Investigating the epigenetic regulation of toll-like receptor 3

Toll-like receptors are a family of pattern recognition receptors (PRRs), which have evolved to recognise structurally conserved components of microorganisms. They form an important part of the innate immune system, yet in spite of their importance, there is still a paucity of information regarding the expression and regulation of these receptors. Epigenetics is a field of genetics which focuses on non-nucleotide based changes in gene expression, and highlights the implications that changes in chromatin structure and function have for gene expression and regulation. We theorised that TLRs might be susceptible to changes in the epigenetic state, and we theorised that epigenetic modifications may alter the regulation and function of TLRs, most notably TLR3. Our studies were centred on two cell lines, epithelial HCT 116 cells and monocytic THP-1 cells, two cell lines which play important but distinct roles in the innate immune system. We targeted two families of enzymes involved in remodelling chromatin structure, DNA methyltransferases (DNMTs) and histone deacetylase complexes (HDACs). We initially showed that knockout of DNA methyltransferases in HCT 116 cells produced significant decreases in TLR3 expression, with less dramatic results seen in other TLRs. This finding was replicated with pharmacological inhibition of DNMTs with 5-aza-2-dc and inhibition of HDACs with SAHA. Inhibition of DNMTs and HDACs also inhibited TLR3 function, with no signalling cascade or cytokine release seen with enzyme inhibition. TLR3 function was markedly different in THP-1 and HCT 116 cells, with THP-1 cells not expressing TLR3. This difference in expression led us to search for a potential regulator common to both cell lines. Literature vi i searches led to IRF8, a potential negative regulator of TLR3, which is known to be highly expressed in THP-1 cells. We determined that high IRF8 expression was correlated with low TLR3 expression, and lack of functional responses. Overexpression of IRF8 in HCT 116 cells, which prevented TLR3 functional responses, suggested that IRF8 may indeed be an epigenetically controlled regulator of TLR3. Our studies show the immunomodulatory capabilities of these epigenetic drugs, with decreased viral receptor function and inhibition of inflammatory cytokine release seen with inhibition of DNMTs and HDACs. Furthermore, our studies suggest a potential role for epigenetic modifications, which may alter IRF8 expression, in indirectly regulating TLR3 expression and function.

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College of Science

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Attribution-NonCommercial-NoDerivs 3.0 Ireland

cbn

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University of Galway Theses (PhD Theses)