Cells of the innate and adaptive immune systems respond to the presence of foreign DNA. Bacterial DNA is recognised by the immune system due to the presence of unmethylated CG dinucleotides within particular sequence contexts. These activating sequences are generally known as CpG (Cytosine-phosphate-Guanine) motifs. In response to the presence of CpG motifs macrophages, DCs and B-cells produce pro-inflammatory cytokines such as TNFV, IL- 6 and IL-12, up-regulate co-stimulatory molecules and have an increased resistance to apoptosis. The immunostimulatory properties of bacterial DNA can be mimicked by synthetic oligonucleotides (ODN) containing CpG motifs. Other motifs in DNA also can modulate the function of the immune system - these include inhibitory motifs and oligo [dG] motifs. This thesis focuses on elucidating the mechanisms responsible for the actions of immunomodulatory DNA, primarily CpG DNA.
Chapter 1 provides background to the roles of immunomodulatory DNA in activation of the immune system and a brief description of their potential use as therapeutic agents. Toll-like receptor 9 (TLR9) has been identified as the major intracellular receptor for immunostimulatory DNA. Chapter 2 examines the potential role for replication protein A (RPA) as a co-receptor for CpG DNA. Previous work in our laboratory had suggested that RPA preferentially bound to ODN containing a CpG motif. Data presented here will show that RPA preferentially binds to oligonucleotides (ODN) with little secondary structure and is unlikely to be involved with directly detecting CpG DNA.
Bacterial DNA is a potent activator of murine macrophages. Whilst short single-stranded CpG-containing ODN are also immunostimulatory, they are much less efficient than bacterial DNA. Chapter 3 demonstrates that the increased activity of bacterial DNA on macrophages does not relate to its strandedness or epigenetic factors, but rather is due to increased length. Long ODN (44 base) were taken up much more efficiently than short ODN (22 base) by macrophages, but not by B-cell lines. The known scavenger receptor ligand dGg strongly inhibited both uptake and activity of long but not short CpG-containing ODN; however, published work suggests a classical scavenger receptor is not responsible for general DNA uptake by macrophages. Overall, results suggest that a length-dependent DNA uptake receptor is restricted to or enriched in macrophages.
Natural DNA contains a phosphodiester (PO) backbone that is susceptible to nuclease degradation. In therapeutic situations, ODN with a nuclease resistant phosphorothioate (PS) backbone are used. Chapters 4 and 5 focus on the effect of PS modification on the immunostimulatory properties of CpG ODN. Chapter 4 presents data showing that small changes to the sequence of PS but not PO-ODN can dramatically change their immunostimulatory activity. Notably the addition of terminal guanines to PS-ODN decreases their activity. Chapter 4 proposes that the formation of PS-ODN specific secondary structure dramatically affects PS-ODN activity.
Published work has shown that mice and humans respond optimally to CG dinucleotides with different flanking sequences. The optimal CpG motif for mice is purine-purine-C-Gpyrimidine- pyrimidine whereas in humans, CG dinucleotides separated by stretches of thymines is preferable. Chapter 5 demonstrates that this species-specific recognition of CpG motifs in dependent on the ODN backbone being PS-modified. Data presented also shows that species-specific responses to PS-ODN can occur in the absence of a CG dinucleotide and these CG-independent responses still require the presence of TLR9.
Finally Chapter 6 utilises cDNA microarray technology to examine the spectrum of genes induced by murine macrophages in response to CpG DNA. CpG DNA was found to induce a range of genes involved in controlling diverse cellular processes. Additionally cDNA microarrays were used to investigate the proteins involved in signalling in response to inhibitory ODN and oligo [dGj. Data presented in Chapter 6 suggests that inhibitory ODN do not induce an inhibitory signal and are likely to act as competitive inhibitors. A final discussion and future directions for this field of study are provided in Chapter 7.