Regulation of oxidoreductase enzymes during inflammation Sonia Louise Ingram September 2018 A thesis submitted to the University of Brighton and the University of Sussex for a programme of study undertaken at the Brighton and Sussex Medical School for the degree of Doctor of Philosophy 1 Abstract Autoimmune diseases such as rheumatoid arthritis (RA) occur when immune responses erroneously target host cells and proteins. The triggers for this dysregulated inflammation are unknown, but Toll-like receptors (TLRs) and oxidative stress are contributing factors (1, 2). Peroxiredoxin (PRDX) and thioredoxin (TXN) are intracellular protein thiol-disulphide oxidoreductase enzymes which link the TLRs to oxidative stress. These enzymes are induced during inflammation and oxidative stress, and are released from cells following inflammatory stimuli. In the extracellular environment they can act as danger signals, triggering TLR activation and perpetuating inflammation. However, knowledge of oxidoreductase regulation and release is limited. The hypotheses of this study were that responses of oxidoreductases vary depending on the TLR stimulated, and that serum oxidoreductases could be used as biomarkers for RA. To investigate TLR-induced oxidoreductase responses, concentrations of TLR agonists required to induce release were examined and tested for effects on tumour necrosis factor (TNF) production and cell viability in RAW 264.7 cells. Cells were stimulated with optimised concentrations in serum free OptiMEM (24h) for cell lysate and supernatant gel electrophoresis and Western blot experiments, and cell culture media (6-18h) for real time Taqman qPCR. PRDX1 and TXN1 release was found to occur primarily following TLR3 and TLR4 stimulation. Each oxidoreductase had a different pattern of gene expression to the TLRs: PRDX1 only increased with TLR4 stimulation, whilst TXN reductase (TXNRD1) increased with TLR4, TLR7 and TLR8, and heme oxygenase (HO1) increased with these TLRs plus TLR1/2 and TLR3. To investigate the role of reactive oxygen and nitrogen species (ROS and RNS respectively) on oxidoreductase release, RAW 264.7 cells were stimulated with PMA or interferon (IFN)γ + lipopolysaccharide (LPS). Griess assay was used to assess nitrite production. IFNγ+LPS increased TXN1 release but abrogated PRDX1 release. PRDX1 protein underwent NO-mediated proteasomal degradation following IFNγ+LPS stimulation. Gene expression of all oxidoreductases investigated increased with IFNγ+LPS stimulation compared to LPS alone, and inhibition of protein synthesis prevented PRDX1 gene expression, suggesting a secondary signal protein is required. 2 To investigate serum PRDX2, serum was isolated from whole blood from RA patients and healthy donors. ELISA was used to quantify both PRDX2 and the inflammatory marker C-reactive protein (CRP). No difference was found between RA and healthy serums, and PRDX2 did not correlate with CRP. The findings from this project show that oxidoreductase regulation and responses are fine-tuned depending on the TLR stimuli, and that serum PRDX2 is not a suitable biomarker for RA. 3 Courage doesn’t always roar. Sometimes courage is the little voice at the end of the day that says “I’ll try again tomorrow” - Mary Anne Radmacher 4 Contents Abstract .............................................................................................................................................. 2 Contents ............................................................................................................................................ 5 List of figures ..................................................................................................................................... 9 List of tables .................................................................................................................................... 12 Abbreviations .................................................................................................................................. 13 Acknowledgements ........................................................................................................................ 16 Declaration ...................................................................................................................................... 17 1. Introduction ................................................................................................................................. 19 1.1 Inflammation and immunity ................................................................................................ 19 1.1.1 The Immune System .................................................................................................... 19 1.1.2 Rheumatoid Arthritis .................................................................................................... 24 1.1.3 Biomarkers of disease ................................................................................................. 27 1.1.4 Inflammation .................................................................................................................. 29 1.1.5 Macrophages ................................................................................................................ 35 1.1.6 Toll-like receptors ......................................................................................................... 38 1.1.7 NF-κB ............................................................................................................................. 46 1.1.8 Nrf2 ................................................................................................................................. 50 1.1.9 Nrf2 and NF-κB cross-signalling ................................................................................ 55 1.2 Reduction-Oxidation (Redox) Biology .............................................................................. 57 1.2.1 Reactive oxygen species ............................................................................................ 57 1.2.2 Reactive nitrogen species ........................................................................................... 61 1.2.3 Reactive sulphur species ............................................................................................ 62 1.2.4 Cysteine and the free thiol .......................................................................................... 63 1.2.5 The reactive species interactome .............................................................................. 65 1.3 Oxidoreductase enzymes ................................................................................................... 66 1.3.1 Thioredoxin .................................................................................................................... 68 1.3.2 Thioredoxin reductase ................................................................................................. 71 1.3.3 Peroxiredoxin ................................................................................................................ 75 1.3.4 Redox and inflammation.............................................................................................. 78 1.3.5 Oxidoreductases and inflammation ........................................................................... 79 Aims .................................................................................................................................................. 82 2. Methods and Materials .............................................................................................................. 84 2.1 Materials ................................................................................................................................ 84 2.1.1 Instruments .................................................................................................................... 84 5 2.1.2 Chemicals and Kits ...................................................................................................... 85 2.1.3 Plastics .......................................................................................................................... 87 2.1.4 Solutions, Buffers and Media ..................................................................................... 87 2.2 Software and Analysis ........................................................................................................ 88 2.2.1 ImageJ 1.48v ................................................................................................................ 88 2.2.2 Microsoft Excel 2010 ................................................................................................... 88 2.2.3 GraphPad Prism 7.03 .................................................................................................. 88 2.3 Cell culture and stimulation ................................................................................................ 89 2.3.1 RAW 264.7 cells ........................................................................................................... 89 2.3.2 RAW 264.7 cell culture ................................................................................................ 89 2.3.3 Cell stimulations ........................................................................................................... 89 2.3.4 Cell viability assay ........................................................................................................ 92 2.4 Ethical approvals, patients and recruitment ...................................................................