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A microarray analysis of gnotobiotic mice indicating that microbial exposure during the neonatal period plays an essential role in immune system development

Masahiro Yamamoto12, Rui Yamaguchi3, Kaori Munakata1, Kiyoe Takashima1, Mitsue Nishiyama2, Kyoji Hioki4, Yasuyuki Ohnishi4, Masao Nagasaki3, Seiya Imoto3, Satoru Miyano3, Atsushi Ishige1 and Kenji Watanabe1*

Author Affiliations

1 Center for Kampo Medicine, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo, 160-8582, Japan

2 Tsumura Research Laboratories, Tsumura & Co., Ami, Ibaraki, 300-1192, Japan

3 Human Genome Center, Institute of Medical Science, University of Tokyo, Tokyo, 108-8639, Japan

4 Central Institute for Experimental Animals, Kawasaki, Kanagawa, 216-0001, Japan

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BMC Genomics 2012, 13:335  doi:10.1186/1471-2164-13-335

Published: 23 July 2012



Epidemiological studies have suggested that the encounter with commensal microorganisms during the neonatal period is essential for normal development of the host immune system. Basic research involving gnotobiotic mice has demonstrated that colonization at the age of 5 weeks is too late to reconstitute normal immune function. In this study, we examined the transcriptome profiles of the large intestine (LI), small intestine (SI), liver (LIV), and spleen (SPL) of 3 bacterial colonization models—specific pathogen-free mice (SPF), ex-germ-free mice with bacterial reconstitution at the time of delivery (0WexGF), and ex-germ-free mice with bacterial reconstitution at 5 weeks of age (5WexGF)—and compared them with those of germ-free (GF) mice.


Hundreds of genes were affected in all tissues in each of the colonized models; however, a gene set enrichment analysis method, MetaGene Profiler (MGP), demonstrated that the specific changes of Gene Ontology (GO) categories occurred predominantly in 0WexGF LI, SPF SI, and 5WexGF SPL, respectively. MGP analysis on signal pathways revealed prominent changes in toll-like receptor (TLR)- and type 1 interferon (IFN)-signaling in LI of 0WexGF and SPF mice, but not 5WexGF mice, while 5WexGF mice showed specific changes in chemokine signaling. RT-PCR analysis of TLR-related genes showed that the expression of interferon regulatory factor 3 (Irf3), a crucial rate-limiting transcription factor in the induction of type 1 IFN, prominently decreased in 0WexGF and SPF mice but not in 5WexGF and GF mice.


The present study provides important new information regarding the molecular mechanisms of the so-called "hygiene hypothesis".

Hygiene hypothesis; Germ-free; Toll-like receptor; Type 1 interferon; MetaGene Profiler