Yamamoto et al. BMC Genomics 2012, 13:335 http://www.biomedcentral.com/1471-2164/13/335 Page 10 of 15 Figure 9 Signal pathways overrepresented in SPL of 0WexGF, 5WexGF and SPF mice compared with GF mice. MGP analysis was performed using signal pathways and chains in the TRANTHPATH database. The pathways/chains were sorted according to the integrated p- value (q) and those with q < 0.001 have been represented. Some pathways/chains were classified by their biological contexts and colored as represented in the figure. comparison of our results with those of Schumann et al. [43] would be particularly valuable because in the latter study, enteric bacteria were eradicated by antibiotic treatment of neonatal rats from postnatal day 7 to 21. In the study by Schumann and colleagues, in addition to significant down-regulation of antigen presentation sys- tems, expression of paneth cell products such as α- defensins, matrylsin, and type IIA phospholipase A2 decreased and expression of mast cell proteases increased as a result of a drastic reduction in enteric bacteria evoked by antibiotics. Similar changes were observed in the SI of GF mice compared with the other 3 colonization models in the present study (data not shown). Furthermore, Schumann et al.. have also found that eradication of enteric bacteria had a greater effect, in terms of the number of affected probe sets, in SI than in the colon. Clustering analysis of the affected probe sets in the study revealed only a few functional categor- ies; i.e., endocytosis and vesicle-mediated transport in the proximal SI, immune response in the distal SI, and ion transport processes in the colon. The present ana- lysis based on MGP identified expression changes for many functional categories, including proton transport and vesicle-mediated transport in the SI (Table 2) and immune responses in the LI (Table 1). In spite of the ex- tensive differences in species, experimental settings, microarray platforms, and approaches to statistical ana- lysis, a detailed comparative examination of these two transcriptome data sets might provide new insights into the impact of commensal microbiota colonization during the neonatal period. Conclusions In summary, the gene expression profiles of mice with bacterial colonization at different times suggest that the encounter with environmental commensal microbiota during the specific time interval within neonatal period is essential for normal development of the immune sys- tem, especially of the LI. Microbiota-mediated develop- ment of regulatory circuits of TLRs and type I IFN seem to play a particularly important role. Rectification of chemokine expression might also be involved in microbiota-related immunological dysregulation because expression of chemokines was activated specifically in mice colonized at pre-pubertal ages. The present study provides important insights for clarification and refine- ment of the so-called "hygiene hypothesis". Methods Animals All animal experiments were performed in Central Insti- tute of Experimental Animals (CIEA; Kanagawa, Japan). IQI/Jic mice were kept under SPF and GF mice were housed in a Trexler-type flexible film isolator in a stand- ard germ-free state and screened on a weekly basis for germ-free status by sterile feces sampling and culturing on MRS-agar plates under aerobic and anaerobic condi- tions. Mice were housed in an air-conditioned room (temperature 24 ± 1 °C) with a controlled light/dark cycle (light on between 6:30 AM and 7:00 PM). Sterile food and water were available ad libitum. The mice were ran- domly divided into 4 groups; 10 male mice were included in each group. GF, SPF, 0WexGF, and 5WexGF mice (Figure 1). For generation of 0WexGF mice, preg- nant GF mice were housed with SPF female mice 1 day before delivery and only male pups were retrieved after weaning. To generate 5WexGF mice, 5-week-old GF male mice were housed with SPF female mice. Mice in all groups were sacrificed at 9 weeks of age. All animal procedures were approved by the institution's ethical committee for care and use of laboratory animals in research.