Bacterial DNA activates cells of the mammalian innate and acquired immune systems. This activity is mediated by the presence of unmethylated Cytosine-phosphate-Guanine (CpG) motifs present in bacterial DNA, and can be mimicked by synthetic single stranded oligodeoxynucleotides (ODNs) containing such unmethylated CpG motifs (otherwise referred to as CpG DNA). CpG DNA has been tested extensively as a immuno-modulator and vaccine adjuvant in animal models. Macrophages are cells of the innate immune system which provide the first line of defence against invading pathogens, and co-ordinate the subsequent pathogen specific adaptive immune response. This thesis is concerned with the activation of murine macrophages by bacterial/CpG DNA.
Chapter 1 provides a brief introduction to the innate immune system, the role of macrophages and biology of bacterial/CpG DNA. Macrophages are produced by the macrophage specific survival, growth and differentiation factor colony stimulating factor-1 (CSF-1). Chapter 2 examines the reciprocal interaction of bacterial/CpG DNA and CSF-1 signalling. Despite blocking CSF-1 signalling, treatment with bacterial/CpG DNA did not result in cell death. Indeed bacterial/CpG DNA treatment promoted independent survival of BMMs. The role of intracellular signalling modules in mediating bacterial/CpG DNA macrophage survival were investigated in Chapter 3. These pharmacological inhibition studies revealed no obligate role for the mitogen activated protein kinase (MAPK) extracellular-signal regulated kinase-1/2 (ERKl/2) pathway. However, the phosphatidyl inositol-3 (PI-3) kinase-Akt pathway is indispensable for macrophage survival mediated by bacterial/CpG DNA.
Protection against nuclease digestion, afforded by phosphorothioate modification of ODNs, is required for CpG DNA to work effectively in vivo as a vaccine adjuvant and immunomodulator. Chapter 4 demonstrates that the phosphorothioate modification of CpG DNA can modulate various aspects of macrophage activation. Most studies on the action of bacterial/CpG DNA on murine macrophages use either BMMs or the murine macrophagelike cell line RAW-264.7 Chapter 5 reports that despite strongly activating numerous responses in the aforementioned cell types, bacterial/CpG DNA was a relatively poor activator of these responses in both resident peritoneal macrophages (RPMs) and inflammatory thioglycolate elicited peritoneal macrophages (TEPMs). This relative insensitivity of RPMs and TEPMs was selective given strong responses were observed after stimulation with the archetypal macrophage activator lipopolysaccharide (LPS).
The Toll-like receptor-9, TLR-9, has been demonstrated to initiate intracellular signalling in immune cells after encountering bacterial/CpG DNA. However, the exact manner in which macrophages physically detect bacterial/CpG DNA is unclear. Identification of replication protein A (RPA) as a CpG specific binding protein is reported in Chapter 6. Chapter 6 also examines the possible contribution of the DNA dependent protein kinase (DNA-PK) in bacterial/DNA detection and responses. These studies revealed that pharmacological inhibition of bacterial/CpG DNA responsiveness by wortmannin, previously interpreted to implicate DNA-PK in bacterial/CpG DNA mediated signalling, actually blocks the uptake of bacterial/CpG DNA by macrophages. Hence, this work provides further evidence of a requirement for internalisation of DNA and ODNs before exerting their immuno-stimulatory activity. Chapter 7 provides a final discussion of the relevance of studies presented within this thesis to current knowledge on the detection and action of bacterial/CpG DNA. Future directions are also discussed which may aid in our understanding of how this novel immune activator functions.