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Phospholipase D: Key Player in Macrophage- Mediated Inflammation and Resolution

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Phospholipase D: Key Player in Macrophage- Mediated Inflammation and Resolution PHOSPHOLIPASE D: KEY PLAYER IN MACROPHAGE- MEDIATED INFLAMMATION AND RESOLUTION A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy By RAMYA GANESAN B.Tech., Anna University, 2011 M.S., Wright State University, 2014 2017 Wright State University i WRIGHT STATE UNIVERSITY GRADUATE SCHOOL Dec 11, 2017 I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY Ramya Ganesan ENTITLED Phospholipase D: Key Player in Macrophage-mediated Inflammation and Resolution BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Doctor of Philosophy. Committee on Final Examination Julian G. Cambronero, Ph.D. Professor Julian G. Cambronero, Ph.D. Thesis Director Nancy J. Bigley, Ph.D. Professor Juliusz A. Kozak, Ph.D. Associate Professor Mill W. Miller, Ph.D. Director, Biomedical Sciences Ph.D. Program Michael P. Markey, Ph.D. Research Associate Professor Yong-jie Xu, Ph.D. Associate Professor Barry Milligan, Ph.D. Interim Dean of the Graduate Gerald M. Alter, Ph.D. School Professor ii COPYRIGHT BY RAMYA GANESAN 2017 iii ABSTRACT Ganesan, Ramya Ph.D., Biomedical Sciences Ph.D. program, Wright State University, 2017. Phospholipase D: Key Player in Macrophage-mediated Inflammation and Resolution Macrophages are central to the inflammatory response and its ability to resolve effectively. They are complex cells that adopt a range of subtypes depending on the tissue type and stimulus. This flexibility allows them to play multiple, sometimes opposing, roles in inflammation and tissue repair. Their central role in the inflammatory process is reflected in macrophage dysfunction being implicated in chronic inflammation and poorly healing wounds. Acute inflammatory response induces an increase in prostaglandins and leukotrienes and leads to chronic inflammation, which is inhibited by resolvins. Additionally, resolvins play a crucial role in wound healing. During inflammation, leukocytes release cytokines, exacerbate inflammation and damage tissues, while neutrophils produce oxygen radicals that worsen the initial inflammation. Atherosclerosis is an inflammatory disease caused by accumulation of foam cells derived from macrophages on blood vessel walls. It is a significant health problem and a major contributor to cardiovascular disease (CVD), which accounts for one in three deaths in the U.S. (Mozaffarian, Benjamin et al. 2015), and continues to rise globally (Field, Hazinski et al. 2010, Townsend, Nichols et al. 2015). Deleterious inflammation is a primary feature of breast cancer. Accumulating evidence demonstrates that macrophages, the most abundant leukocyte population in mammary tumors, have a critical role at each stage of cancer progression. iv Phospholipase D (PLD) is a cell membrane remodeling and signaling protein implicated in the pathology of chronic inflammation. As PLD is also central to macrophage cell migration, we investigated the molecular basis of PLD’s involvement and regulation in macrophage-initiated inflammation (atherosclerosis) and resolution. We have found that PLD is associated with signaling proteins and positively affects cell movement, phagocytosis and NADPH-initiated release of Reactive Oxygen Species (ROS). We found that PLD2 but not PLD1 is important for foam cell formation that causes atherogenesis. We have also found a novel way of inducing macrophage (MØ) class-switch (polarization) by PLD overexpression. PLD induces a macrophage M1 to M2 class-switch that accelerates resolution of inflammation and limits damage to blood vessels and affected tissues during atherosclerosis and other inflammatory conditions. We also investigated a new molecular pathway for MØ class-switch (M1-to-M2) by overexpressed PLD resulting in inflammation by bacterial phagocytosis or resolution of inflammation by efferocytosis. In order to understand the physiological relevance of PLD’s role in inflammation and resolution, we studied the effect of resolvins, a class of specialized proresolving lipid mediators (SPMs), on PLD expression and activity in the different macrophage populations taking into consideration the time course of inflammation and resolution. We found that RvD5 upregulates PLD activity and expression in M2 macrophages confirming a molecular mechanism for PLD’s role in resolution of inflammation. v TABLE OF CONTENTS INTRODUCTION ............................................................................................................ 1 Phospholipase D.............................................................................................................. 1 Inflammation ................................................................................................................... 8 Resolution of Inflammation .......................................................................................... 12 Resolvins ....................................................................................................................... 16 Role of Macrophages in Inflammation ......................................................................... 20 Macrophage Phenotypes ............................................................................................... 21 Atherosclerosis .............................................................................................................. 24 Inflammation in Atherosclerosis ................................................................................... 27 Role of PLD2 in Atherosclerosis .................................................................................. 28 Inflammation and Resolution Involving Macrophages ................................................. 29 MATERIALS AND METHODS ................................................................................... 31 Materials ....................................................................................................................... 31 Animals ......................................................................................................................... 31 Ischemia-reperfusion-induced second-organ injury ..................................................... 33 Macrophage and PMN phagocytosis and efferocytosis ................................................ 33 PLD activity assay ........................................................................................................ 35 Real-time (quantitative) Reverse Transcription-PCR ................................................... 35 SDS-PAGE and Western blot analyses ......................................................................... 36 Myeloperoxidase (MPO) Assay .................................................................................... 37 Isolation of bone marrow-derived monocytes .............................................................. 38 vi Differentiation of bone marrow cells into bone marrow-derived macrophages (BMDM) ....................................................................................................................................... 39 Preparation of aggregated, oxidized LDL particles (Agg-Ox-LDL) ............................ 39 Phagocytosis of Agg-Ox-LDL by macrophage foam cells ........................................... 40 Phagocytosis of Zymosan ............................................................................................. 41 Immunofluorescence microscopy of phagocytosis and key proteins ........................... 41 Human artery specimens ............................................................................................... 41 Computational transcriptomic analysis ......................................................................... 42 PLD inhibitors ............................................................................................................... 43 Co-immunoprecipitation ............................................................................................... 43 Statistical Analysis ........................................................................................................ 45 RESULTS ........................................................................................................................ 46 Chapter I: To study the regulation of Phospholipase D by resolvins in macrophage mediated inflammation-resolution ...................................................... 46 1.1. PLD activity and expression are altered in anti- and pro-inflammatory macrophages ............................................................................................................. 46 1.2. Effect of resolvins on PLD and S6 Kinase gene expression .............................. 49 1.3. Effect of resolvins on anti- and pro-inflammatory macrophage PLD activity .. 56 1.4. Effect of resolvins on PLD-mediated inflammation resolution ......................... 58 1.5. Effect of PLD and resolvins on macrophage functions .................................... 61 DISCUSSION ............................................................................................................... 65 Chapter II: To study the role of Phospholipase D in macrophage polarization ... 68 2.1. Effect of PLD on macrophage function ............................................................. 68 vii 2.2. Macrophage polarization in inflammation ......................................................... 71 2.3. PLD affects human M0 macrophage polarization ............................................. 73 2.4. M1 to M2 macrophage class-switch induced by PLD ......................................
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