The so-called "hygiene hypothesis" suggests that reduced exposure of children to microbes is associated with increased prevalence of common allergies in developed countries [1–3]. At birth, the gastrointestinal tract is sterile and the neonatal immune response is characterized by a polarized T helper 2 (Th2) cytokine profile [4, 5]. During gut colonization by commensal microorganisms, the gut immune system is constantly challenged by a myriad of bacterial and food antigens. Gut colonization apparently plays a major role in driving the initial Th2-skewed immune response toward a more finely balanced Th1/Th2 response, by boosting counterregulatory Th1 immune responses . Numerous studies using animal models have suggested the possible involvement of immunoregulatory lymphocytes, e.g., regulatory T cells (Treg) and/or interleukin-10 (IL-10) producing B cells and cytokines (IL-10 and transforming growth factor-β) in intestinal homeostasis, which are driven by the intestinal bacterial burden [7–9]
Many epidemiological studies suggest that there is an inverse relationship between infections in early childhood and the subsequent development of allergic diseases [10, 11]. Therapy with broad-spectrum antibiotics is frequently performed in pediatric practice and children receiving this therapy within their first year of life are particularly prone to develop allergic diseases later in life [12–14]. Studies in animal models, such as GF animals, have also suggested that microbiota play a critical role in normal development of the immune system . Oyama et al.  reported that antibiotic use during infancy in mice promotes a shift in the Th1/Th2 balance toward Th2-dominant immunity. Further, they demonstrated that GF mice do not develop oral tolerance, which was restored by microbial reconstitution in neonatal (3-week-old) mice but not in older mice . Impairment of immune tolerance has been shown to augment disease in various models of allergy and/or autoimmunity, including diabetes onset in NOD mice [18, 19], collagen-induced arthritis [20, 21], and experimental colitis . Taken together, these findings provide new perspectives on the pathogenesis and recurrence of these diseases [15, 23, 24]
In the present study, to elucidate the impact of microbes on the immune system during the neonatal period, we performed microarray analysis of LI, SI, LIV, and SPL of mice with or without enteric microbiota, and of GF mice reconstituted with microbiota at different ages.
In order to extract useful information from the massive amount of gene expression data obtained by microarray assay, we employed a gene set enrichment analysis approach in the present study. This type of analysis uses predetermined aggregations of genes (alternatively called gene sets, metagenes, gene modules, etc.) rather than individual genes to assess for coordinated expression in the samples. Single gene analysis may miss important effects on signaling because cellular processes often affect sets of genes acting in concert, with moderate effects on the strength of expression. Subtle but orchestrated changes of internally-related genes have often been found to be more important than a dramatic increase/decrease of a single gene. Therefore, it may be important to evaluate the statistical significance of changes in a gene aggregate or gene set, rather than in an individual gene. Since Subramanian et al..  initially proposed “Gene Set Enrichment Analysis (GSEA)”, a number of algorithms optimized for this type of analysis have been developed [26–33], and in the present study, we employed “MetaGene Profiler (MGP)” developed by Gupta et al. . Unlike most existing methods, the main characteristic of MGP is that it evaluates statistical data for a set of genes independently from data of other gene sets. Because of the advantage of the analysis, it is logical to compare the results of tests for the same set of genes observed under different conditions, such as case–control experiments with multiple cases and time-course experiments, because the statistical evidence is evaluated using the same standard. MGP was therefore suitable for the present study, which required multiple cross comparisons of the same samples . This study is part of a research project using GF and SPF mice of the IQI strain, which has been established as an inbred strain from ICR mice at the Central Institute for Experimental Animals (Kawasaki, Japan). We previously reported some of the results of microarray analyses conducted for the project. These studies indicated that 1) activation of the IFN-α system in LI differs significantly between GF and SPF mice , and 2) there is a striking commonality in transcriptome profiles between GF LI and SI, while the profiles of SPF LI and SI share almost nothing in common . The present study, despite the use of different cohorts of animals, different versions of GeneChip arrays, and completely different strategies and algorithms for bioinformatics analysis from previous studies, has given support to our earlier findings. Furthermore, we demonstrated that the lack of neonatal encounter with commensal microorganisms may result in profound alteration of certain signaling pathways including TLRs, Rac 1, and type-1 IFN, which cannot be restored by later exposure to microbiota. These findings may provide important insights into the molecular events underlying the interaction between neonatal immune systems and commensal microbiota.