Macrophages are critical cellular mediators of innate immunity and also participate in maintaining tissue homeostasis. However, dysregulated macrophage activation and/or function contributes to pathology in a number of acute and chronic inflammatory diseases. Macrophages utilise families of pattern recognition receptors to sense foreign and endogenous danger signals; the most widely studied of these are the Toll-like receptors (TLRs), which are key activators of inflammatory pathways. The signalling pathways downstream of TLRs, as well as their secreted products, are therefore targets for anti-inflammatory therapies. One potential anti-inflammatory approach is inhibition of histone deacetylases (HDACs), a family of 18 enzymes that post-translationally modify proteins by catalysing removal of acetyl groups on lysine residues. Broad-spectrum inhibitors that target all members of the HDAC family have shown therapeutic effects in several models of inflammatory diseases, however the specific enzymes that drive inflammatory pathways are still being elucidated This thesis explored effects of HDAC inhibitors on macrophage inflammatory pathways, and also identified specific HDAC enzymes regulating TLR pathways in macrophages.
Shown in Chapter 3, broad-spectrum HDAC inhibitors did not affect monocyte and macrophage numbers in vivo, but did affect macrophage activation. HDAC inhibitors suppressed the production of a subset of lipopolysaccharide (LPS)-inducible inflammatory mediators such as Endothelin-1 (Edn1), interleukin (IL)-12p40 and IL-6, whereas another subset (Cyclooxygenase-2, Plasminogen activator inhibitor-1) was amplified, indicating that HDACs can both positively and negatively regulate inflammatory genes in macrophages. HDAC inhibitors did not affect the LPS-induced activation of the master transcriptional regulator nuclear factor-κB as assessed by promoter-reporter assays. The Edn1 gene was used as a model to investigate alternative mechanisms by which HDAC inhibitors target TLR responses. LPS-induced activation of an Edn1 promoter-reporter required HDAC activity and a functional binding site for the transcription factor hypoxia-inducible factor-1 (HIF-1)-α. In addition, a broad-spectrum HDAC inhibitor abrogated LPS-inducible HIF-1α mRNA and protein expression. Use of class-selective inhibitors suggested that Class II HDACs promoted LPS-induced inflammatory mediators, and the functions of HDAC6 and HDAC7 (Class II enzymes) were therefore investigated in further studies.
Whilst LPS/TLR4 responses were unimpaired in Hdac6-/- macrophages (Chapter 6), several lines of evidence implicated Hdac7 in macrophage function (Chapter 4). Hdac7, but not other Hdacs enhanced LPS-induced Edn1 promoter activity and Hdac7 synergised with HIF-1α to promote this response. Hdac7 mRNA expression was elevated in inflammatory macrophages compared to resting macrophages and the mouse macrophage RAW264 cell line. Furthermore, over-expression of Hdac7 in RAW264 cells amplified LPS-induced expression of HDAC-dependent mediators such as Edn1 and IL12p40. This effect was restricted to a specific isoform of Hdac7 that lacked the first 22 amino acids (Hdac7-s), whilst full-length Hdac7 (Hdac7-l) did not have this activity. The disparity between isoforms was not simply due to differential interaction with HIF-1α as both isoforms co-immunoprecipitated with HIF-1α.
Evidence is also presented for a second role of Hdac7 in TLR4 pathways (Chapter 5). In contrast to the selective effect of Hdac7-s in promoting inflammatory gene expression, both Hdac7-s and Hdac7-l were lost from the nucleus of RAW264 cells in response to LPS. Both Hdac7 isoforms repressed the activity of the vascular cell adhesion molecule (Vcam)-1 promoter. The Sry-related HMG box (Sox)-18 transcription factor upregulated Vcam-1 promoter activity and Hdac7 inhibited this response. It is proposed that nuclear export of Hdac7 permits inducible expression of another set of TLR target genes. Hence, targeting of Hdac7, either by blocking Hdac7-s deacetylase function, or by preventing Hdac7 nuclear export, represent candidate approaches for the development of novel anti-inflammatory agents targeting macrophage function.