Masato Tsuda

Epigenetic control of the host gene by commensal bacteria in large intestinal epithelial cells.

Background: Intestinal epithelial cells (IECs) express low levels of TLR4 and are hyporesponsive to commensal bacteria. Results: TLR4 gene is methylated in IECs, and this process is dependent on commensal bacteria in the large intestine. Conclusion: Commensal bacteria control epigenetic modification of the host gene. Significance: This study shows a novel mechanism underlying the maintenance of intestinal symbiosis. Intestinal epithelial cells (IECs) are continuously exposed to large numbers of commensal bacteria but are relatively insensitive to them, thereby averting an excessive inflammatory reaction. We have previously reported that the hyporesponsiveness of a human IEC line to LPS was primarily the result of a down-regulation of TLR4 gene transcription through epigenetic mechanisms. In the present study we show that DNA methylation in the 5′ region of the TLR4 gene is significantly higher in IECs than in splenic cells in vivo. The methylation was shown to be dependent on the differentiation state of the IECs, as the differentiated IEC population that expressed higher levels of intestinal alkaline phosphatase (IAP) also displayed greater methylation and lower expression of the TLR4 gene than the undifferentiated population. The IAPhigh, differentiated population also showed less abundant expression of CDX2, the transcription factor required for the development of the intestine, than the IAPlow, undifferentiated population. Overexpression of CDX2 in an IEC line decreased the methylation level of the TLR4 gene, increased transcriptional promoter activity of the gene, and increased responsiveness to the TLR4 ligand. Furthermore, the methylation level of the TLR4 gene was significantly lower in IECs of the large intestine of germ-free mice than in those of conventional mice, whereas the level in IECs of the small intestine was almost equal between these mice, indicating that commensal bacteria contribute to the maintenance of intestinal symbiosis by controlling epigenetic modification of the host gene in the large intestine.

IgA production in the large intestine is modulated by a different mechanism than in the small intestine: Bacteroides acidifaciens promotes IgA production in the large intestine by inducing germinal center formation and increasing the number of IgA+ B cells

It has been demonstrated that intestinal commensal bacteria induce immunoglobulin (Ig) A production by promoting the development of gut-associated lymphoid tissues in the small intestine. However, the precise mechanism whereby these bacteria modulate IgA production in the large intestine, which harbors the majority of intestinal commensals, is poorly understood. In addition, it is not known which commensal bacteria induce IgA production in the small intestine and which induce production in the large intestine. To address these issues, we generated gnotobiotic mice mono-associated with different murine commensal bacteria by inoculating germ-free (GF) mice with Lactobacillus johnsonii or Bacteroides acidifaciens. In GF mice, IgA production was barely detectable in the small intestine and was not detected in the large intestine. Interestingly, total IgA secretion in the large intestinal mucosa of B. acidifaciens mono-associated (BA) mice was significantly greater than that of GF and L. johnsonii mono-associated (LJ) mice. However, there was no difference in total IgA production in the small intestine of GF, LJ and BA mice. In addition, in the large intestine of BA mice, the expression of IgA(+) cells and germinal center formation were more remarkable than in GF and LJ mice. Furthermore, B. acidifaciens-specific IgA was detected in the large intestine of BA mice. These results suggest that the production of IgA in the large intestine may be modulated by a different mechanism than that in the small intestine, and that B. acidifaciens is one of the predominant bacteria responsible for promoting IgA production in the large intestine.

TGF-β–Mediated Foxp3 Gene Expression Is Cooperatively Regulated by Stat5, Creb, and AP-1 through CNS2

Foxp3 plays an important role in the development and the function of regulatory T cells (Treg). Both the induction and maintenance of Foxp3 gene expression are controlled by several regulatory regions including two enhancers in the conserved noncoding sequences (CNS). The functions of Enhancer 1 in CNS1 are well established, whereas those of Enhancer 2 in CNS2 remain unclear. Although CNS2 contains enhancer activity, methylated CpG sequences in this region prevent Foxp3 gene expression in Foxp3− T cells. These sequences are, however, demethylated in Foxp3+ Treg by mechanisms as yet unknown. To investigate the role of CNS2, we have determined the Enhancer 2 core sequence by luciferase reporter assays in the absence of methylation to exclude the inhibitory effect and shown that transcription factors AP-1, Stat5, and Creb cooperate in regulating Enhancer 2 activity. We have then determined the methylation sensitivity of each of the transcription factors. AP-1 was found to be methylation sensitive as has previously been described for Creb. However, Stat5 was active even when its binding site in CNS2 was methylated. Stat5 binding to Enhancer 2 occurred early and preceded that of AP-1 and Creb during Treg induction. In addition, Stat5 activation is itself dependent on TGF-β signaling through Smad3-mediated blockade of Socs3 expression. These findings suggest that Stat5 is a key regulator for opening up the CNS2 region during induced Treg induction, whereas AP-1 and Creb maintain Enhancer 2 activity.

Bacteroides Induce Higher IgA Production Than Lactobacillus by Increasing Activation-Induced Cytidine Deaminase Expression in B Cells in Murine Peyer's Patches

The gut mucosal immune system is crucial in host defense against infection by pathogenic microbacteria and viruses via the production of IgA. Previous studies have shown that intestinal commensal bacteria enhance mucosal IgA production. However, it is poorly understood how these bacteria induce IgA production and which genera of intestinal commensal bacteria induce IgA production effectively. In this study, we compared the immunomodulatory effects of Bacteroides and Lactobacillus on IgA production by Peyer’s patches lymphocytes. IgA production by Peyer’s patches lymphocytes co-cultured with Bacteroides was higher than with Lactobacillus. In addition, the expression of activation-induced cytidine deaminase increased in co-culture with Bacteroides but not with Lactobacillus. We found that intestinal commensal bacteria elicited IgA production. In particular, Bacteroides induced the differentiation of Peyer’s patches B cell into IgA+ B cells by increasing activation-induced cytidine deaminase expression.

Commensal bacteria promote migration of mast cells into the intestine.

Mast cells differentiate from hematopoietic stem cells in the bone marrow and migrate via the circulation to peripheral tissues, where they play a pivotal role in induction of both innate and adaptive immune responses. In this study, the effect of intestinal commensal bacteria on the migration of mast cells into the intestine was investigated. Histochemical analyses showed that germ-free (GF) mice had lower mast cell densities in the small intestine than normal mice. It was also shown that GF mice had lower mast cell proportion out of lamina propria leukocytes in the small intestine and higher mast cell percentages in the blood than normal mice by flow cytometry. These results indicate that migration of mast cells from the blood to the intestine is promoted by intestinal commensal bacteria. In addition, MyD88⁻/⁻ mice had lower densities of intestinal mast cells than CV mice, suggesting that the promotive effect of commensals is, at least in part, TLR-dependent. The ligands of CXC chemokine receptor 2 (CXCR2), which is critical for homing of mast cells to the intestine, were expressed higher in intestinal tissues and in intestinal epithelial cells (IECs) of normal mice than in those of GF or MyD88⁻/⁻ mice. Collectively, it is suggested that commensals promote migration of mast cells into the intestine through the induction of CXCR2 ligands from IECs in a TLR-dependent manner.

Intestinal commensal bacteria promote T cell hyporesponsiveness and down-regulate the serum antibody responses induced by dietary antigen

Colonization of the gut by commensal bacteria modulates the induction of oral tolerance and allergy. However, how these intestinal bacteria modulate antigen-specific T cell responses induced by oral antigens remains unclear. In order to investigate this, we used germ-free (GF) ovalbumin (OVA)-specific T cell receptor transgenic (OVA23-3) mice. Conventional (CV) or GF mice were administered an OVA-containing diet. Cytokine production by CD4(+) cells from spleen (SP), mesenteric lymph nodes (MLN) and Peyers patches (PP) was evaluated by ELISA, as was the peripheral antibody titer. T cell phenotype was assessed by flow cytometry. CD4(+) cells from the SP and MLN of CV and GF mice fed an OVA diet for 3 weeks produced significantly less IL-2 than the corresponding cells from mice receiving a control diet, suggesting that oral tolerance could be induced at the T cell level in the systemic and intestinal immune systems of both bacterial condition of mice. However, we also observed that the T cell hyporesponsiveness induced by dietary antigen was delayed in the systemic immune tissues and was weaker in the intestinal immune tissues of the GF mice. Intestinal MLN and PP CD4(+) T cells from these animals also produced lower levels of IL-10, had less activated/memory type CD45RB(low) cells, and expressed lower levels of CTLA-4 but not Foxp3 compared to their CV counterparts. Furthermore, GF mice produced higher serum levels of OVA-specific antibodies than CV animals. CD40L expression by SP CD4(+) cells from GF mice fed OVA was higher than that of CV mice. These results suggest that intestinal commensal bacteria promote T cell hyporesponsiveness and down-regulate serum antibody responses induced by dietary antigens through modulation of the intestinal and systemic T cell phenotype.

Gene Expression in the Gitr Locus Is Regulated by NF-κB and Foxp3 through an Enhancer

Glucocorticoid-induced TNFR (Gitr) and Ox40, two members of the TNFR superfamily, play important roles in regulating activities of effector and regulatory T cells (Treg). Their gene expression is induced by T cell activation and further upregulated in Foxp3+ Treg. Although the role of Foxp3 as a transcriptional repressor in Treg is well established, the mechanisms underlying Foxp3-mediated transcriptional upregulation remain poorly understood. This transcription factor seems to upregulate expression not only of Gitr and Ox40, but also other genes, including Ctla4, Il35, Cd25, all critical to Treg function. To investigate how Foxp3 achieves such upregulation, we analyzed its activity on Gitr and Ox40 genes located within a 15.1-kb region. We identified an enhancer located downstream of the Gitr gene, and both Gitr and Ox40 promoter activities were shown to be upregulated by the NF-κB–mediated enhancer activity. We also show, using the Gitr promoter, that the enhancer activity was further upregulated in conjunction with Foxp3. Foxp3 appears to stabilize NF-κB p50 binding by anchoring it to the enhancer, thereby enabling local accumulation of transcriptional complexes containing other members of the NF-κB and IκB families. These findings may explain how Foxp3 can activate expression of certain genes while suppressing others.

Intestinal Bifidobacterium association in germ-free T cell receptor transgenic mice down-regulates dietary antigen-specific immune responses of the small intestine but enhances those of the large intestine

Bifidobacterium is a dominant bacterial species among commensals in the human intestine and is thought to have probiotic immunomodulatory effects. In this study, we investigated the effect of the association with Bifidobacterium pseudocatenulatum JCM 7041 (Bp) on dietary ovalbumin (OVA)-specific immune responses using germ-free OVA-specific T cell receptor transgenic mice (OVA23-3 mice). We established germ-free OVA23-3 mice, and then associated with Bp (BIF group) or without (CONT group) and additionally associated with segmented filamentous bacteria (SFB) and clostridia in both groups. BIF and CONT mice were fed an egg-white diet containing OVA for 1 week. Cytokine production in response to OVA by cells of Peyers patches (PPs) and lamina propria (LP) from the small and large intestine was measured. Interferon (IFN)-gamma and interleukin (IL)-6 production by PP cells from BIF group mice was lower than that of the CONT group. The proportion of PP cells expressing CD4+CD62L(low), an activated/memory T cell phenotype, was higher in BIF group mice than the CONT group. Furthermore, LP cells from the small intestine in Bp-associated mice showed a tendency to produce slightly lower IFN-gamma and IL-6, while the cells from large intestine produced markedly higher IFN-gamma, IL-5 and IL-6 than those in the CONT group. The pattern of cytokine production by PP in BIF animals was similar to those isolated from conventional mice. These results suggest that intestinal association with Bp might down-regulate excessive immune responses to dietary antigens of the small intestine but enhance those of the large intestine.

Commensal microbiota-induced microRNA modulates intestinal epithelial permeability through the small GTPase ARF4.

The intestinal tract contains many commensal bacteria that modulate various physiological host functions. Dysbiosis of commensal bacteria triggers dysfunction of the intestinal epithelial barrier, leading to the induction or aggravation of intestinal inflammation. To elucidate whether microRNA plays a role in commensal microbiome-dependent intestinal epithelial barrier regulation, we compared transcripts in intestinal epithelial cells (IECs) from conventional and germ-free mice and found that commensal bacteria induced the expression of miR-21-5p in IECs. miR-21-5p increased intestinal epithelial permeability and up-regulated ADP ribosylation factor 4 (ARF4), a small GTPase, in the IEC line Caco-2. We also found that ARF4 expression was up-regulated upon suppression of phosphatase and tensin homolog (PTEN) and programmed cell death 4 (PDCD4), which are known miR-21-5p targets, by RNAi. Furthermore, ARF4 expression in epithelial cells of the large intestine was higher in conventional mice than in germ-free mice. ARF4 suppression in the IEC line increased the expression of tight junction proteins and decreased intestinal epithelial permeability. These results indicate that commensal microbiome-dependent miR-21-5p expression in IECs regulates intestinal epithelial permeability via ARF4, which may therefore represent a target for preventing or managing dysfunction of the intestinal epithelial barrier.

Post-Transcriptional Regulation of Toll-Interacting Protein in the Intestinal Epithelium

Immune responses against gut microbiota should be minimized to avoid unnecessary inflammation at mucosal surface. In this study, we analyzed the expression patterns of Toll-interacting protein (Tollip), an inhibitor of TLRs and IL-1 family cytokine-related intracellular signaling, in intestinal epithelial cells (IECs). Comparable mRNA expression was observed in murine small and large IECs (S-IECs and L-IECs). However, Tollip protein was only detected in L-IECs, but not in S-IECs. Similar results were obtained in germ-free mice, indicating that L-IEC-specific TOLLIP expression does not depend on bacterial colonization. Next, to understand the mechanisms underlying the post-transcriptional repression of Tollip, 3´-UTR-mediated translational regulation was evaluated. The region +1876/+2398 was responsible for the repression of Tollip expression. This region included the target sequence of miR-31. The inhibition of miR-31 restored the 3´-UTR-meditaed translational repression. In addition, miR-31 expression was significantly higher in S-IECs than in L-IECs, suggesting that miR-31 represses the translation of Tollip mRNA in S-IECs. Collectively, we conclude that the translation of Tollip is inhibited in S-IECs, at least in part, by miR-31 to yield L-IEC-specific high-level expression of the Tollip protein, which may contribute to the maintenance of intestinal homeostasis.