Epigenetic Regulation of TLR4 Gene Expression in Intestinal Epithelial Cells for the Maintenance of Intestinal Homeostasis

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. In this study, we show that the low responsiveness of human IEC lines to LPS was mainly brought about by a down-regulation of TLR4 gene transcription. Additionally, the presence of an IEC-specific repressor element in the 5′ region of the TLR4 gene and binding of a NF to the element was shown. The transcription factor ZNF160, which was expressed more abundantly in a LPS-low responder IEC line than in a LPS-high responder IEC line, repressed TLR4 gene transcription. ZNF160 is known to interact with the scaffold protein KAP1 via its N terminus to recruit histone deacetylase. Histone deacetylation, as well as DNA methylation, at the 5′ region of the TLR4 gene was significantly higher in LPS-low responder IEC lines than in a monocyte line or a LPS-high responder IEC line. It was demonstrated that TLR4 gene transcription was repressed by these epigenetic regulations, which were, at least in part, dependent on ZNF160. Down-regulaton of TLR4 gene expression by these mechanisms in IECs possibly contributes to the maintainance of homeostasis in the intestinal commensal system.

Epigenetic Control of the Host Gene by Commensal Bacteria in Large Intestinal Epithelial Cells

“…Alternatively, cells were cotransfected with pcDNA3.1-CDX2 or pcDNA3.1-empty and the TLR4 siRNA expression plasmid pBAsi-TLR4 or the control plasmid pBAsi-cont (14). Fortyeight hours later, cells were stimulated with Ultra Pure LPS (Invivogen) for 20 -22 h. Secreted IL-8 in the culture supernatant was concentrated by Amicon Ultra centrifugal filters (nominal molecular weight limit, 3000) (Millipore, Billerica, MA) and then quantified with a Quantikine Human CXCL/ IL-8 system (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.…”

“…Reporter Gene Assay-Cells were cotransfected with the reporter plasmid pGL-TLR4Ϫ1013/ϩ118 (14) and the CDX2 expression plasmid or an empty control vector using FuGENE HD Transfection Reagent (Roche Diagnostics). The reporter plasmid pGL-TLR4Ϫ1013/ϩ118 carries the 5Ј region of the human TLR4 gene (nucleotides Ϫ1013/ϩ113), which contains cis-acting elements to activate transcription, upstream of the luciferase gene.…”

“…Increased expression of TLR4 and an elevated response to commensal organisms are often observed in patients with inflammatory bowel disease (12,13). We have previously reported using human IEC lines that low responsiveness of IECs to LPS is mainly brought about by down-regulation of the TLR4 gene and that transcriptional repression of this gene in IECs is mediated by epigenetic mechanisms including DNA methylation and histone deacetylation (14). In this study we analyzed DNA methylation of the TLR4 gene in IECs in vivo, determining the involvement of intestinal commensal bacteria in this process.…”

Biomarkers Induced by the Immunomodulatory Bacterial Extract OM-85: Unique Roles for Peyer’s Patches and Intestinal Epithelial Cells

“…The finding of a lack of IEC response against pathogen-derived molecules may be a protective mechanism to avoid excessive inflammation induced by commensal microorganisms, thereby aiding the maintenance of intestinal homeostasis [38][39][40]. Indeed, IECs are recognized as central mediators of immune homeostasis in the intestine by maintaining a commensal bacteria permissive environment while responding to excessive inflammation induced by pathogens [41].…”

Erase–Maintain–Establish: Natural Reprogramming of the Mammalian Epigenome

“…ZFP57 plays a conserved role in imprinting maintenance in humans and mice. Diverse ZFPs/ZFNs (mouse/human) are involved in retrovirus silencing and other physiological functions (Okumura et al 1997;Zheng et al 2000;Li et al 2008;Takahashi et al 2009;Wolf and Goff 2009;Quenneville et al 2011;Nishitsuji et al 2012Nishitsuji et al , 2015Tan et al 2013;Castro-Diaz et al 2014;Jacobs et al 2014;Wolf et al 2015b). (Messerschmidt 2012;Messerschmidt et al 2012).…”