Function and regulation of TLR9

Macrophages, dendritic cells and B cells detect bacterial DNA containing unmethylated CpG motifs via Toll-like receptor 9 (TLR9) and respond by producing proinflammatory mediators such as IL-6, IL-12 and TNF, and upregulating cell surface expression of co-stimulatory molecules. These properties can be mimicked by synthetic oligonucleotides (ODNs) that contain an unmethylated CG dinucleotide that is flanked by appropriate 5’ and 3’ sequences, but other motifs in DNA can actually inhibit cellular responses to stimulatory CpG DNA. Chapter 3 aimed to characterize the inhibitory effects of different classes of inhibitory ODNs by investigating their mode of action and their specificity for TLR9 versus other TLR family members. G-rich inhibitory ODNs had the greatest specificity as TLR9 inhibitors, but these ODN also partially antagonized responses to the TLR1/2 ligand, Pam3Cys. Whilst it is well documented that TLR9 is required for anti-viral responses and is the receptor for bacterial DNA, at the commencement of this thesis no studies had assessed the function of TLR9 in bacterial infection models. Chapter 4 investigated the function of TLR9 in the macrophage response to S. typhimurium. Whilst Salmonella-induced cytokine production did not depend on TLR9, intracellular S. typhimurium bacterial loads were enhanced in TLR9-deficient bone marrow-derived macrophages (BMMs), implying a role in the anti-microbial response. G-rich inhibitory ODN, which suppressed TLR9 signalling, also greatly enhanced intracellular bacterial loads, but intriguingly this effect was TLR9-independent. Thus, inhibitory ODNs, which have been proposed as therapeutic agents for the treatment of chronic inflammatory disease, have TLR9-independent effects that may compromise the host response to bacterial pathogens. Due to their ability to potently activate the innate immune system and direct development of the acquired response, bacterial products have therapeutic potential as vaccine adjuvants. CpG DNA has been widely pursued as a vaccine adjuvant because it activates innate immune cells, primes Th1 responses and is relatively non-toxic in vivo. The major macrophage growth factor, macrophage colony stimulating factor (MCSF/ CSF-1) is a potent inhibitor of TLR9 expression and CpG DNA responsiveness in mouse macrophages. Chapter 5 therefore explored the hypothesis that antagonism of CSF-1 action would enhance CpG DNA efficacy. AFS98, a monoclonal antibody against the CSF-1R, amplified TLR9 expression in CSF-1-replete BMM and amplified expression of CSF-1-repressed genes in vivo. AFS98 also amplified CpG DNA responses in vivo, although this effect was highly variable between experiments. To examine if antagonizing CSF-1 action in human cells mimics the effects that were apparent in the mouse, the regulation of TLR9 expression by CSF-1 in human monocytes and macrophages was also assessed. The effects were variable, but at least in some donors, TLR9 mRNA expression was inhibited by CSF-1 in human monocytes and during monocyte to macrophage differentiation. In summary, this thesis explored the hypotheses that TLR9 function could be manipulated for therapeutic applications and that TLR9 plays a role in the host response to bacterial pathogens. v