University of Plymouth PEARL https://pearl.plymouth.ac.uk 04 University of Plymouth Research Theses 01 Research Theses Main Collection 2018 The role of Lysophosphatidylcholine acyltransferase-2 (LPCAT-2) in inflammatory responses Alrammah, Hanaa http://hdl.handle.net/10026.1/11291 University of Plymouth All content in PEARL is protected by copyright law. Author manuscripts are made available in accordance with publisher policies. Please cite only the published version using the details provided on the item record or document. In the absence of an open licence (e.g. Creative Commons), permissions for further reuse of content should be sought from the publisher or author. The role of Lysophosphatidylcholine acyltransferase-2 (LPCAT-2) in inflammatory responses By HANAA SALIH ABD ALI ALRAMMAH A thesis submitted to Plymouth University in partial fulfilment for the degree of DOCTOR OF PHILOSOPHY University of Plymouth March 2018 Copyright statement This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with its author and that no quotation from the thesis and no information derived from it may be published without the author's prior consent. ii The role of Lysophosphatidylcholine acyltransferase-2 (LPCAT-2) in inflammatory responses Hanaa Salih Abd Ali Alrammah Abstract Sepsis is the overwhelming inflammatory response to infection, especially bacterial infection and associated bacterial products. It has major healthcare impacts, affecting an estimated 19-30 million persons/year worldwide with a mortality of 30-70%. Despite intense research, no specific therapy has been established for sepsis and in addition to the high mortality, the associated economic costs are very high. For example, recent data shows that the annual cost of patients with sepsis is more than $20 billion in the USA, and £2.5 billion in the UK. Therefore, novel targets and new therapies for sepsis are required which will have an important impact on both mortality and economic benefits. Recent work has demonstrated that the phopsholipid modifying enzyme, LPCAT, has a role in the regulation of inflammatory responses to bacterial infections. However, the mechanism of action in this regard is not well understood. This project aimed to identify the role of LPCAT-2 in inflammatory response to infections. This project has utilized the RAW264.7 murine macrophage cell line as an experimental model and LPS or Pam3CSK4 as infectious stimuli to investigate the role of overexpressing LPCAT-2 as well as silencing the over-expressed LPCAT-2 using siRNA technique. RAW264.7 cells transiently or stably transfected with the LPCAT-2 gene were used to study the role of LPCAT-2 in the inflammatory responses of macrophages. LPCAT-2 was successfully over-expressed in RAW264.7 cells and the overexpression was successfully confirmed with real time polymerase chain reaction (RT-PCR) and western blotting. The overexpression of LPCAT-2 significantly upregulated the pro-inflammatory cytokines TNF-α and IL-6, at both gene expression, and protein level, while the anti-inflammatory cytokine, IL-10, was down regulated in these cells. Moreover, overexpression of LPCAT- 2 significantly decreased the expression of TLR4, peroxisome proliferator-activated receptors –gamma (PPARγ) and CD206 (a marker of M2 macrophages) while it significantly increased CD14, TLR2, COX-2 and iNOS (M1 markers). LPCAT-2 gene expression was also increased when PPARγ was blocked with the selective (PPAR-γ) antagonist T0070907. Importantly, silencing the transiently over-expressed murine LPCAT-2 resulted in a significant reduction in TNF-alpha and a significant increase in IL-10 gene expression. Both the transient and stably transfected RAW264.7 cells have been used to study the role of LPCAT-2 in regulating inflammatory responses in macrophages. The results have significantly added to knowledge of the role of LPCAT-2 in the inflammatory response and will aid in the development of novel therapies for inflammatory conditions such as sepsis. iii List of content. Copyright statement ……………………...………………………………..……………ii Abstract………………………………………………………………………………....iii List of content .................................................................................................................. iv List of tables. .................................................................................................................. xiii List of figures. ................................................................................................................ xiv List of figures of chapter 3. ........................................................................................... xiv List of figures of chapter 4. ........................................................................................... xiv List of figure of chapter 5 ............................................................................................ xvii Acknowledgments. ......................................................................................................... xix Dedication. ................................................................................................................... xxiii Author's Declaration ................................................................................................... xxiv List of abbreviations ..................................................................................................... xxx Chapter 1: General introduction & literature review. .................................................... 1 1. General introduction and literature review. ................................................................ 2 1.1. Sepsis. ........................................................................................................................ 2 1.1.1. Definitions and Incidence. ..................................................................................... 2 1. 2. Pathophysiology of Sepsis. ....................................................................................... 8 iv 1.2.1. Lipopolysaccharide. ............................................................................................... 8 1. 3. The innate immune system and the response to LPS. .......................................... 12 1.3.1. The role of Macrophages. .................................................................................... 12 1.3. 2. Toll-like receptors. ............................................................................................... 14 1.3.3. NOD- Like receptors (NLRs). .............................................................................. 22 1.4. Proinflammatory cytokines. .................................................................................... 25 1.4.2. Tumour Necrosis Factor Alpha (TNF-α). ........................................................... 25 1.4.3. Interleukin-1. ........................................................................................................ 26 1.4.4. Interleukin-8. ........................................................................................................ 27 1.4.5. Peroxisome proliferator-activated receptors –gamma (PPAR-γ), Cyclo- oxygenase enzymes (COX-2), iNOS and CD206. .......................................................... 27 Figure 6. Schematic picture of the network of M1/M2 – macrophage polarization. Adapted from Martinez & Gordon, 2014. ..................................................................... 31 1.5. Phospholipid metabolism and Cell membranes. .................................................... 31 1.5.1. Phospholipid turnover. ......................................................................................... 31 1.5.2. The LPCAT family of enzymes. ........................................................................... 32 Figure 8, Land Cycle (the remodeling pathway) Membrane phospholipids ............... 36 1.5.4. LysoPAF Acetyltransferase activity. .................................................................... 37 1.6. The biological roles and expression of LPCAT in pro and anti-inflammatory processes. ........................................................................................................................ 39 v 1.6.1. Inflammatory cells. .............................................................................................. 39 1.6.4. Lungs and surfactant production. ....................................................................... 40 1.6.5. Brain. .................................................................................................................... 41 1.7. The LPCAT enzyme as a novel target for anti-sepsis therapy. .............................. 42 1.8. Experimental models. .............................................................................................. 44 1.8.1. LPS and Pam3CSK4. ........................................................................................... 44 1.8.2. Macrophages, the murine RAW264.7 cell line. .................................................. 44 1.8. Rational for the study. ............................................................................................. 48 1.9. Hypothesis, Aims and objectives of this project. .................................................... 52 1.9.1. Hypothesis. ........................................................................................................... 52 1.9.2. Aims. ....................................................................................................................