Epigenetic modifications influence TLR3 expression and activity

Abstract

BackgroundToll‐like receptors (TLRs) are a family of pattern recognition receptors (PRRs) which recognise conserved pathogen associated molecular patterns (PAMPs) present in bacteria and viruses e.g. TLR3 recognises dsRNA from viruses. TLRs are expressed on a variety of cells including intestinal epithelial cells and play an important role in the immune response by stimulating cytokine release via phosphorylation of signalling proteins, but it is not known how their expression is regulated. We hypothesised that epigenetic mechanisms such as DNA methylation and histone deacetylation may play a role. In order to investigate this, we studied the effect of inhibition and/or genetic knockout of the enzymes responsible for these events (DNA methyltransferases (DNMT) or histone deacetylase complexes (HDAC)) on TLR3 expression and signalling in HCT‐116 intestinal epithelial cells.MethodsHCT‐116 (both wild‐type (WT) and DNMT 1/3B double knockout (DKO) cells) cells were either untreated or treated with the pan‐HDAC inhibitor suberanilohydroxamic acid (SAHA, 10μM, 48hrs), or the DNMT inhibitor 5‐aza‐2‐deoxycytidine (500nM, 72hrs), followed by stimulation with the TLR3 ligand Poly I:C (10μg/ml, 6 or 24 hrs). Cytokine mRNA expression was measured using quantitative polymerase chain reaction (QPCR) while protein release was measured using ELISA of the cell culture supernatant. Intracellular signalling protein phosphorylation was measured using Western blotting. Statistical analyses was performed in SPSS and in all cases n ≥ 3.ResultsTreatment of HCT‐116 WT cells with Poly I:C resulted in a significant increase in TLR3 expression (p<0.001) and in the mRNA expression of cytokines (p<0.001) and also the release of cytokines (p<0.001). Poly I:C treatment also resulted in increased phosphorylation of signalling proteins (NF‐κB, IκBα, JNK, p38, IRF3). Pre‐treatment with SAHA, 5‐aza‐2‐deoxycytidine and genetic knockout of DNTM1/3b prevented this Poly I:C induced increase in TLR3 expression and cytokine mRNA expression, cytokine release and signalling protein phosphorylation.ConclusionInhibition of HDACs or DNMTs by pharmacological means or genetic knockout prevents TLR3 signalling and the activation of the TLR3 inflammatory pathway. This suggests that viral induced inflammation may be controlled using epigenetic modifying drugs.Support or Funding InformationCollege of Science Scholarship, NUI Galway