Secretory antibodies in breast milk promote long-term intestinal homeostasis by regulating the gut microbiota and host gene expression

Abstract

Maintenance of intestinal homeostasis requires a healthy relationship between the commensal gut microbiota and the host immune system. Breast milk supplies the first source of antigen-specific immune protection in the gastrointestinal tract of suckling mammals, in the form of secretory IgA (SIgA). SIgA is transported across glandular and mucosal epithelial cells into external secretions by the polymeric Ig receptor (pIgR). Here, a breeding scheme with polymeric Ig receptor-sufficient and -deficient mice was used to study the effects of breast milk-derived SIgA on development of the gut microbiota and host intestinal immunity. Early exposure to maternal SIgA prevented the translocation of aerobic bacteria from the neonatal gut into draining lymph nodes, including the opportunistic pathogen Ochrobactrum anthropi. By the age of weaning, mice that received maternal SIgA in breast milk had a significantly different gut microbiota from mice that did not receive SIgA, and these differences were magnified when the mice reached adulthood. Early exposure to SIgA in breast milk resulted in a pattern of intestinal epithelial cell gene expression in adult mice that differed from that of mice that were not exposed to passive SIgA, including genes associated with intestinal inflammatory diseases in humans. Maternal SIgA was also found to ameliorate colonic damage caused by the epithelial-disrupting agent dextran sulfate sodium. These findings reveal unique mechanisms through which SIgA in breast milk may promote lifelong intestinal homeostasis, and provide additional evidence for the benefits of breastfeeding.

Fig. 1.

Passive and active delivery of SIgA into the gastrointestinal tract. (A) Breeding scheme for generation of mice receiving passive and/or active SIgA. (B) Sections of colons from representative 10-d-old Pigr^−/− pups from Pigr^+/− and Pigr^−/− dams, stained for IgA (green) and nuclei (blue). Dotted lines indicate the boundary between the epithelium and lumen. (Scale bars, 50 μm.) (C) Levels of fecal IgA from Pigr^+/− and Pigr^−/− offspring of Pigr^+/− and Pigr^−/− dams (mean ± SEM, n = 9). Two-way ANOVA: a, significant effect of maternal Pigr genotype; b, significant effect of offspring Pigr genotype (P < 0.05).

Fig. 2.

Lack of passive SIgA in breast milk results in translocation of aerobic bacteria to MLNs in nursing mice. (A) MLN homogenates from 21-d-old mice were cultured for 4 d under anaerobic or aerobic conditions for quantification of cfus; each symbol represents an individual mouse. Differences in cfu were analyzed by two-way ANOVA; N.S., not significant. (B) Representative plates showing aerobically grown colonies harvested from MLNs.

Fig. 3.

The composition of the intestinal microbiota is shaped by passive SIgA. Fecal samples were collected from Pigr^+/− offspring of Pigr^+/− and Pigr^−/− dams at 21 d of age (weanling) and from the same mice at 70 d of age (adult). (A) Interactive Tree of Life maps showing relative abundance (weighted Unifrac distances) of OTUs as quantified by PhyloChip analysis of the bacterial 16S rRNA gene. OTUs are defined as groups of bacteria that share >99% sequence identity in the 16S rRNA gene. Each of the inner rings represents an individual offspring of Pigr^−/− dams, and each of the outer rings represents an individual offspring of Pigr^+/− dams. (B) 2D ordination plot generated by principal coordinate analysis of the fecal microbiota of Pigr^+/− offspring of Pigr^+/− and Pigr^−/− dams (sampled at 21 and 70 d of age), based on the presence or absence of 80 individual OTUs (unweighted Unifrac distances) that were uniquely characteristic of each group (i.e., present in the fecal microbiota of 60% of mice in a given group and absent in all mice in every other group). Symbols represent individual mice. (C) Hierarchical clustering of the 80 unique OTUs described in B. Bars represent samples from individual mice. 2D ordination and hierarchical clustering plots based on the abundance of bacterial OTUs (weighted Unifrac distances) are shown in SI Appendix, Fig. S5 E and F.

Fig. 4.

Gene expression in colonic ECs and responses to acute inflammation are shaped by both passive and active sources of SIgA. Seventy-day-old Pigr^+/− and Pigr^−/− offspring of Pigr^+/− and Pigr^−/− dams were left untreated or given 2% DSS in drinking water for 8 d. Global gene expression was quantified by microarray analysis of RNA from colonic ECs. RNA was collected from six mice in each treatment category, then was pooled into two samples from three mice per sample. The statistical analyses of the microarray data are described in detail in SI Appendix, Fig. S7. (A) Total number of genes significantly altered by each effect or combination (P < 0.01). (B) Relative changes in expression levels for the 1,130 differentially regulated genes described in A. (Left) Specific effects of early exposure to passive SIgA in breast milk were calculated by comparing gene expression in Pigr^+/− offspring of Pigr^+/− vs. Pigr^−/− dams in the absence of DSS. (Center) Specific effects of endogenous, actively transported SIgA were calculated by comparing gene expression in Pigr^+/− vs. Pigr^−/− offspring of Pigr^+/− dams in the absence of DSS. (Right) Specific effects of DSS were calculated by comparing gene expression in Pigr^+/− offspring of Pigr^+/− dams in the presence or absence of DSS. Dots above the red dashed line represent genes that were significantly altered by the indicated factor (P < 0.01). (C) Selected genes regulated by SIgA in mouse colonic ECs that are orthologous to human gene loci associated with CD, UC, both CD and UC (IBD), early-onset IBD, and CelD. SI Appendix, Table S2 provides a complete list of disease-associated genes that were regulated by passive SIgA, active SIgA, and DSS. (D) Multifactorial PCA was conducted using expression levels of the 1,130 differentially regulated genes described in A. Each sample represents pooled RNA from three mice, for a total of six mice per treatment group. Data for mRNA transcript levels from each sample were reduced to two PCs (PC1 and PC2). Scores were calculated as described in SI Appendix, Table S4. The points within the green oval represent the unique pattern of gene expression in mice that received both passive and active SIgA. (E) Colon pathology scores were determined for individual mice (six mice per group), and the average for each group was plotted against the average PC1 and PC2 scores from D.