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, 25 (5), 1449-60

Colonic Mucosal DNA Methylation, Immune Response, and Microbiome Patterns in Toll-like Receptor 2-knockout Mice

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Colonic Mucosal DNA Methylation, Immune Response, and Microbiome Patterns in Toll-like Receptor 2-knockout Mice

Richard Kellermayer et al. FASEB J.

Abstract

The connection between intestinal microbiota and host physiology is increasingly becoming recognized. The details of this dynamic interaction, however, remain to be explored. Toll-like receptor 2 (Tlr2) is important for its role in bacterial recognition, intestinal inflammation, and obesity-related metabolic changes. Therefore, we sought to determine the epigenomic and metagenomic consequences of Tlr2 deficiency in the colonic mucosa of mice to gain insights into biological pathways that shape the interface between the gut microbiota and the mammalian host. Colonic mucosa from wild type (WT) and Tlr2(-/-) C57BL/6 mice was interrogated by microarrays specific for DNA methylation and gene expression. The mucosal microbiome was studied by next-generation pyrosequencing of bacterial 16S rRNA. The expression of genes involved in immune processes was significantly modified by the absence of Tlr2, a number of which correlated with DNA methylation changes. The epigenomic and transcriptomic modifications associated with alteration in mucosal microbial composition. Several bacterial species, including members of the Firmicutes were significantly different in abundance between WT and Tlr2(-/-) animals. This manuscript highlights the intimate interrelationships between expression of immune-related genes and immunity pathways in the host with compositional and functional differences of the mammalian microbiome.

Figures

Figure 1.
Figure 1.
Tlr2-dependent DNA methylation changes at Anpep correlate with its expression. A) Methylation of the Anpep-associated SmaI/XmaI interval (calculated from both flanking sites; see Materials and Methods) increased in Tlr2−/− colonic mucosa (WT, n=8; Tlr2−/−, n=9; P<0.0001). B) Anpep expression decreased in Tlr2−/− colonic mucosa (WT, n=10; Tlr2−/−, n=11; P=0.017). C) Methylation at Anpep inversely correlated with expression (r=−0.8; n=13; P=0.008).
Figure 2.
Figure 2.
Methylation of the Anxa-associated 3′ SmaI/XmaI site is decreased in Tlr2−/− colonic mucosa (P=0.0154; n=10/group).
Figure 3.
Figure 3.
Tlr2-dependent DNA methylation changes at Ifit2 correlate with its expression. A) Average methylation of 2 CpG sites in the downstream vicinity (see Materials and Methods) of the IRF3 transcription factor binding site in the Ifit2-promoter region increased in Tlr2−/− colonic mucosa (n=10/group; P=0.0026). B) Ifit2 expression increased in Tlr2−/− colonic mucosa (WT, n=9; Tlr2−/−, n=10; P=0.0001). C) Methylation at Ifit2 positively correlates with expression (r=0.5; n=13; P=0.04).
Figure 4.
Figure 4.
Validation of transcript level changes by real time RT-PCR. A) Fas expression increased in Tlr2−/− mucosa, with a trend approaching significance (n=10/group). B–D) Significant expression changes were confirmed in Tlr2−/− colonic mucosa for Lgals2 (WT, n=9; Tlr2−/−, n=10; B), Spon2 (n=10/group; C), and Stat1 (WT, n=10; Tlr2−/−, n=8; D).
Figure 5.
Figure 5.
Dual hierarchical clustering dendogram of the most predominant and ubiquitous 50 genera among mucosal samples. Heat map depicts the relative percentage of each genus for each sample; color scale for heat map at top left. Tlr2−/− animals were from 3 different litters (samples Tlr1 and Tlr2; Tlr3 and Tlr5; and Tlr4, respectively), while WT animals were from 2 different litters (samples 11 and 12; and 16, 19, and 20, respectively). Therefore, interindividual (within the same litter) variation exceeded that of interlitter variation within the two groups of animals.
Figure 6.
Figure 6.
Principal component analysis of UniFrac distance metric. Three-dimensional visualization of the PCA analysis using the top 3 vectors clearly shows the significant clustering distance between the two groups. Red balls represent Tlr2−/− samples; blue balls represent WT samples.
Figure 7.
Figure 7.
“Omic” Venn diagram of large organic categories involved in IBD pathogenesis in the background of Tlr2 deficiency. This study confirmed significant colonic epithelium-associated epigenomic, transcriptomic, and microbiome changes in Tlr2−/− mice (bold and larger font) compared to WT.

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