Elucidation of genes up-regulated by hypoxia and lipopolysaccharide in human macrophages

Macrophages accumulate in inflamed tissues such as atherosclerotic plaques, malignant tumours, and wounds, which are characterised by hypoxia, and respond by altering expression of genes which enhance adaptation to hypoxia. In addition to hypoxia, lipopolysaccharide (LPS) can be present in infected tissues due to contamination by microbes. The hypothesis in this thesis was that synergy between hypoxia and LPS could affect macrophage gene expression in sites such as infected wounds which contain both factors. Using cDNA microarrays, investigation was carried out into which genes were induced by hypoxia and LPS in primary human macrophages. Gene expression profiles changed markedly: 55 genes were up-regulated by hypoxia, 277 by LPS and 384 by hypoxia plus LPS, suggesting significant cross talk or synergy between hypoxia and LPS. Two synergistically regulated genes, TRAIL (TNF-related apoptosis-inducing ligand) and DDIT4 (DNA-damage-inducible transcript 4) were studied in more detail. Proximal TRAIL promoter luciferase reporter constructs were prepared, but were not inducible, suggesting involvement of more distal promoter elements. The hypoxia-inducible factor HIF-stabilizing agents desferrioxamine and cobalt chloride were used to investigate the role of HIF-1 in up-regulation of DDIT4 mRNA. Both agents up-regulated DDIT4 expression, and were capable of synergistic up-regulation when combined with LPS, suggesting that, for DDIT4, HIF-1 is responsible for the hypoxia and LPS synergy. This report also shows for the first time that DDIT4 is induced by LPS alone. DDIT4 mRNA induction by hypoxia was slightly down-regulated by the PI3-kinase inhibitors LY294002 and wortmannin, also suggesting a role for PI3-K. Immunoblotting showed DDIT4 protein up-regulation in response to hypoxia and LPS, which was consistent with mRNA levels determined by arrays and RT-PCR. In conclusion, this thesis suggests that hypoxia may also be able to synergise with a variety of stimuli to alter macrophage gene expression in a range of pathological conditions.