Restricting microbial exposure in early life negates the immune benefits associated with gut colonization in environments of high microbial diversity

Abstract

Background: Acquisition of the intestinal microbiota in early life corresponds with the development of the mucosal immune system. Recent work on caesarean-delivered infants revealed that early microbial composition is influenced by birthing method and environment. Furthermore, we have confirmed that early-life environment strongly influences both the adult gut microbiota and development of the gut immune system. Here, we address the impact of limiting microbial exposure after initial colonization on the development of adult gut immunity.

Methodology/principal findings: Piglets were born in indoor or outdoor rearing units, allowing natural colonization in the immediate period after birth, prior to transfer to high-health status isolators. Strikingly, gut closure and morphological development were strongly affected by isolator-rearing, independent of indoor or outdoor origins of piglets. Isolator-reared animals showed extensive vacuolation and disorganization of the gut epithelium, inferring that normal gut closure requires maturation factors present in maternal milk. Although morphological maturation and gut closure were delayed in isolator-reared animals, these hard-wired events occurred later in development. Type I IFN, IL-22, IL-23 and Th17 pathways were increased in indoor-isolator compared to outdoor-isolator animals during early life, indicating greater immune activation in pigs originating from indoor environments reflecting differences in the early microbiota. This difference was less apparent later in development due to enhanced immune activation and convergence of the microbiota in all isolator-reared animals. This correlated with elevation of Type I IFN pathways in both groups, although T cell pathways were still more affected in indoor-reared animals.

Conclusions/significance: Environmental factors, in particular microbial exposure, influence expression of a large number of immune-related genes. However, the homeostatic effects of microbial colonization in outdoor environments require sustained microbial exposure throughout development. Gut development in high-hygiene environments negatively impacts on normal succession of the gut microbiota and promotes innate immune activation which may impair immune homeostasis.

Figure 1. Body weight of sow-reared and isolator-reared animals.

Body weight (BW) of all experimental animals at day 28 and 56 is shown. At day 28, a significant reduction in BW was observed for animals housed in isolators compared to sow-reared animals. InIs and OIs animals were significantly heavier than IN animals at day 56.

Figure 2. Gut morphology of sow-reared and isolator-reared animals.

Representative microscopy images of haematoxylin and eosin–stained cross-sections of tissue taken at 75% of the length of the small intestine from pigs at: (A) day 5 (original magnification 10×), (B) villus tips at day 5 (original magnification 20×), (C) day 28 (original magnification 10×), and (D) day 56 (original magnification 10×).

Figure 3. Bacterial diversity of the mucosa-adherent microbiota.

DGGE microbial profile at days 5, 28 and 56 in indoor pigs reared in isolators (A) and outdoor pigs reared in isolators (B) at days 5, 28 and 56. The overall DGGE profile showed a highly diverse microbiota at all three sampling time-points, increasing from day 5 towards day 56. Comparisons of DGGE pattern profiles in InIs animals (C) and OIs animals (D) were performed using Dice's similarity coefficient and visualized by dendrograms using the UPGMA algorithm.

Figure 4. Ileum transcriptome differences between indoor isolator-reared and outdoor isolator-reared animals.

Venn diagram of differentially expressed genes at each time-point is shown for the two treatment groups (P<0.01, −2≤ fold change ≥2).

Figure 5. Pathway analysis of genes differentially expressed in indoor isolator-reared and outdoor isolator-reared animals.

Differentially expressed genes (P<0.05) were imported into GeneGo MetaCore analytical software to determine significantly enriched canonical pathways in each group. Data represent the distribution in cell process categories of statistically significantly enriched pathways (P<0.05) between the two treatment groups.

Figure 6. Heatmap of IFN-induced gene subset.

A heatmap of IFN-related genes was generated from a subset extracted from the list of significantly expressed genes. Columns represent individual arrays, while rows represent specific genes of interest. The Z-score depicts a measure of distance, in standard deviations, away from the mean. The relative value for each gene is depicted by color intensity, with green indicating upregulated and red indicating downregulated genes.