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

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 isolatorreared 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.

Establishment of normal gut microbiota is compromised under excessive hygiene conditions

Background: Early gut colonization events are purported to have a major impact on the incidence of infectious, inflammatory and autoimmune diseases in later life. Hence, factors which influence this process may have important implications for both human and animal health. Previously, we demonstrated strong influences of early-life environment on gut microbiota composition in adult pigs. Here, we sought to further investigate the impact of limiting microbial exposure during early life on the development of the pig gut microbiota. Methodology/Principal Findings: Outdoor- and indoor-reared animals, exposed to the microbiota in their natural rearing environment for the first two days of life, were transferred to an isolator facility and adult gut microbial diversity was analyzed by 16S rRNA gene sequencing. From a total of 2,196 high-quality 16S rRNA gene sequences, 440 phylotypes were identified in the outdoor group and 431 phylotypes in the indoor group. The majority of clones were assigned to the four phyla Firmicutes (67.5% of all sequences), Proteobacteria (17.7%), Bacteroidetes (13.5%) and to a lesser extent, Actinobacteria (0.1%). Although the initial maternal and environmental microbial inoculum of isolator-reared animals was identical to that of their naturally-reared littermates, the microbial succession and stabilization events reported previously in naturally-reared outdoor animals did not occur. In contrast, the gut microbiota of isolator-reared animals remained highly diverse containing a large number of distinct phylotypes. Conclusions/Significance: The results documented here indicate that establishment and development of the normal gut microbiota requires continuous microbial exposure during the early stages of life and this process is compromised under conditions of excessive hygiene.

Gut microbiome modulates the toxicity of hydrazine: a metabonomic study

The influence of gut microbiota on the toxicity and metabolism of hydrazine has been investigated in germ-free and ‘conventional’ Sprague Dawley rats using 1H NMR based metabonomic analysis of urine and plasma. Toxicity was more severe in germ-free rats compared with conventional rats for equivalent exposures indicating that bacterial presence altered the nature or extent of response to hydrazine and that the toxic response can vary markedly in the absence of a functional microbiome.

Environmentally-acquired bacteria influence microbial diversity and natural innate immune responses at gut surfaces

Background: Early microbial colonization of the gut reduces the incidence of infectious, inflammatory and autoimmune diseases. Recent population studies reveal that childhood hygiene is a significant risk factor for development of inflammatory bowel disease, thereby reinforcing the hygiene hypothesis and the potential importance of microbial colonization during early life. The extent to which early-life environment impacts on microbial diversity of the adult gut and subsequent immune processes has not been comprehensively investigated thus far. We addressed this important question using the pig as a model to evaluate the impact of early-life environment on microbe/host gut interactions during development. Results: Genetically-related piglets were housed in either indoor or outdoor environments or in experimental isolators. Analysis of over 3,000 16S rRNA sequences revealed major differences in mucosa-adherent microbial diversity in the ileum of adult pigs attributable to differences in earlylife environment. Pigs housed in a natural outdoor environment showed a dominance of Firmicutes, in particular Lactobacillus, whereas animals housed in a hygienic indoor environment had reduced Lactobacillus and higher numbers of potentially pathogenic phylotypes. Our analysis revealed a strong negative correlation between the abundance of Firmicutes and pathogenic bacterial populations in the gut. These differences were exaggerated in animals housed in experimental isolators. Affymetrix microarray technology and Real-time Polymerase Chain Reaction revealed significant gut-specific gene responses also related to early-life environment. Significantly, indoorhoused pigs displayed increased expression of Type 1 interferon genes, Major Histocompatibility Complex class I and several chemokines. Gene Ontology and pathway analysis further confirmed these results.