The current study sought to investigate the effects of environmental hygiene on microbial colonization and composition of the gut microbiota. Additionally, transcriptomic profiling was performed to assess the impact of environmental hygiene on gene expression, in particular those genes and pathways associated with immune function. Both indoor and isolator (representing urban lifestyle and high-hygiene status, respectively), and outdoor (representing rural lifestyle and low-hygiene status) conditions were compared using pigs as an experimental model.
Using extensive analysis of 16S rRNA gene libraries our study categorically revealed that early-life environment has a major impact on microbial diversity and that these differences are sustainable throughout adult life. Many of the bacterial phylotypes identified in our study are commonly found in the human and animal gastrointestinal tract [28–30]. Our results also identified that only 3.3% of the clones had less than 97% sequence similarity to existing database entries.
A major finding of the current study was the significant increase in the Firmicutes phylum in sow-reared pigs housed in outdoor environments compared to littermates housed in isolators with daily antibiotic treatment. Within the Firmicutes phylum, the most compelling observation was the abundance of lactobacilli in animals reared in the outdoor environment. Lactobacilli are often associated with the suckling pig and early stages of colonization in the gastrointestinal tract. In this study, the high abundance of lactobacilli in the fecal samples obtained from truly adult sows identified lactobacilli as normal colonizers of the adult pig microbiota in the outdoor environment. Leser et al.  found similar high-abundance phylotypes associated with the ileum, including L. amylovorous, L. johnsonii and L. reuteri, in pigs from different rearing environments. Our study further revealed that an increase in hygiene status in pigs housed both indoor and in isolators with antibiotic administration was associated with a significant decrease in mucosa-adherent lactobacilli. Affected species included L. reuteri, L. delbrueckii, L. amylovorous, L. johnsonii and L. mucosae.
The reduced microbial diversity in outdoor animals compared to indoor and isolator housed groups was a somewhat surprising outcome. These outdoor animals were exposed to a huge variety of different bacterial species, as well as fungi, Archaea and viruses, originating from both maternal and environmental sources. The soil especially is hugely abundant in micro-organisms, and estimates of soil diversity show the presence of at least 32 phyla, the dominant members of which are Proteobacteria, Bacteroidetes and Firmicutes . Soil ecosystems potentially provide an important source of microbes for gut colonization of outdoor animals. However, only a selective subset of environmental bacteria colonize the intestine, since we noted that the pig gut microbiota was comprised of a restricted number of phyla, dominated by Bacteroidetes and Firmicutes, consistent with published findings on the diversity of the adult human gut . Current thinking has focussed on the benefits of a highly diverse gut microbiota, as it has long been considered that this confers greater plasticity of the bacterial community to respond to perturbations within the gut ecosystem . Paradoxically, we found that exposure to a large variety of environmental microbes in early life does not generate greater diversity in the adult gut but rather leads to a microbiota that is dominated by a limited number of phyla composed of bacteria with proven health-promoting properties.
Lactobacilli have long been known for their health-promoting effects and they directly limit the prevalence of several intestinal pathogens including E. coli and salmonella [32–34]. In this study, L. reuteri was one of the most abundant members of the mucosa-adherent microbiota of the outdoor group. Reuterin, a broad-spectrum antimicrobial substance, is produced by L. reuteri  and inhibits most intestinal bacteria with the exception of Lactobacillus strains . Importantly, the greater abundance of L. reuteri in the outdoor animals may contribute to the enhanced presence of other Lactobacillus species as well as the decreased microbial diversity observed in these animals. A further point meriting comment is the reduced presence of potentially pathogenic phylotypes in outdoor-housed pigs. These phylotypes were clearly present in both indoor and isolator housed animals, although animals showed no overt signs of infection. The specific reduction in Firmicutes, in particular lactobacilli, in these pigs may affect the normal mechanisms of colonization resistance that control potentially pathogenic populations within the gut ecosystem.
Although there has been a major focus on health-promoting probiotic actions of lactobacilli following their introduction as oral supplements, significantly less attention has been paid to the effects of naturally-acquired, gut-colonizing (autochthonous) lactobacilli. Given that immune modulation is dependent on gut colonization, close proximity to the mucosa and host adaptation, naturally-acquired lactobacilli clearly deserve greater attention. Of those species studied, L. casei, L. johnsonii and L. plantarum are strong inducers of IL-12 and/or INF-γ, thereby favouring a Th1 cytokine profile [37, 38]. Conversely, L. reuteri inhibits the induction of IL-12 and TNF-α and also attenuates L. casei-induced IL-12 . A fine balance between Th1-polarising lactobacilli strains and those which counterbalance such responses may be an important factor in maintaining mucosal immune homeostasis and explain the lack of overt Th1 or Th2 responses in outdoor-housed pigs in the current study.
While there was no evidence of Th1/Th2 pathways being affected, we found significant effects of environment on the Type 1 interferon (IFN) signalling pathways. Isolator-reared pigs exhibited increased gene expression levels of the IFNα/β transcription/signalling factors IRF7 and USP18. Type 1 IFN signalling induces the expression of a large number of target genes, which in the current study included MX2, G1P2, ISG20, FAM14A, IFIT2 and IFIT3. Three Type 1 IFN-inducible genes (IFRD1, OAS1 and IFIT2) were increased in indoor-housed animals compared to outdoor-housed animals, indicating that the IFNα/β pathway is directly affected by the housing environment. A number of recent studies further support our data describing the influence of the gut microbiota on the Type 1 IFN pathway. For example, conventionalized pigs exhibited increased expression of IRF7, STAT1 and STAT2 when compared with their germ-free counterparts . Conversely, bacterial colonization of germ-free mice led to a decreased expression of the IFN-related genes IRF7, ISGF3G, IFIT1 and STAT1. Our study further qualifies these findings by establishing that specific microbial composition, rather than the microbiota as such, influences Type 1 IFN signalling during early colonization and development.
Type 1 IFNs have many biological properties, including innate, cellular and humoral adaptive immune responses . Much evidence has focussed on their central role in pathogen resistance, especially viral immunity through recognition of dsRNA. The significance of Type 1 IFNs in response to bacterial colonization and infection is receiving increasingly more attention [42, 43]. IFN expression is induced in numerous cell lineages, including macrophages and plasmacytoid dendritic cells, by bacterial components such as LPS and CpG DNA [44–46]. It is worth noting that the transcriptome analysis was performed on whole ileal tissue samples, rather than on a specific cell subset. In this study we elected to study interactions and contributions of all cell lineages present in the gut to comprehensively characterize the transcriptomic changes induced by different microbiota compositions. However, the contribution of individual lineages such as plasmacytoid dendritic cells (DCs), which naturally produce Type 1 IFN, will be addressed in subsequent studies.
IFN-α/β has profound effects on immune cell development  by regulating the differentiation of B and T cells, myeloid DCs and natural killer cells. Activation of immature DCs by IFN-α/β upregulates major histocompatibility complex (MHC) class I. Consistent with this, we found that antigen presentation by MHC class I was also affected by the microbiota and was upregulated in indoor reared animals which also displayed increased Type 1 IFN levels. MHC class I molecules are Type 1 IFN-inducible genes whose promoter regions contain typical IFN-stimulated response elements (ISREs). MHC class I molecules are specialized for presentation of endogenously synthesized proteins, including self-proteins, to the TCR of CD8+ T-cells . The cross-presentation of antigens on MHC class I molecules, the induction of CTL responses and the subsequent memory CD8+ T cell survival are also dependent on IFN-α/β.
Increased expression of MHC class I in the indoor environment was accompanied by the upregulation of a plethora of chemokines, including CCL2, CCL8, CCL28, CCR1, CXCR4 and CXCL12. Chemokines are chemotactic cytokines that function during immune responses to recruit effector cells to sites of inflammation and infection. They are involved in the pathophysiology of many diseases. Numerous chemokines have been implicated in the pathology and perpetuation of tissue destructive inflammatory processes in patients with IBD, including CCL2  and CCL8 . Increased expression of these chemokines in the indoor-housed animals indicates the presence of an immune-activated gut microenvironment. This contrasts with the lack of innate and pro-inflammatory gene expression in the outdoor-housed animals, which may be indicative of a more immune-tolerant and homeostatic mucosal immune system in these animals. Further studies are required to assess the impact of the microbiota, immune gene transcription and immune cell lineages on specific tolerance towards food and environmental antigens and long-term predisposition to infection, food intolerance and allergy.