Lipopolysaccharide (LPS) and Cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODNs) cause macrophages to produce the proinflammatory cytokines IL-12, IL-6 and TNF-α. Pre-treatment of the macrophages with ES-62, an anti-inflammatory glycoprotein secreted by the parasitic filarial nematode Acanthocheilonema viteae suppresses the production of these cytokines.Although able to prevent disease development in mouse models of allergy and autoimmunity, ES-62 is not suitable for drug therapy due to its potential immunogenicity. A library of small molecule analogues (SMAs) therefore was designed and tested for the previously mentioned inhibitory effects. SMAs 11a and 12b among library members were found to mimic ES-62’s anti-inflammatory effects. These findings rationalised further testing in order to determine their mechanism of action based on their effects on the metabolome of primary macrophages.From analysis of cellular extracts using hydrophilic interaction chromatography in combination with high resolution mass spectrometry it could be seen that stimulation of macrophages with either LPS or CpG produced metabolic changes in various pathways. Stimulation of macrophages with LPS or CpG in the presence of SMAs 11a and 12b revealed that many of the metabolic shifts were the same as observed with LPS and CpG alone. However, there were clear effects of the SMAs in producing downregulation in creatine metabolism/uptake and upregulation in glutathione biosynthesis.By downregulating creatine metabolism/uptake, the SMAs may be controlling the availability of creatine for transporting high energy phosphate from the mitochondria to where it is required for biological functions including cell signalling, phagocytosis and motility. By causing an upregulation of the glutathione biosynthesis pathway the SMAs may be protecting the cells from oxidative stress and of note SMA12b has been previously linked to increased activity of the Nrf2/ARE/HO-1 anti-oxidant pathway. The SMAs may be downregulating the availability of the energy produced by oxidative phosphorylation in general without targeting the TCA cycle directly since they do not affect NADH levels in comparison with LPS or CpG stimulation alone. Finally, in the process of examining the response of the cells to LPS and CpG additional potential anti-inflammatory targets were revealed.
|Date of Award||21 Jun 2018|
- University Of Strathclyde
|Supervisor||William Harnett (Supervisor) & David Watson (Supervisor)|