Macrophages respond to a range of different cell products during the innate and acquired immune response. Of these, interferon-γ (IFNγ) is among the most important. Amongst its diverse array of activities, IFNy "primes" macrophages for more rapid and heightened responses to pathogen-associated molecular patterns such as lipopolysaccharide (LPS), a cell wall constituent of gram-negative bacteria, and unmethylated CpG motifs present in bacterial DNA (CpG DNA). Many mechanisms have been elucidated for IFNγ priming of macrophage responses to LPS, and involve reciprocal cross-regulation of signalling molecules between the IFNγ and LPS signalling pathways. These mechanisms include induction of the LPS signal transducer, TLR4, induction of MyD88 and MD2 signalling machinery, potentiation of LPS-induced NFKB activity and transcription factor synergy at the promoters of target genes. However, no mechanisms explaining IFNy priming of macrophage CpG DNA responses have been described.
This thesis offers a detailed analysis of the molecular mechanisms by which IFNγ primes macrophage CpG DNA responses. Two mechanisms were examined in detail, (i) lFNy-dependent induction of mRNA for tlr9, the CpG DNA receptor, via promoter activation; and (ii) CpG DNA-induced activation of signal transducer and activator of transcription- 1 (STAT 1), the canonical signalling molecule of the IFNγ pathway.
Prior to this study, it was documented that both IFNy and LPS could synergise with CpG DNA to enhance pro-inflammatory gene expression and effector function in murine macrophages. This thesis demonstrates that both LPS and IFNγ upregulated tlr9 mRNA in murine bone marrow-derived macrophages (BMM). The ability of LPS and IFNγ to induce tlr9 mRNA expression in BMM was dependent upon the presence of the growth factor, colony stimulating factor- 1 (CSF1) that suppressed tlr9 expression. However, there were clear mechanistic differences in tlr9 mRNA induction by LPS and IFNγ. LPS stimulation rapidly removed the CSFIR from the cell surface and thereby blocked CSF1 -mediated transcriptional repression, resulting in indirect induction of tlr9 mRNA expression. In contrast, IFNγ did not dramatically regulate cell surface CSFIR expression.
The mechanisms of regulated tlr9 gene expression were examined. The mouse tlr9 promoter was identified, cloned and promoter elements necessary for gene regulation determined by transfection analysis using promoter-reporter constructs in the macrophage-like cell line RAW 264.7. A composite PU.l/interferon regulatory factor (IRF) binding site overlapping the major transcription start site was identified, and was found to be a key mediator of constitutive and IFNγ-inducible tlr9 expression in murine rnacrophages. The lack of conservation of this IRF site in the human promoter, and the striking differences in promoter architecture between mice and humans, may partially account for the divergent patterns of tlr9 expression apparent between these species. Induction of tlr9 mRNA by IFNγ was also likely to occur via an autocrine loop involving IFNβ, which regulated tlr9 mRNA expression distally to the composite IRF/PU.l site.
The IFNγ signalling molecule, STAT1, can modulate gene regulation when phosphorylated at tyrosine 701. However, full transactivator potential is only unleashed when STAT1 is also phosphorylated at the serine S727 residue. STAT1 is a point of convergence of the IFNγ and TLR4 signalling pathways, as it has been shown by others that LPS induced both S727 and Y701 STAT1 phosphorylation. Many similarities exist between the signalling pathways engaged by TLR4 and TLR9, and the pathways responsible for STAT1 phosphorylation by LPS were reported by others to be activated by CpG DNA. For this reason, STAT1 phosphorylation in response to CpG DNA was investigated.
This thesis describes CpG DNA-dependent activation of STAT1 by S727 but not Y701 phosphorylation, and aimed to assess the contribution of STAT 1 S727 phosphorylation to IFNγ priming of CpG DNA responses. STAT 1 serine phosphorylation was found to be a mediator of some responses to CpG DNA in the absence of IFNγ priming, and provides the first report of a serine phosphorylation-dependent, but tyrosine phosphorylation-independent activity of STATI in signalling engaged by Toll-like receptors. This pathway also relieved the negative feed-back loop that regulates expression of tlr4
and thereby may have functions in relieving TLR tolerance.
The intersection of signalling pathways at the STAT1 S727 residue offers a potential mechanism for IFNγ priming of TLR4 and TLR9 responses. The STAT1 S727 residue is likely to allow the potentiation of IFN signalling when appropriate stress signals (e.g. TLR agonists) are present, and as such, STAT1 functions as an environmental sensor that integrates multiple input signals to give an appropriate cellular response.