Methylation of TNFRSF13B and TNFRSF13C in duodenal mucosa in canine inflammatory bowel disease and its association with decreased mucosal IgA expression

Abstract

Although decreased intestinal IgA expression has been reported in dogs with inflammatory bowel disease (IBD), the mechanism underlying this decrease is unknown. Transmembrane activator and calcium-modulating cyclophilin–ligand interactor (TACI) and B cell-activating factor of the TNF family (BAFF) receptor (BAFF-R) are key receptors for T cell-independent IgA class switching by the binding of IgA-inducing cytokine a proliferation-inducing ligand (APRIL) and BAFF. Here we show decreased TACI and BAFF-R mRNA expression and hypermethylation of their corresponding genes TNFRSF13B and TNFRSF13C, respectively in the duodenal mucosa of dogs with IBD. To examine whether DNA methylation of the TNFRSF13B and TNFRSF13C influences the mRNA expression of TACI and BAFF-R, respectively, we first analyzed methylation and mRNA expression levels in vitro using 2 canine B lymphoid cell lines, GL-1 and CLBL-1. Methylation profiles in the cells were examined by bisulfite sequencing and methylation-specific PCR (MSP) with primer pairs specific to methylated or unmethylated sequences. These methylation analyses revealed hypermethylation of the CpG islands of both TNFRSF13B and TNFRSF13C in GL-1, but not in CLBL-1 cells. The mRNA expression levels of TACI and BAFF-R were significantly lower in GL-1 than in CLBL-1 cells. Treatment with 5-aza-2′-deoxycytidine significantly increased TACI and BAFF-R mRNA expression in GL-1 cells through demethylation of TNFRSF13B and TNFRSF13C, respectively. These results suggest that the mRNA expression of TACI and BAFF-R is regulated through methylation of their genes in canine B cells. Quantitative real-time MSP showed significant hypermethylation of the CpG islands of TNFRSF13B and TNFRSF13C in the duodenal mucosa of dogs with IBD. Furthermore, duodenal mRNA expression levels of TACI and BAFF-R were significantly lower in dogs with IBD than in healthy controls. The mRNA expression levels of TACI positively correlated with intestinal IgA expression, whereas the methylation level of its gene (TNFRSF13B) negatively correlated with IgA expression. The present results suggest the role of TACI in the regulation of mucosal IgA expression through epigenetic modifications.