Investigation of the Role of DNA Methylation in Regulating Toll-Like Receptor Activity

Morgane Clarke, Conor Hennessy, Declan McKernan. NUI Galway, Galway, Ireland

Introduction: Toll-like receptors (TLRs) are pattern recognition receptors (ten known in humans) that recognise components of bacteria and viruses. TLR signalling resulting from receptor stimulation culminates in the production of a variety of cytokines, including Tumour Necrosis Factor alpha (TNFα), which orchestrate the inflammatory response. TLRs are expressed on a wide variety of cells, including peripheral immune cells such as monocytes. The expression and activity of these receptors varies with cell type and with inflammatory disease progression (1). DNA methylation is an epigenetic mechanism that can regulate gene expression. DNA methyltransferases (DNMT) catalyses the addition of a methyl (-CH3) group from S-adenosylmethionine to the 5’ position of cytosine in the context of CG dinucleotides in a DNA sequence (2). Recently, DNMT inhibitors such as 5-aza-2’-deoxycytidine (5azadC) have been developed. Our hypothesis is that DNA methylation may regulate of TLR activity and as a result, inflammation.

Methods: In this study, we examined the mRNA expression of TNFα following stimulation for 6 hours with or without TLR 2, 4 and 5 ligands (heat-killed Listeria monocytogenes (HKLM), lipopolysaccharide (LPS) and Flagellin, respectively) using the THP-1 monocytic cell line. Prior to stimulation, cells were treated were treated with or without 5azadC (500 nM for 72 hours; n=3 for all groups). Relative mRNA expression was quantified with quantitative PCR and analysis performed using the 2(-Delta Delta C(T)) method (4). Statistical analysis involved using Two-way Analysis of Variance (ANOVA) with a Bonferroni post-hoc test (data was expressed as mean (relative units) +/- SEM with p < 0.05 considered significant).

Results: Treatment with 5azadC (0.002 +/- 0.003) resulted in a significant increase in the basal expression of TNF compared with untreated cells (0.0002 +/- 0.0005; p = 0.0009). Stimulation of cells with the TLR2 ligand HKLM resulted in a significant increase in TNF expression (0.0107 +/- 0.0015; p < 0.0001) when compared to untreated cells (0.0002 +/- 0.0005), which was further amplified by pre-treatment with 5azadC (0.0109 +/- 0.0012; p = 0.0107). Stimulation of cells with the TLR4 ligand LPS resulted in a significant increase in TNF expression (0.0037 +/- 0.0005; p < 0.0001) compared to untreated cells (0.0009 +/- 0.0002) and was further amplified by pre-treatment with 5azadC (0.014 +/- 0.0013; p = 0.0002). Stimulation of cells with the TLR5 ligand Flagellin resulted in a significant increase in TNF expression (0.0029 +/- 0.00015; p < 0.0001) compared to untreated cells (0.0002 +/- 0.00003) and was further amplified by pre-treatment with 5azadC (0.008 +/- 0.0009; p = 0.0033).

Conclusion: Our findings indicate that DNA methylation may regulate the activity of certain TLRs. Therapeutic targeting of the epigenome might provide new opportunities for targeting the innate immune system for halting the progression of chronic inflammatory disorders.

References

(1) Akira S et al. (2006) Pathogen recognition and innate immunity. Cell 124:783-801.

(2) Bestor TH (2000). The DNA methyltransferases of mammals. Hum Mol Genet. 9:2395-402.

(3) Livak KJ & Schmittgen TD (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402-8.