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

Abstract

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 IAP(high), differentiated population also showed less abundant expression of CDX2, the transcription factor required for the development of the intestine, than the IAP(low), 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.

FIGURE 1.

The 5′ region of the TLR4 gene is highly methylated in IECs in vivo. A, expression of cytokeratin, a marker of epithelial cells, in SIECs and LIECs prepared from mice was confirmed by immunostaining. Cells were stained with FITC-conjugated anti-pan cytokeratin antibody (upper panels) or FITC-conjugated isotype control antibody (lower panels). Left panels show fluorescent images under confocal laser scanning microscopy, and right panels show transmission images under phase-contrast microscopy. A representative staining pattern of SIECs is shown. Similar staining patterns were obtained for LIECs. B, methylation frequencies of CpG motifs existing in the 5′ region of the TLR4 gene were compared among mouse SIECs, LIECs, and splenic cells (SP). DNA methylation of 7 CpG motifs in the 5′ region (nucleotides −102/+202) was analyzed by the bisulfite conversion reaction. The modified DNA from 6 mice was amplified by PCR, cloned, and sequenced for 15–17 independent clones. A methylation pattern of 7 CpG motifs (–7) for each clone is shown in the order of methylation frequency. Filled squares indicate methylated CpG motifs, whereas open squares indicate unmethylated CpG motifs. C, mean numbers of methylated CpG motifs ± S.D. are represented in the graph. * and **, significantly different (*, p < 0.05; **, p < 0.0001).

FIGURE 2.

TLR4 gene expression is repressed by epigenetic mechanisms during the differentiation of IECs. Differentiated (Fr1) and undifferentiated (Fr2) mouse SIECs were separately prepared. A, expression of IAP, a differentiation marker of IECs, was quantified by qRT-PCR in each SIEC population. After normalizing IAP expression using GAPDH mRNA levels, the relative expression level of IAP in Fr2 to that in Fr1 was calculated. Results are expressed as the mean ± S.D. of four independent experiments. B, methylation of CpG motifs in the 5′ region of the TLR4 gene in Fr1 and Fr2 SIECs was analyzed by a bisulfite conversion reaction. Nucleotide sequences after the conversion reaction were determined for 22 and 25 independent clones, respectively. Mean numbers of methylated CpG motifs ± S.D. are shown. C, TLR4 mRNA expression in each SIEC population was determined by qRT-PCR. Relative values normalized using GAPDH mRNA levels are shown. Results are expressed as the mean ± S.D. of four independent experiments. *, **, and ***, significantly different (*, p < 0.05; **, p < 0.01; ***, p < 0.0001).

FIGURE 3.

CDX2 decreases methylation of the TLR4 gene and increases LPS sensitivity in undifferentiated IECs. A, expression of CDX2 in differentiated (Fr1) and undifferentiated (Fr2) SIECs prepared from mice was determined by qRT-PCR. After normalizing CDX2 expression using GAPDH mRNA levels, relative expression levels of CDX2 in Fr2 to that in Fr1 is calculated. Results are expressed as the mean ± S.D. of four independent experiments. *, significantly different (p < 0.05). B, the IEC line Caco-2 and the monocyte line THP-1 were cotransfected with the CDX2 expression plasmid (pcDNA3.1-CDX2) or empty control vector (pcDNA3.1-empty) and a reporter plasmid carrying the 5′ region of the TLR4 gene (pGL-TLR4−1013/+118) for transient expression assays. The -fold increase in luciferase activity relative to that of cells transfected with the reporter plasmid alone is shown. Results are expressed as mean ± S.D. of three independent experiments. ** and ***, significantly different from control transfected with an empty vector (**, p < 0.001; ***, p < 0.0001). C, expression of the transcription factor SOX9 in differentiated (Fr1) and undifferentiated (Fr2) SIECs was determined by qRT-PCR. After normalizing SOX9 expression using GAPDH mRNA levels, the relative expression level of SOX9 in Fr2 to that in Fr1 was calculated. Results are expressed as the mean ± S.D. of two independent experiments. D, Caco-2 cells were cotransfected with the SOX9 expression plasmid (pcDNA3.1-SOX9) or empty control vector and a reporter plasmid carrying the 5′ region of the TLR4 gene for transient expression assays. The -fold increase in luciferase activity relative to that of cells transfected with the reporter plasmid alone is shown. Results are expressed as mean ± S.D. of two independent experiments. E, the CDX2 expression plasmid or empty control vector was introduced into Caco-2 cells. After selecting the transfected cells with G418, methylation of CpG motifs in the 5′ region of the TLR4 gene was analyzed by bisulfite conversion reaction. Mean numbers ± S.D. of methylated CpG motifs of 16–19 independent clones are shown. *, significantly different (p < 0.05). F and G, Caco-2 cells were transfected with the CDX2 expression plasmid or the empty vector (F). For RNAi experiments, the TLR4 siRNA expression plasmid (pBAsi-TLR4) or the control plasmid (pBAsi-cont) was additionally introduced into cells (G). Cells were then stimulated with 0.1–1000 ng/ml LPS for 18–22 h. Secretion of IL-8 into the culture supernatant was measured by ELISA. Results are expressed as mean ± S.D. of three independent experiments. N.D., not detected.

FIGURE 4.

Commensal bacteria are essential for TLR4 gene methylation in LIECs. Methylation frequencies of the TLR4 gene in SIECs and LIECs were compared between wild-type mice under CV conditions (black bars), wild-type mice under GF conditions (white bars), and MyD88^−/− mice under conventional conditions (MyD88^−/−, gray bars). DNA methylation was analyzed by the bisulfite conversion reaction using SIECs and LIECs prepared from 6–9 mice. Nucleotide sequences of 17–27 independent clones were determined. Mean numbers ± S.D. are represented. * and **, significantly different (*, p < 0.0005; **, p < 0.00005).

FIGURE 5.

Two axes regulate TLR4 gene expression. A–C, TLR4 mRNA expression in SIECs, LIECs, and splenic cells (SP) of CV (A), GF (B), and MyD88^−/− (C) mice was quantified by qRT-PCR. Relative values normalized using GAPDH mRNA levels are shown (blue circles). Results are expressed as the mean ± S.D. of 3–6 independent experiments. The frequency of methylated CpG motifs in SIECs, LIECs and splenic cells of CV, GF, and MyD88^−/− mice is also represented in the graphs (red squares). D and E, TLR4 mRNA expression levels in SIECs (D) and LIECs (E) of CV and MyD88^−/− mice relative to that of GF mice (blue bars) and the frequency of methylated CpG motifs (red squares) are shown as the mean ± S.D. a–h, significantly different (a, p < 0.05; f, p < 0.005; h, p < 0.0005; b–d, p < 0.0001; g, p < 0.00005; e, p < 0.00001). A–G, significantly different (A, B, C, D, F, and G, p < 0.05; E, p < 0.005).

FIGURE 6.

Mechanisms underlying the regulation of TLR4 gene expression in IECs. Mechanisms underlying the regulation of TLR4 gene expression in IECs are schematically drawn. TLR4 gene expression is repressed due to block of transcription by epigenetic mechanisms in differentiated villus IECs to prevent triggering an excessive inflammatory reaction against commensal bacteria ([A]). However, when invading microbes reach the crypt, undifferentiated IECs in this region expressing TLR4 at a higher level may respond to them. TLR4 expression is beyond control of epigenetic repression and is increased by stimulation with commensal bacteria in undifferentiated crypt IECs ([B]). Commensal bacteria contribute to epigenetic repression of TLR4 gene expression in IECs of the large intestine.