In the present study, transcriptome analysis was performed on colon tissue. This approach to profile transcription in whole tissues may be limited in that the detected changes are derived from composite changes in plural cell types. However, previous transcriptome studies that have focused on intestinal epithelial cells (IECs) [20, 28] did not appear to adequately characterize the microflora-induced immunological changes in terms of transcriptional profiling, even in the small IECs, the physiology of which is supposed to be profoundly affected by a variety of GALT-derived cells and/or mediators. Although recent studies have revealed a wide array of immune-oriented functions of IECs [35, 36], the spectrum of immune functions carried out by IECs is limited. By analyses of gene expression using whole colons from commensal GF mice, certain bacteria have been reported to increase the expression of IFN-γ-related genes and immunoglobulins [14, 15, 21].
In the present study, the expression of CRSs was increased by commensal bacteria. CRSs are cationic peptides that have a pro-region with high similarity to the pro-region of α-defensins. Although mature CRS peptides have no sequence homology or structural homology to any other known anti-microbial peptides, CRSs have been reported to co-localize with cryptdins in the granules of Paneth cells in similar amounts to cryptdins and to have potent microbial bacteriacidal activity [37–39]. A previous study using the differential display technique showed higher expression of CRS4C in the small intestines of SPF ICR mice than of GF ICR mice . If CRSs function as the first-line defense against microbial invasion, like other various intestinal anti-microbial peptides, it is reasonable that the expression of CRSs in the small and large intestines of SPF mice is higher than that of GF mice. However, apparently normal colons do not have Paneth cells, which are the main sites of cryptdin and CRS localization in the small intestine. Although metaplastic Paneth cells expressing antimicrobial proteins such as cryptdins and lysozymes are known to appear in inflammed colons [41, 42], histological examination detected no such cells in the large intestines of SPF mice (data not shown). Because cryptdins have been reported to also be present in the epithelial cells of the small intestines , CRSs may be expressed in the colonic epithelium. The localization and biological implication of CRSs remains to be elucidated in future research.
The most interesting finding of the present study is the possible importance of IFN-α in the defense systems of the large intestine. The most prominent difference in the expression of professional immune molecules was observed for genes involved in the IFN-α induction pathway. Many of them, such as Ifit1, 2 , Ifi47 , Tgtp , Gbp2 and 4 , Irgm . Lgals9 , and Rsad2  were originally identified as IFN-α-inducible genes. One molecule, Irf7, is a rate limiting transcription factor located in the center of a self-amplifying positive feedback loop of massive IFN-α production . Microbial infection induces phosphorylation of IRF7 protein, and phosophorylated IRF7 is transported into nuclei to strongly induce the expression of IFN-αs and Irf7 itself. In the presence of kinase activated by infectious agents, newly synthesized IRF7 continues to activate the loop resulting in the explosive production of IFN-αs. The inducibility of IFN-α production in various tissues/cells and their steady state level of IRF7 proteins are known to be closely correlated . Isgf3g (Irf9) and Stat1 are 2 components of ISGF3, a signaling complex that transduces the signal from receptors of IFN-α/β and induces the expression of IFN-α-related molecules including double-stranded RNA-dependent kinase Eif2ak2 (Prkr) , 2'-5' oligozdenylate synthetase 1A (Oas1a or Oas1g) , ISG15 ubiquitin-like modifier (G1p2 or Isg15) , and especially, Stat1 and Irf7 . Changes in the expression of Stat2, the other component of ISGF3, have also been observed. In contrast, the expression of other interferon regulatory factors such as Irf1, Irf2, and Irf8 did not show differences, and expression of end-products such as the IFN-αs, a2 and a4 was not detected. These data suggest that, although GF and SPF mice usually do not express IFN-α, or do so at extremely low levels, once the stimulatory signals are triggered, they might produce different levels of IFN-α due to the difference in basal expression levels of rate-limiting regulatory factors of IFN-α production.
It is unknown, among the wide array of signaling molecules involved in IFN-α production, why only the relatively confined members showed differences in the present study. One possible explanation was provided by the results of IFN-α induction experiments. Administration of oral IFN-α inducers resulted in elevated tissue IFN-α content in both the small and large intestines. However, in spite of the high basal levels of IFN-α-related genes, including Irf7, the production of IFN-α in the small intestine occurred later than in the large intestines. The extreme rapidness of IFN production in the large intestine suggests a possible involvement of Type 1 interferon producing cells (IPCs), which are virtually identical to plasmacytoid dendritic cells (pDCs) . IPCs express extremely high levels of Irf7 mRNA and protein constitutively, and can produce 100 to 1000 times more IFN than other blood cell types within several hours following stimulation.
This assumption was further supported by our in situ hybridization analysis using specific probes against the IFN-α-inducible genes, Ifnal, Ifit, Irf7, and Oas1g, and the plasmacytoid dendritic cell marker Tlr7. In the small intestine, histological analysis clearly indicated that the cells which had positive signals for type 1 IFN-related genes and Tlr7 were mainly Paneth cells. In the large intestine, all signals were located in the mononuclear cells scattered predominantly in the lamina propria, although further examination is necessary to determine whether these signals were generated from the same cells. In accordance with the results of our IFN-α ELISA, the number of Ifna1-positive cells increased in the large intestine but not in the small intestine at 4 hours. The increase in Irf7-positive cells in the small intestine may reflect amplification of the IRF7 pool, which is a prerequisite for massive IFN production in most cells types except for IPCs. In good agreement with the results of IFN-α ELISA at 20 hours, the numbers of IFN-α- and Irf7-positive cells increased in the small intestine. The increase in the number of these cells in the large intestine ceased at this time point, suggesting that the detected immunoreactive IFN-α might represent circulating IFN-α in the blood.
These data, collectively, address the possibility that IFN production and biological defense by IFN in the large intestine is borne mainly by IPCs, presumably recruited from the bloodstream to the intestinal lamina propria.
Finally, we performed immunohistochemical analysis to confirm whether the cells responsible for IFN-α production in the present study are IPCs/pDCs. We have screened several antibodies raised against type 1 IFN-related genes and found that the antibody for ISG15, a well-known IFN-stimulated gene [55, 57], stained the cells in the lamina propria of the colon at 4 hours after IFN-α inducer treatment. Multiple staining by a combination of anti-ISG15 antibody and various CD markers has demonstrated that ISG15+ cells contains CD11b+ cells and mPDCA1+ cells but not CD11c+ cells. Conventionally, pDCs (or IPCs) have been identified as CD11c+B220+Gr-1+ cells or mPDCA1+ cells. However, recent studies suggested the phenotype of pDCs, especially in the peripheral tissues, may have a wide variation [58, 59]. Takenaka S. et al  reported that, in the colons of BALB/c and C57BL/6 mice, many mPDCA1+ cells exist but they are neither CD11c+ nor CD11b+, and no typical CD11c+B220+Gr-1+ pDCs were present. These findings have also been obtained in the colons of IQI mice in our study (data not shown): 1) CD11b+ cells were predominant in the colonic lamina propria; 2) the majority of mPDCA1+ cells was stained by neither anti-CD11b nor anti-CD11c antibodies; 3) typical pDCs (i.e., CD11c+B220+Gr-1+ cells) were virtually absent. We have found that some CD11b+ cells and mPDCA1+ cells were co-stained with anti-ISG15 antibody, but more than half of ISG15+ cells were double-negative for CD11b and mPDCA1. Colonic IPC may therefore be comprised of multiple cell populations with unique phenotypic characteristics distinct from conventional pDCs/IPCs. Accordingly, IFN-α production in IBD model mice has been noted in both CD11b+ and CD11c+ dendritic cells in colonic lamina propria . However, the sensitivity and specificity of immunohistochemistry are limited and further extensive studies are necessary to clarify the functional and phenotypic characteristics of colonic IPCs.
Among the immune-related genes whose expression differed in the presence and absence of intestinal flora in the present GeneChip analysis, the IFN-related genes, MHC-related genes, some complements, and immunoglobulins were expressed at lower levels in SPF mice than in GF mice. This seems somewhat paradoxical because it is plausible that contact with high levels of microorganisms in SPF mice may result in enhancement of certain immunological defense systems in the large intestine. Along this line, Chowdhury et al.  reported that the expression of IFN-related genes including Irf7, Stat1, Stat2 and Tap1 in the small intestines was increased by the colonization of GF piglets with adult conventional swine feces. The discrepancy between our data and theirs may be explainable by the differences in various experimental settings such as the host animal (mouse vs. swine), RNA source (large intestine tissue vs. small intestinal epithelial cell), microbial status (SPF vs. conventional), and age of animals (9 vs. 2 weeks old). However, we think that the difference in the period after colonization may be responsible for the difference in expression of IFN-related genes between our study and theirs. The inflammation induced by microbial colonization of GF animals has been known to be only temporary and cease within several weeks [62, 63]. After the inflammation is terminated, histological findings return to a state apparently indistinguishable from the normal intestines of mice genuinely harboring the microbes. Therefore, the up-regulation of IFN-related genes in the midst of or shortly after inflammation, and their down-regulation after a long period of resumption of integrity of tissues and adaptation to enteric microbes may be quite compatible and it appears that the IFN system may play an extraordinary role in the immunological confrontation, negotiation and reconciliation between microbiota and host animals. The immune systems of the gastrointestinal tract are known to have potent anti-inflammatory and regulatory properties. In the intestinal immune systems, constant exposure to bacteria-derived immunostimulating molecules may crowd up the threshold of activation of the inflammatory aspect of the immune system. Alternatively, the immune system may be programmed to develop anti-inflammatory and/or regulatory responses to microbial stimuli as a default setting, unless certain specialized machinery such as toll-like receptors provide the additional signature indicating the stimuli are derived from specific dangerous pathogens. The down-regulation of immune-related genes may represent certain acquired characteristics of the intestinal immune systems to adapt to circumstances in which immune cells are continually exposed to vast amounts of commensal bacteria.