patient is graphed in a different color and R-squared value is based on a linear regression and Pearson’s correlation coefficient.
Figure 3
Figure 3. IgA is a necessary component of breast milk to prevent the development of experimental NEC.
a) Experimental NEC mouse model where wild-type pups are fed by dams that either can (C57BL/6; n=31) or cannot produce IgA (Rag1−/−; n=22 or Igha−/−; n=14). Formula fed mice (n=48) are used as a positive control b) Representative IgA staining of the fecal matter of 8 day old pups from a), shows absence of IgA-bound bacteria in dam breast-fed and formula-fed pups. C57BL/6 (n=19), Rag1−/− (n=9), Igha−/− (n=8) and formula fed (n=23) c) Representative images of H&E staining of small intestine of pups undergoing murine NEC protocol; pups fed by C57BL/6 (n=23), Rag1−/− (n=17), Igha−/− (n=17) and formula fed (n=40) d) Histology scores of the small intestines of pups from c), one-way ANOVA with multiple comparisons; Mean±SEM. e) Percent number of pups from a) surviving the NEC protocol at different timepoints; statistics determined by Log-rank (Mantel-Cox) test. f) Weights of pups from a), statistical difference calculated by one-way ANOVA with multiple comparisons on weights at experimental completion point (day 4); Mean±SEM. Data shown in panel b-f is grouped together from 3 individual experiments with minimum n=5 pups in each group for every experiment.
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