Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

56728_Elizabeth_Gallagher_ElizabethGallagher_Research_Project_with_No_references_497985_389335805

207 views

Published on

  • Be the first to comment

  • Be the first to like this

56728_Elizabeth_Gallagher_ElizabethGallagher_Research_Project_with_No_references_497985_389335805

  1. 1. “Investigating the Effects of Novel Marine Extracts on Inflammation” A thesis submitted in partial fulfilment for the degree of B.Sc in Analytical Science Completed by Elizabeth Gallagher Under the Supervision of Professor Christine Loscher 12378191 B.Sc. in Analytical Science 2015/2016
  2. 2. Declaration on Plagiarism Assignment Submission Form This form must be filled in and completed by the student(s) submitting an assignment Name: Elizabeth Gallagher Programme: AS4 Module Code:BE494 Assignment Title: Investigating the Effects of Novel Marine Extracts on Inflammation. Submission Date: 13/05/2016 Supervisor: Prof. Christine Loscher I declare that this material, which I now submit for assessment, is entirely my own work and has not been taken from the work of others, save and to the extent that such work has been cited and acknowledged within the text of my work. I understand that plagiarism, collusion, and copying are grave and serious offences in the university and accept the penalties that would be imposed should I engage in plagiarism, collusion or copying. I have read and understood the Assignment Regulations. I have identified and included the source of all facts, ideas, opinions, and viewpoints of others in the assignment references. Direct quotations from books, journal articles, internet sources, module text, or any other source whatsoever are acknowledged and the source cited are identified in the assignment references. This assignment, or any part of it, has not been previously submitted by me or any other person for assessment on this or any other course of study. I have read and understood the referencing guidelines found at: http://www.dcu.ie/info/regulations/plagiarism.shtml , https://www4.dcu.ie/students/az/plagiarism and/or recommended in the assignment guidelines. Name: ________________________________ Date: ____________
  3. 3. Acknowledgments Firstly I would like to thank Prof. Christine Loscher for all her support throughout my research project. It has been an absolute privilege to work under the supervision of someone who is so passionate about the research being carried out. Thank you for your guidance and encouragement which have made my research project one of my fondest memories of my academic time in DCU. I would like to thank my supervisor Dr..Izabela Marszalowska. I have learned so much from you over the last 3 months. It been an absolute pleasure working with someone with such a high level of expertise in the area. Thank you for being so enthusiastic everyday, and for providing me with an opportunity to grow my independence as a scientist. I absolutely stuck gold in terms of the lab I joined for my research project. Laura, Kim, Niamh, Kathy, Alisha and Conor thank you so much for helping me maintain some level of sanity over the last three months, thank you for the chats and laughs and for always being available to provide ideas and troubleshooting. Finally I can say with 99.9% certainty that I would not be in a position to submit this thesis if it wasn’t for the support of my family, especially my mam. Thank you for being my number one supporter from day one. Thank you for supporting me emotionally, financially and in some instances literally physically. Thank you for pushing me to give it another go in first year. Who would have thought? Here I am, I did it mam!
  4. 4. Contents ABBREVIATIONS ................................................................................................................................................................6 ABSTRACT .............................................................................................................................................................................7 1.0 INTRODUCTION ...........................................................................................................................................................1 1.1 AIMS OF PROJECT ......................................................................................................................................................6 2.0 METHODS AND MATERIALS .................................................................................................................................7 2.1 CULTURE OF J774 MACROPHAGE CELLS ....................................................................................................................9 2.2 VIABILITY ASSAY (MTS ASSAY) ...................................................................................................................................9 2.3 ELISA PROTOCOL..............................................................................................................................................................9 2.4 FLOW CYTOMETRY FOR RECEPTOR EXPRESSION.................................................................................................10 2.5 PHAGOCYTOSIS ASSAY.................................................................................................................................................11 3.0 RESULTS.........................................................................................................................................................................13 3.1: THE ANALYTICAL AND SYNTHESIZED STANDARDS DID NOT AFFECT CELL VIABILITY.............................................13 3.2: THE MARINEANALOGUESHAD VARIOUSPRO- AND ANTI– INFLAMMATORY EFFECTSON IL-12P40 SECRETION IN MACROPHAGE CELLS...............................................................................................................................................................13 3.3: THE MARINEANALOGUESHAD VARIOUSPRO AND ANTI – INFLAMMATORYEFFECTSON TNFΑSECRETION IN MACROPHAGE CELLS...............................................................................................................................................................14 3.4: THE MARINEANALOGUESHAD VARIOUSPRO AND ANTI – INFLAMMATORYEFFECTSON IL-6SECRETION IN MACROPHAGE CELLS...............................................................................................................................................................14 3.5: THE MARINEANALOGUESHAD VARIOUSPRO AND ANTI – INFLAMMATORYEFFECTSON IL-10 SECRETION IN MACROPHAGE CELLS...............................................................................................................................................................15 3.6: THE MARINEANALOGUESHAD VARIOUSEFFECTSON THE MHC II CELL RECEPTOR EXPRESSION ON MACROPHAGE CELLS...............................................................................................................................................................15 3.7: THE MARINEANALOGUESHAD VARIOUSEFFECTSON THE CD40CELLRECEPTOR EXPRESSION ON MACROPHAGE CELLS.........................................................................................................................................................................................16 3.8: THE MARINEANALOGUESHAD VARIOUSEFFECTSON THE CD86CELLRECEPTOR EXPRESSION ON MACROPHAGE CELLS.........................................................................................................................................................................................16 3.9: THE MARINEANALOGUESHAD VARIOUSEFFECTSON THE CD80CELLRECEPTOR EXPRESSION ON MACROPHAGE CELLS.........................................................................................................................................................................................17 4.0: THE MARINE ANALOGUES ALL HAD A SUPPRESSIVE EFFECT ONPHAGOCYTOSIS......................................................17 5.0 DISCUSSION.................................................................................................................................................................29 6.0 CONCLUSION..............................................................................................................................................................34 7.0 REFERENCES ..........................................................................................ERROR! BOOKMARK NOT DEFINED. 8.0 APPENDICES ................................................................................................................................................................35
  5. 5. Table of Figures Figure 1.0 Activation of Macrophage Cell; ..............................................................................5 Figure 3.1: The analytical and synthesized standards did not affect cell viability. ................18 Figure 3.2: The marine analogues had various pro- and anti – inflammatory effects on IL- 12p40 secretion in macrophage cells. ......................................................................................19 Figure 3.3: The marine analogues had various pro and anti – inflammatory effects on TNFα secretion in macrophage cells. .................................................................................................20 Figure 3.4: The marine analogues had various pro and anti – inflammatory effects on IL-6 secretion in macrophage cells. .................................................................................................21 Figure 3.5: The marine analogues had various pro and anti – inflammatory effects on IL-10 secretion in macrophage cells. .................................................................................................22 Figure 3.6: The marine analogues had various effects on the MHC II cell receptor expression on macrophage cells.................................................................................................................23 Figure 3.7: The marine analogues had various effects on the CD40 cell receptor expression on macrophage cells.................................................................................................................24 Figure 3.8: The marine analogues had various effects on the CD86 cell receptor expression on macrophage cells.................................................................................................................25 Figure 3.9: The marine analogues had various effects on the CD80 cell receptor expression on macrophage cells.................................................................................................................26 Figure 4.0: The marine analogues all had a suppressive effect on phagocytosis. ..................27
  6. 6. Abbreviations APCs- Antigen Presenting Cell BC- Before Christ BSA- Bovine Serum Albumin CD- Crohns Disease CD80- Cluster of Differentiation 80 CD86- Cluster of Differentiation 86 DMEM- Dulbecco’s Modified Eagles Medium ELISA- Enzyme-Linked Immunosorbent Assay FACS- Fluorescence Activated Cell Sorting FCS- Foetal Calf Serum FDA- Food and Drug Administration HIV- Human Immunodeficiency Virus HRP- Horse Radish Peroxidase IFN-γ- Interferon Gamma IL- Interleukin LPS- Lipopolysaccharide MHC- Major Histocompatability Complex MNPs- Marine Natural Products NPs- Natural Products NK- Natural Killer PAMPs- Pathogen-Associated Molecular Patterns pH- Per Hydrogen Ion PRRs- Pattern Recognition Receptors RA- Rheumatoid Arthritis RD- Reagent Diluent RPM – Rotations per minute TLRs- Toll-like Receptors TMB- Tetramethylbenzine TNFa- Tumour Necrosis Factor alpha TNFR- Tumour Necrosis Factor Receptor
  7. 7. Abstract Marine analogues synthesized from compounds derived from a marine species have exhibited anti-inflammatory properties. Auto-inflammation and its associated diseases are a current study as they can have devastating effects on the individual and a relapse is always a possibility. To date the mode of treatment used in inflammatory diseases is often impairment of the immune response through the use of immuno-suppressants. The demand for a more target specific drugs is high as it is often unfavourable to leave a patient immunosuppressed for an extended period of time. In this project fifteen marine analogues were screened on macrophage cells. Cytokine secretion, cell receptor expression and phagocytosis were all studied in a bid to assess their potential effect on inflammation. It was found that each of the 15 analogues housed advantageous properties and through this an ability to mediate the inflammatory response elicited by macrophage cells.
  8. 8. 1 1.0 Introduction Planet earth, a planet rich in biodiversity is home to an ever growing 7.4 billion humans (Worldometers). Planet earth is also home to 8.7 million eukaryotic species (DiscoveryEducation) These species, marine and terrestrial inhabit every corner of the earth. As the ocean spans over seventy one percent of the planet, it is not surprising that the ocean is home to almost 1 million known species and an estimated 9 million undiscovered species (DiscoveryEducation). It has been known for many years by researchers that the ocean is abundant in medicinally relevant marine organisms with potent therapeutic activities. Furthermore it has been estimated that areas of the ocean are richer in biodiversity than the tropical rainforests of the world (Haefner, B. 2003). The earth’s ocean has provided a trove of potential for modern medicine. Exploitation of these marine organisms has led to the discovery of new anti-viral, anti-inflammatory and anti-bacterial agents, which are derived by isolating active compounds from these marine organisms. This is illustrated by the discovery of the anti-HIV activity of chemicals isolated from Tectitethya crypta in 1951 by Bergmann and Feeney (Rangel and Falkenberg 2015). Tectitethya crypta is a large sea sponge found in the shallow waters of the Caribbean. Discovery of the medicinal potential of the derivatives isolated from Tectitethya crypta provided a window for the creation of Azidothymidine, a therapeutic used to treat HIV. Similar to Azidothymidine, many therapeutics have also been sourced from the sea such as Yandelis, an anti-cancer therapeutic used in relapse cases of ovarian cancer (Cassier et al. 2008). The adaptability of these marine organisms to their environment has been the driving force behind their production of chemical diversity within the marine organism community. Consequently, it has been possible to produce various types of therapeutics from extracts of these marine organisms. As highlighted in a paper by Montaser and Luesch in 2011 the adaptability of these marine organisms is extraordinary in a bid to building a tolerance the extreme environments which they inhabit (Montaser and Luesch 2011). One of the most extreme examples includes the hydrothermal vents which are present in the deepest parts of the ocean harbouring temperatures up to 400°C, darkness, high pressure, normally toxic chemical levels and severe pH levels. (Montaser and Luesch 2011). These harsh environments have actually been shown to harbour highly populated communities of marine organisms whom are biologically diverse and possess specifically distinct metabolic characteristics (Montaser and Luesch 2011). The ability of these marine organisms to
  9. 9. 2 produce biologically active secondary metabolites as a defence mechanism to protect themselves from their toxic environments is their distinguishing trait from other organisms and their main attraction as an ongoing target for drug development. The use of natural sources in drug development stretches back to the years of traditional Chinese medicine which is believed to have begun in the second century BC. In today’s modern society natural products are favoured, in the pharmaceutical and beauty industry, consumers prefer using natural products and the research potential is vast. Many potent metabolites have been isolated and to date 60% of the drugs available on the market originate from natural sources (Martins et al. 2014.). This is a considerable advancement as now the marine pharmaceutical market has gained FDA approval for eight drugs and there are a vast number of drugs still in clinical trials (Martins et al. 2014). In 2004 Ziconotide, Cytarabine, omega-3-acid ethyl esters and Vidarabine were the only MNPs entering the pharma market (Martins et al. 2014). Whereas today in 2016 seven marine-derived therapeutics have achieved FDA approval and twenty-three marine derived therapeutics are being tested in phase I to III trials (Marine Pharmacology). Based on this information it may be deduced that the demand for natural products is increasing everyday. Unfortunately many of the marine organisms from which these compounds are derived are small. Therefore it is not possible to continue extracting these organisms from the ocean for many reasons. These reasons include the devastating damage to the ocean’s ecosystem and the exhaustion of the supply resulting in a cease in drug development and or species extinction. Because of these limitations the production of synthetic marine analogues of these marine extracts proves a productive and sustainable method for drug development. Future drug development using marine extracts will prove advantageous in relation to cases such as the malfunctioning of the immune system. This dysregulation is commonly seen in the process of inflammation. Inflammation is an action carried out by the immune system of the host in response to the introduction of foreign antigen (Germain and Schwartzberg 2011). The hallmarks of Inflammation are heat, pain, swelling and redness in response to the invasion of the body by a foreign antigen (Germain and Schwartzberg 2011). Whilst this is the anticipated reaction to the presence of a foreign antigen, in cases of auto-inflammatory diseases and many auto-immune diseases, dysregulation of the inflammatory response has
  10. 10. 3 devastating effects on the patient. In many auto-immune conditions, the reason for this dysregulation of the immune response is unknown. The immune system, which is broken up into two responses, the non-specific (innate) and the specific (adaptive) which are both essential. The immune response relies on both responses and would not be functional if it not be for the coordinated efforts of both the innate and the adaptive responses (Filiano et al. 2015). The innate immune response acts majorly in the recognition and clearance of microbes and the process of inflammation. Cells involved in the innate immune response include cells such as macrophage, dendritic cells and neutrophils. These primary defence cells act as the next line of the defence following physical barriers such as the skin, saliva and cilia. (Filiano et al. 2015). Pivotal to the immune response is the macrophage cell, as it is involved in both, non-specific recognition of pathogens, clearance of foreign antigens by phagocytosis and activation of adaptive immune response by presenting the foreign antigen to T cells. Phagocytosis is the process of which an invading agent or host cells which have been marked for destruction are ingested into the macrophage cells were they are encased in a phagolysosome. In this encasement the invading agent is broken down in order to destroy the agent. In the immune response macrophage cells play leading roles in the defending the host. They do this by their ability to recognise, engulf and kill microorganisms. The action of phagocytosis for cell destruction is the primary actions performed by the human macrophage, however macrophage are also responsible for the stimulation of the cells of the adaptive immune response through acting as antigen presenting cells (APCs). The digested components of the invading antigen may be expressed on the macrophage cell surface without harming the macrophage. This carried out by ingestion of the antigen by phagocytosis and expression of its cellular contents on the MHC II on the macrophage cell surface. Hence macrophage play a secondary role as APCs as well as they primary role in phagocytosis for antigen destruction and by doing so they activate the secretion of pro- inflammatory cytokines and co-stimulation of the cells of the adaptive immune response. The secretion of cytokines is the driving force behind the mounting immune response. Cytokines are proteins secreted by cells and have a specific effect on cell interaction and communication. During infection mainly macrophage secrete proinflammatory cytokines during the upregulation of the inflammatory response. Table 1.0 below outlines cytokines secreted and how they’re involved in the mounting of an immune response. Through this secretion the cells and cytokines of the immune response may play a destructive role seen in the chronic inflammation of the joints in Rheumatoid Arthritis (RA) or destruction of the intestinal wall as seen in Crohn’s Disease (CD). Efforts have been made throughout research
  11. 11. 4 for many years to find a mechanism to block the pathways that lead to this chronic inflammation without leaving the individual completely immuno-compromised. Table 1.0: Cytokines secreted from Macrophage and their Function in the immune response. (Table adapted from Zhang and An 2007) Cytokine Cell Function IL-6 Activated Th2 cells, APCs, other somatic cells B cell proliferation, synergistic relationship with IL-1 and TNF on T cells. IL-10 Activated Th2 cells, CD8+ T and B cells, macrophages Anti-inflammatory. IL-10 inhibits cytokine production, stimulates B cell proliferation, antibody production and dampens cellular immunity. TNF alpha macrophages, mast cells, NK cells, sensory neurons Inflammation, pain and cell destruction. IL-12p40 B cells, macrophages Proliferation of Natural Killer cells, Interferon production, Stimulates cell- mediated immune functions. IL-23 Controls Th17 function and proliferation. Stimulates CD8+ memory T-cells to proliferate. IL-27 Regulation of innate and adaptive immunity. Essential to the process of antigen display are receptors that macrophage expresses on its surface upon activation receptors such as CD80, MHC II, CD40 and CD86 are expressed on the cell surface. Macrophage cells also possess many cell surface features including toll like receptors (TLRs) which are a type of pattern recognition receptors (PRRs) (Kawai and Akira. 2009). [10] These aforementioned PRRs are the receptors Which recognise distinctive features of pathogens, called pathogen-associated molecular patterns (PAMPs). Which recognise distinctive features of pathogens, called pathogen-associated molecular patterns (PAMPs). These PAMPs are bound by the TLRs expressed on the surface of the macrophage and thus
  12. 12. 5 introduced into the macrophage allowing it to act as an antigen presenting cell (Kawai and Akira. 2009). There is currently 11 known TLRs. The engaging of the TLRs with the PAMPs results in the cells ability to elicit an innate immune response causing a secretion of cytokines in many cells including macrophage (Kawai and Akira. 2009). This causes many direct and downstream stimulatory actions including the activation of the adaptive immune response and creation a “memory” of the pathogen using T-cells and B- cells in order to elicit a faster response if the body is ever invaded by the same pathogen (Filiano et al. 2015). Each cell of the immune response has an exclusive role to play, macrophage cells reside in the tissues of the body and are in some cases the first responders to the invasion by an antigen. Furthermore, activated macrophage cells secrete cytokines that are essential to shape an adaptive immune response, including the secretion of the pro- inflammatory IL-12 family of cytokines, TNF alfa, IL-6 and anti-inflammatory IL-10. Figure 1.0 Activation of Macrophage Cell; Showing the consequential event which take place after the activation of a macrophage cell by a stimulus. The stimulus shown above is the toll ligand LPS which is recognised by the TLR4 on the cell surface of macrophage cells. Following this 3 events take place; cytokine release, receptor expression and the process of phagocytosis (Image created for research project presentation).
  13. 13. 6 1.1 Aims of Project In this project, novel marine-derived compounds were screened for anti-inflammatory properties using macrophage cell line. Specifically, the effect of these marine-derived compounds on activation of the macrophage cell, including cell secretion, cell surface receptor expression and phagocytosis. The aim of the project was to identify the compound that demonstrated suppression of pro-inflammatory cytokines IL-12p40, IL-6 and maintained levels of TNF alfa and IL-10, and furthermore supressed expression of cell surface receptors MHC II, CD40, CD80 and CD86 and supressed phagocytosis levels of activated macrophage. Following screening approach was used:  Parent compound, commercially-bought compound and 15 parent-derived compounds were used to stimulate cells prior to activation with LPS for 24 h. ELISA was used to estimate levels of cytokine secretion.  Five most promising parent-derived compounds were brought forward to examine their role in cell surface receptor expression and phagocytosis using flow cytometry
  14. 14. 7 2.0 Methods and Materials Table 1.1: Cell Culture Materials and Corresponding Sources Material Source Centrifuge Tubes 15mL and 50mL Sarstedt Brightline Haemocytometer Invivogen™ Foetal Bovine Serum (FBS) Invivogen™ LPS (E.coli serotype R515) Enzo Lifesciences Penicillin Streptomycin/ Glutamine Gibco® T25cm2 and T75cm2 Tissue culture flasks Nunc™ Tissue culture plates 6, 24 and 96 well Nunc™ Trypan Blue (0.4% w/v) Sigma-Aldrich® DMEM Media Invitrogen™ Table 1.2: MTS assay Materials and Corresponding Sources Material Source CellTitre 96 Aqueous One Solution Promega™ 96-well micro titre plate Nunc™ Table 1.3: ELISA Materials and Corresponding Sources Material Source 96-well micro titre plate Nunc™ Bovine serum albumin (BSA) Sigma-Aldrich® Dulbecco’s Phosphate Buffered Saline (PBS) Gibco® DuoSet® ELISA kits R&D systems Strepdavidin-HRP R&D systems VersaMax™ microplate reader Molecular Devices 3,3’,5,5’-tetramethyl-benzidine Sigma-Aldrich® Tween®20 Sigma-Aldrich®
  15. 15. 8 Table 1.4: Dilutions of the Antibodies and Standards used in the ELISAs Table 1.5 Flow Cytometry Materials for Both Receptor Expression and Phagocytosis Assay. Material Source Sodium Azide Sigma-Aldrich® EDTA Sigma-Aldrich® Anti-CD40 Anti-CD80 Anti-CD86 Latex Beads carboxylate-modified polystyrene, fluorescent yellow green. Sigma-Aldrich® Foetal Calf Serum Gibco™ 37% Formaldehyde Sigma-Aldrich® Table 1.6: Dilutions of Antibodies used in Cell Receptor Expression Analysis using Flow Cytometry. Antibody Dilution used CD40 1:100 CD80 1:100 CD86 1:250 Cytokine Capture antibody dilution Detection Antibody dilution Top standard concentration (pg/mL) IL-6 1:120 1:60 1000 IL-10 1:120 1:60 2000 IL-12p40 1:120 1:60 2000 TNFα 1:120 1:60 2000 IL-23 1:120 1:60 2500 IL-27 1:120 1:60 1000
  16. 16. 9 MHC II 1:250 2.1 Culture of J774 Macrophage cells As J774A.1 cells were the main cell use in analysis throughout this experiment the correct culturing and viability of these cells was essential from the onset. All cell culture work was carried out in class II biological safety cabinet. J774 cells were cultured in a NUNC T75 flask (See table 1.1). The media used to sustain their viability was DMEM medium supplemented with Foetal Calf Serum and 2% (v/v) Penicillin Streptavidin. The J774 cells were stored in incubation at 37°C at 95% humidity and 5% CO2. The cells were split at 90% confluence. 2.2 Viability Assay (MTS assay) Viability assay was carried out to determine the dose response of the macrophage cells for the analytical and marine standard. Varying concentrations were used, these included 5µM, 10µM, 25µM, 50µM, 100µM and 200µM which were made up to working concentration of in ethanol. Ethanol and cells minus any standard were also plated as internal controls. Macrophage cells were diluted to a concentration of 1.0x106 cells/mL. At this concentration the cells were then plated using a volume of 100µl per well in a 96 well plate. The 96 well plates were then incubated to allow the cells to settle for 1 hour at 37°C at 5% CO2 atmosphere. Following this the analytical and marine standards and ethanol were then added, the standards were added in increasing concentration. The plate was then incubated for 24 hours at 37°C at 5% CO2 atmosphere. Post incubation 20µl of CellTitre 96 Aqueous One Solution (see table 1.2) was then added to each well. The plate was again incubated at the same conditions outlined above for 1 hour and read using the Molecular Device VersaMax Microplate reader at 490nm. The plate was then stored in the incubator at the above conditions for reading at T= 2 hours and T=3 hours. The viability was then assessed using Microsoft Excel by comparing the analytical and marine standard treated cells to the vehicle control of cells at 100% viability. 2.3 ELISA Protocol Carried out for IL-6, IL-10, IL-12p40, IL-23, IL-27 and TNFα. All ELISAs were carried out using a 3 day formula. See Table 1.3
  17. 17. 10 Day one included the coating of a NUNC 96 well plate with capture antibody for each cytokine respectively. The capture antibody had been previously prepared in its working concentration using 5ml of non-sterile PBS and the volume of cytokine stated on the guidelines supplied with the ELISA kits (see table 1.4). Day one entailed adding 50µl of the capture antibody to each well covering the 96 well plate with cling film and tin foil and storing a room temperature overnight. Day two began with the preparation of standards and samples, necessary dilutions etc. Following this the 96 well plate was washed three times using an ELISA wash (see appendix A) and patted on clean tissue paper. The plate was then blocked using reagent diluent (see appendix A) at a volume of 300µl per well for at least 1 hour to prevent non-specific binding. Following the above blocking step the plate was washed three times again using ELISA wash buffer. Each well of the plate was then coated with 25µl of reagent diluent. The standards were then added to the wells of the ELISA plates followed by the samples to be measured. The plate was again wrapped in cling film and tin foil and stored at 4°C overnight. Day three entailed the preparation of detection antibody for each respective cytokine. See table 1.4 for the volumes used in the preparation of the detection antibody. The plate was then washed three times using the ELISA wash buffer. The plate was then coated with 50µl of detection antibody and covered with cling film and tin foil for 2 hours at room temperature. During this 2 hours a 1:40 concentration of Streptavidin conjugated horse-radish peroxidise was prepared using reagent diluent and Streptavidin conjugated horse-radish peroxidise. The plate which contained the detection antibody was then washed three times with ELISA wash and coated with 50µl per well of Streptavidin conjugated horse-radish peroxidise and reagent diluent (1:40) covered with cling film and tin foil for 20 minutes at room temperature. Following this the 96-well plate was washed three times using the ELISA wash. Following the washing step 50µl of tetramethylbenzine (TMB) substrate was added to each well and the plates were stored in the dark drawer for approximately 20 minutes based on the speed of the colour change. To stop the reaction 25µl of stop solution (2N sulphuric acid) was added to each well. A colour change from blue to yellow was noted upon application of the stop solution. The 96 well plates then were read at 450nm on a Molecular Device VersaMax Mircroplate Reader. 2.4 Flow Cytometry for Receptor Expression J774A.1 macrophage cells were plated on a 6 well plate at a concentration of 0.5x106 cells/mL.
  18. 18. 11 The cells were allowed to settle in the incubation (37°C, 5% CO2). Following this a 1:250 dilution of the marine analogues were added to the wells and 1:250 dilution of both the analytical standard and synthesized standard were also added to the appropriate wells. The plates were then again placed in incubation for 1hr at 37°C, 5% CO2 and 95% humidity. During this time LPS was prepared as a 1:100 dilution (see appendix A). Final stimulation concentration of 100 ng/ml of LPS was added to each of the LPS positive wells and the plates were again incubated at 37°C, 5% CO2 and 95% humidity for 24 hours. After 24 hours the 6 well plates were scraped using a 3mL transfer pipette and the contents were transferred to a 15mL falcon. Post transfer 2mL of FCS was added to each falcon to block non-specific binding for 15mins. During this time FACs buffer was prepared (see appendix A) The falcons were then centrifuged at 2000rpm for 5mins. The supernatant was removed and the pellet was resuspended in 1mL of FACs buffer. Using a 96 round bottom well plate 0.2x106 of the cells in FACs buffer were added to the wells. The plate was then centrifuged at 2000rpm for 5mins. (see table 1.5) The corresponding antibodies were then prepared in a dilution of 1:100 (CD80 and CD40) and 1:250 (CD86 and MHC II) (see table 1.6). The supernatants were then removed and care was taken not to lose the cells. The cells were then resuspended in 100µL of the antibody, the plate was then incubated at 4°C for 30 mins. The plate was then centrifuged again at 2000rpm for 5mins at 4°C and washed three times to remove antibody solution (by 200µL of FACs buffer followed by 2000rpm for 5mins at 4°C). The cells were then preserved as reading was to take place 2 days later. 1% formaldehyde fixing buffer was prepared (see appendix A). Cells were resuspended in 100µL of fixing buffer and left for 15mins at room temperature within the fumehood. The plate was then centrifuged at 2000rpm for 5mins and following this a final washing step took place. The cells were then resuspended in 100µL of FACs buffer and stored overnight at 4°C. For reading: cells were transferred into tubes filled with 400µL of FACs buffer. The cells were then read using the FACsAria instrument. 2.5 Phagocytosis Assay J774 macrophage cells were plated on a 6 well plate at a concentration of 0.5x106cells/mL. These cells were allowed to settle in the incubation (37°C, 5% CO2). Following this a 1:250 dilution of the marine analogues were added to the wells and 1:250 dilution of both the
  19. 19. 12 analytical standard and synthesized standard were also added to the appropriate wells. The plates were then again placed in incubation for 1hr at 37°C, 5% CO2 and 95% humidity. During this time LPS was prepared as a 1:100 dilution (See appendix A). Final stimulation concentration of 100 ng/ml of LPS was added to each of the LPS positive wells and the plates were again incubated at 37°C, 5% CO2 and 95% humidity for 24 hours. Latex beads (see table 1.7) then were added at a concentration of 6x106 beads/mL this was left for 1hr in incubation at 37°C, 5% CO2 and 95% humidity. Following incubation, the wells were scraped. The media was then transferred to 15mL falcon tubes and centrifuged for 5min at 2000rpm. Following this the supernatant was removed and disposed of and the pellet was resuspended in 1ml of FACS buffer the cells were transferred to tubes and then read using FACsAria 2 (Beckton Dickinson) flow cytometry instrument.
  20. 20. 13 3.0 Results 3.1: The analytical and synthesized standards did not affect cell viability. As outlined in figure 3.1 macrophage cells were plated at a concentration of 0.1x106 cells/mL. The cells were treated with varied increasing concentrations of the analytical and synthesized standard and ethanol. Untreated cells were used as a vehicle control. This was to assess the dosage response of the cells to the analytical and synthesized standard. There was no previous data recorded to assess the viability of macrophage cells when treated with the analytical and synthesized standards. If the standards had proven to be toxic to the cells there would be no therapeutic use for them as future drugs. Hence assessment of the effect of this on cell viability was imperative. Following treatment the cells were then incubated at 37°C with 5% CO2 atmosphere for 24hrs. Post incubation the cells were treated with CellTitre 96 Aqueous One Solution (see table 1.2) and incubated for 1hr. As the cells were only to be treated with the analogues for one hour in future experiments the reading taken at T=1hr was crucial. Three readings were recorded corresponding to T=1hr, T=2hrs and T=3hrs respectively. As can be seen in figure 3.1 the analytical and synthesized standard did not affect cell viability. (see figure 3.1). 3.2: The marine analogues had various pro- and anti – inflammatory effects on IL- 12p40 secretion in macrophage cells. IL-12p40 is a pro-inflammatory cytokine secreted by B-cells and macrophage in response to a stimulus, in this case Lipopolysaccride (Zhang and An 2007). IL-12 consists of the heterodimer of p35 and p40. It is suggested that IL-12p40 acts as an antagonist of IL-12 function. In order to assess IL-12p40 secretion by macrophage cells an ELISA was carried out. Macrophage cells were plated at a concentration of 0.5x106 cells/well, treated with the analytical, marine standards as internal controls and then the appropriate marine analogues. Following an hour incubation period the cells were stimulated with LPS at a concentration of 100ng/ml and incubated for a further 24 hours. The supernatants were then removed and underwent ELISA as per the protocol (see section 2.3). The results concluded that the marine analogues suppressed the secretion of IL-12p40 in
  21. 21. 14 macrophage cells with the exception of analogue 0.6, 0.7, 0.8, 0.9 and 1 which appeared to induce the secretion of IL-12p40. (see figure 3.2). 3.3: The marine analogues had various pro and anti – inflammatory effects on TNFα secretion in macrophage cells. TNFα is a proinflammatory cytokine secreted by mast cells, NK cells and macrophage in response to a stimulus. TNFα is commonly associated with the development of pathological pain through inflammation (Zhang and An 2007). However a level of TNFα is required to avoid complete immunosuppression in the host. To assess this macrophage cells were plated at a concentration of 0.5x106 cells/well, treated with the analytical, marine and ethanol standards as internal controls and then the appropriate marine analogues. Following an hour incubation period the cells were stimulated with LPS at a concentration of 100ng/ml and incubated for a further 24 hours. The supernatants were then removed and underwent ELISA as per the protocol (see section 2.3). By doing so it was concluded that the marine analogues had a varied effect on TNFα suppression. The ideal analogue would sustain the level of TNFα – this was only noted in analogues 0.5, 0.7, 0.8, 1.3 and 1.4. Suppression of TNFα was seen in analogues 0.1, 0.2,0.3,0.4,0.9 and 1. Unfortunately no suppression of TNFα was also observed with the use of analogues 0.6, 1.1, 1.2 and 1.5. (see figure 3.3). 3.4: The marine analogues had various pro and anti – inflammatory effects on IL-6 secretion in macrophage cells. IL-6 is a pro-inflammatory cytokine secreted by APCs such as macrophage cells. IL-6 plays a role in B-cell proliferation and the secretion of IL-1 and TNF in T-cells (Zhang and An 2007). To asses this macrophage cells were plated at a concentration of 0.5x106 cells/well, treated with the analytical, marine and ethanol standards as internal controls and then the appropriate marine analogues. Following an hour incubation period the cells were stimulated with LPS at a concentration of 100ng/ml and incubated for a further 24 hours. The supernatants were then removed and underwent ELISA as per the protocol (see section 2.3). By doing so it was observed that IL-6 suppression was achieved by all the marine analogues with the exclusion of analogue 1.1 and 1.5 which an increase in secretion was noted. (see figure 3.4).
  22. 22. 15 3.5: The marine analogues had various pro and anti – inflammatory effects on IL-10 secretion in macrophage cells. IL-10 is an anti-inflammatory cytokine secreted by CD8+ T and B cells and macrophage. IL- 10 is an anti-inflammatory cytokine as it suppresses the production of pro-inflammatory cytokines. IL-10 also stimulates B-cell proliferation and dampens cellular immunity (Zhang and An 2007). In order to assess IL-10 secretion J774A.1 macrophage cells were plated at a concentration of 0.5x106 cells/well in a 96 well plate. They were then treated with the analytical, marine and ethanol standards as internal controls and then the appropriate marine analogues. Following an hour incubation period the cells were stimulated with LPS at a concentration of 100ng/ml and incubated for a further 24 hours. The supernatants were then removed and underwent ELISA as per the protocol (see section 2.3). From this assessment it was deduced that only marine analogues 0.2, 0.3, 0.4, 1.2, 1.3 and 1.5 increased or maintained the secretion of IL-10 in the macrophage cells. The remaining marine analogues displayed a visible suppression of IL-10 secretion. (see figure 3.5). 3.6: The marine analogues had various effects on the MHC II cell receptor expression on macrophage cells. MHC II is a family of molecules found on the cell surface of professional APCs of the immune system. The main function of major histocompatibility complex (MHC) class II molecules is to present processed antigens (Holling et al. 2004). For this reason MHC II expression on the cell surface is increased in inflammation. To assess the magnitude of this upregulation flow cytometry to determine receptor expression was used. J774A.1 macrophage cells were plated at a concentration of 0.5x106 cells/well, treated with the analytical, marine and ethanol standards as internal controls and then the appropriate marine analogues. Following an hour incubation period the cells were stimulated with LPS at a concentration of 100ng/ml and incubated for a further 24 hours. The supernatants were then removed and underwent flow cytometry for receptor expression as per protocol (see section 2.4). The observed effects of the analogues were MHC II expression was supressed in macrophage cells with the treat of analogues 0.7, 0.9, 1 and 1.5. MHC II expression was not
  23. 23. 16 supressed by analogue 0.5. Interestingly analogue 0.5 was noted to cause in increase in the expression of MHC II. (see figure 3.6). 3.7: The marine analogues had various effects on the CD40 cell receptor expression on macrophage cells. CD40 is a co-stimulatory protein found on the cell surface of APCs and is vital for their activation. CD40 is commonly associated with inflammatory diseases and is upregulated in the presence on TNFα (Liang et al 2008). In order to determine the effect the marine analogues had on CD40 expression receptor expression was assessed using flow cytometry. J774A.1 macrophage cells were plated at a concentration of 0.5x106 cells/well, treated with the analytical, marine and etanol standards as internal controls and then the appropriate marine analogues. Following an hour incubation period the cells were stimulated with LPS at a concentration of 100ng/ml and incubated for a further 24 hours. The supernatants were then removed and underwent flow cytometry for receptor expression as per protocol (see section 2.4). By doing so it was deduced that the marine analogues all suppressed the expression of CD40 with the exception of analogue 0.5. (see figure 3.7). 3.8: The marine analogues had various effects on the CD86 cell receptor expression on macrophage cells. CD86 is a protein found expressed on the cell surface of APCs. CD86 is partially responsible for the activation and survival of T-cells in the adaptive immune response. The binding of CD86 to CD28 on the surface of T-cells acts as an activation mechanism (Woldai 2014). Hence the expression of CD86 should be ideally supressed in the treatment of dysregulation of the inflammatory response. .To assess this J774A.1 macrophage cells were plated at a concentration of 0.5x106 cells/well, treated with the analytical, marine and etthanol standards as internal controls and then the appropriate marine analogues. Following an hour incubation period the cells were stimulated with LPS at a concentration of 100ng/ml and incubated for a further 24 hours. The supernatants were then removed and underwent flow cytometry for receptor expression as per protocol (see section 2.4). It was observed that in the case of CD86 expression suppression was achieved by marine analogues 0.5, 0.9 and 1. Unfortunately suppression was not successful with treatment with analogues 0.7 and 1.5. (see figure 3.8).
  24. 24. 17 3.9: The marine analogues had various effects on the CD80 cell receptor expression on macrophage cells. CD80, alike CD86 is a protein found on the cell surface on APCs. CD80 expression is upregulated in a mounting immune response in order to stimulate and maintain T-cells. To assess the suppressive properties of the marine analogues J774A.1 macrophage cells were plated at a concentration of 0.5x106 cells/well. They were then treated with the analytical, marine and etOH standards as internal controls and then the appropriate marine analogues. Following an hour incubation period the cells were stimulated with LPS at a concentration of 100ng/ml and incubated for a further 24 hours. The supernatants were then removed and underwent flow cytometry for receptor expression as per protocol (see section 2.4). As a result of analogue treatment the expression of CD80 was successfully suppressed by marine analogues 0.5 and 1.5. Successful suppression was not achieved by analogues 0.7, 0.9 or 1. (see figure 3.9). 4.0: The marine analogues all had a suppressive effect on phagocytosis. Phagocytosis is the process whereby macrophage cells engulf and destroy foreign agents. Macrophage do so by ingesting the foreign agent and digesting it within a phagolysosome. Phagocytosis is a process used in the innate immune response hence it would be expected to see an increase in this process in an activated macrophage. To assess the level of macrophage actively engulfing the latex beads the following was carried out; J774A.1 macrophage cells were plated at a concentration of 0.5x106 cells/well. Following this they were treated with the analytical, marine and ethanol standards as internal controls and then the appropriate marine analogues. Following an hour incubation period the cells were stimulated with LPS at a concentration of 100ng/ml and incubated for a further 24 hours. Post incubation the cells were treated with Latex beads in order to assess the magnitude of phagocytosis. The cells were again incubated for 1 hr. The supernatants were then removed and underwent flow cytometry for phagocytosis as per protocol (see section 2.5). Interestingly the process of phagocytosis was successfully suppressed in the case of each analogue. (see figure 4.0)
  25. 25. 18 V ia b ility (T = 2h rs) C e lls E tO H 5 u M 1 0 u M 2 5 u M 5 0 u M 1 0 0 u M 2 0 0 u M 5 u M 1 0 u M 2 5 u M 5 0 u M 1 0 0 u M 2 0 0 u M 0 25 50 75 100 125 %ViableCells (expressedaspercentageofcontrol) M arine S td. A nalytical S td. Ethanol V e hicle control V ia b ility (T = 3h rs) C ellsE tO H 5u M10u M25u M50u M100u M200u M 5u M10u M25u M50u M100u M200u M 0 25 50 75 100 125 %ViableCells (expressedaspercentageofcontrol) M arine S td. A nalytical S td. V e hicle control Figure 3.1: The analytical and synthesized standards did not affect cell viability. Macrophage cells were plated at a concentration of 0.1x106 cells/mL. The cells were treated with varied increasing concentrations of the analytical and synthesized standard and ethanol. Untreated cells were used as a vehicle control. The cells were then incubated at 37°C with 5% CO2 atmosphere for 24hrs. Post incubation the cells were treated with CellTitre 96 Aqueous One Solution (see table 1.2) and incubated for 1hr. Following this three readings were recorded corresponding to T=1hr, T=2hrs and T=3hrs respectively. As can be seen in figure 3.1 the analytical and synthesized standard did not affect cell viability. V ia b ility (T = 1 h r) C e lls E tO H 5 u M 1 0 u M 2 5 u M 5 0 u M 1 0 0 u M 2 0 0 u M 5 u M 1 0 u M 2 5 u M 5 0 u M 1 0 0 u M 2 0 0 u M 0 25 50 75 100 125 %ViableCells (expressedaspercentageofcontrol) A n a lytica l S td . M a rin e S td . E thanol V e h ic le co n tro l
  26. 26. 19 IL -1 2 p 4 0 C e lls E tO H A n a ly tic a l S ta n d a rd S y n th e s is e d S ta n d a rd 0 .1 0 .2 0 .3 0 .4 1 .5 0 5 0 1 0 0 1 5 0 - L P S + L P S (1 0 0 n g /m L ) Concentrationpg/ml IL -1 2 p 4 0 C e lls E tO H A n a ly tic a l S ta n d a rd S y n th e s is e d S ta n d a rd 0 .7 0 .8 0 .9 1 0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 - L P S + L P S (1 0 0 n g /m L ) Concentrationpg/ml IL -1 2 p 4 0 C e lls E tO H A n a ly tic a l S ta n d a rd S y n th e s is e d S ta n d a rd 0 .5 0 .6 0 .7 0 .8 0 5 0 1 0 0 1 5 0 2 0 0 2 5 0 - L P S + L P S (1 0 0 n g /m L ) Concentrationpg/ml Figure 3.2: The marine analogues had various pro- and anti – inflammatory effects on IL-12p40 secretion in macrophage cells. Macrophage cells were plated at a concentration of 0.5x106 cells/well, treated with the analytical, marine standards as internal controls and then the appropriate marine analogues. Following an hour incubation period the cells were stimulated with LPS at a concentration of 100ng/ml and incubated for a further 24 hours. The supernatants were then removed and underwent ELISA as per the protocol.
  27. 27. 20 T N F  C e lls E tO H A n a ly tic a l S ta n d a rd S y n th e s is e d S ta n d a rd 0 .1 0 .2 0 .3 0 5 0 0 1 0 0 0 1 5 0 0 2 0 0 0 - L P S + L P S (1 0 0 n g /m L ) Concentrationpg/ml T N F  C e lls E tO H A n a ly tic a l S ta n d a rd S y n th e s is e d S ta n d a rd 0 .4 0 .5 0 .6 0 500 1000 1500 - L P S + L P S Concentrationpg/ml T N F  C e lls E tO H A n a ly tic a l S ta n d a rd S y n th e s is e d S ta n d a rd 0 .8 0 .9 1 0 2 0 0 0 4 0 0 0 6 0 0 0 -L P S + L P S (1 0 0 n g /m L ) Concentrationpg/ml T N F  C e lls E tO H A n a ly tic a l S ta n d a rd S y n th e s is e d S ta n d a rd 1 .1 1 .2 1 .3 1 .4 1 .5 0 1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0 5 0 0 0 - L P S + L P S (1 0 0 n g /m L ) Concentrationpg/ml Figure 3.3: The marine analogues had various pro and anti – inflammatory effects on TNFα secretion in macrophage cells. Macrophage cells were plated at a concentration of 0.5x106 cells/well, treated with the analytical, marine and etOH standards as internal controls and then the appropriate marine analogues. Following an hour incubation period the cells were stimulated with LPS at a concentration of 100ng/ml and incubated for a further 24 hours. The supernatants were then removed and underwent ELISA as per the protocol.
  28. 28. 21 IL -6 C ells E tO H A n alyticalS tan d ard S yn th esised S tan d ard 0.1 0.2 0.3 0.4 0 2000 4000 6000 - L P S + L P S (1 0 0 n g /m L ) Concentrationpg/ml IL -6 C e lls E tO H A n a ly tic a l S ta n d a rd S y n th e s is e d S ta n d a rd 0 .5 0 .6 0 .7 0 .8 0 .9 1 0 1000 2000 3000 4000 - L P S + L P S (1 0 0 n g /m L ) Concentrationpg/ml IL -6 C e lls E tO H A n a ly tic a l S ta n d a rd S y n th e s is e d S ta n d a rd 0 .7 0 .8 0 .9 1 0 1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0 -L P S + L P S (1 0 0 n g /m L ) Concentrationpg/ml IL -6 C e lls E tO H A n a ly tic a l S ta n d a rd S y n th e s is e d S ta n d a rd 1 .1 1 .2 1 .3 1 .4 1 .5 0 1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0 - L P S + L P S (1 0 0 n g /m L ) Concentrationpg/ml Figure 3.4: The marine analogues had various pro and anti – inflammatory effects on IL-6 secretion in macrophage cells. Macrophage cells were plated at a concentration of 0.5x106 cells/well, treated with the analytical, marine and etOH standards as internal controls and then the appropriate marine analogues. Following an hour incubation period the cells were stimulated with LPS at a concentration of 100ng/ml and incubated for a further 24 hours. The supernatants were then removed and underwent ELISA as per the protocol.
  29. 29. 22 IL -10 C e lls E tO H A n a ly tic a l S ta n d a rd S y n th e s is e d S ta n d a rd 0 .1 0 .2 0 .3 0 .4 1 .5 0 500 1000 1500 2000 - L P S + LP S (1 00 ng /m L) Concentrationpg/ml IL -10 C e lls E tO H A n a ly tic a l S ta n d a rd S y n th e s is e d S ta n d a rd 0 .7 0 .8 0 .9 1 0 500 1000 1500 - L P S + LP S (1 00 ng /m L) Concentrationpg/ml IL -10 C e lls E tO H A n a ly tic a l S ta n d a rd S y n th e s is e d S ta n d a rd 0 .4 0 .5 0 .6 0 .7 0 .8 0 .9 1 0 200 400 600 - L P S + LP S (1 00 ng /m L) Concentrationpg/ml IL -1 0 C e lls E tO H A n a ly tic a l S ta n d a rd S y n th e s is e d S ta n d a rd 1 .1 1 .2 1 .3 1 .4 0 500 1000 1500 2000 2500 - L P S + L P S (1 0 0 n g /m L ) Concentrationpg/ml IL -1 0 C e lls E tO H A n a ly tic a l S ta n d a rd S y n th e s is e d S ta n d a rd 0 .1 0 .2 0 .3 0 1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0 - L P S + L P S (1 0 0 n g /m L ) Concentrationpg/ml Figure 3.5: The marine analogues had various pro and anti – inflammatory effects on IL-10 secretion in macrophage cells. Macrophage cells were plated at a concentration of 0.5x106 cells/well, treated with the analytical, marine and etOH standards as internal controls and then the appropriate marine analogues. Following an hour incubation period the cells were stimulated with LPS at a concentration of 100ng/ml and incubated for a further 24 hours. The supernatants were then removed and underwent ELISA as per the protocol.
  30. 30. 23 Figure 3.6: The marine analogues had various effects on the MHC II cell receptor expression on macrophage cells. Macrophage cells were plated at a concentration of 0.5x106 cells/well, treated with the analytical, marine and ethanol standards as internal controls and then the appropriate marine analogues. Following an hour incubation period the cells were stimulated with LPS at a concentration of 100ng/ml and incubated for a further 24 hours. The supernatants were then removed and underwent flow cytometry for receptor expression as per protocol (see section 2.4) Cells Synthesized Std. Analytical Std. 0.5 0.7 0.9 1 1.5
  31. 31. 24 Figure 3.7: The marine analogues had various effects on the CD40 cell receptor expression on macrophage cells. Macrophage cells were plated at a concentration of 0.5x106 cells/well, treated with the analytical, marine and etOH standards as internal controls and then the appropriate marine analogues. Following an hour incubation period the cells were stimulated with LPS at a concentration of 100ng/ml and incubated for a further 24 hours. The supernatants were then removed and underwent flow cytometry for receptor expression as per protocol (see section 2.4). Cells Synthesized Std. Analytical Std. 0.5 0.7 0.9 1 1.5
  32. 32. 25 Figure 3.8: The marine analogues had various effects on the CD86 cell receptor expression on macrophage cells. Macrophage cells were plated at a concentration of 0.5x106 cells/well, treated with the analytical, marine and etOH standards as internal controls and then the appropriate marine analogues. Following an hour incubation period the cells were stimulated with LPS at a concentration of 100ng/ml and incubated for a further 24 hours. The supernatants were then removed and underwent flow cytometry for receptor expression as per protocol (see section 2.4). Cell Synthesized Std. Analytical Std. 0.5 0.7 0.9 1.0 1.5
  33. 33. 26 Figure 3.9: The marine analogues had various effects on the CD80 cell receptor expression on macrophage cells. Macrophage cells were plated at a concentration of 0.5x106 cells/well, treated with the analytical, marine and etOH standards as internal controls and then the appropriate marine analogues. Following an hour incubation period the cells were stimulated with LPS at a concentration of 100ng/ml and incubated for a further 24 hours. The supernatants were then removed and underwent flow cytometry for receptor expression as per protocol (see section 2.4). Cells Synthesized Std. Analytical Std. 0.5 0.7 0.9 1.0 1.5
  34. 34. 27 Figure 4.0: The marine analogues all had a suppressive effect on phagocytosis. Macrophage cells were plated at a concentration of 0.5x106 cells/well, treated with the analytical, marine and etOH standards as internal controls and then the appropriate marine analogues. Following an hour incubation period the cells were stimulated with LPS at a concentration of 100ng/ml and incubated for a further 24 hours. Post incubation the cells were treated with Latex beads in order to assess the magnitude of phagocytosis. The cells were again incubated for 1 hr. The supernatants were then removed and underwent flow cytometry for phagocytosis as per protocol (see section 2.5). Cells Synthesized Std. Analytical Std. 0.5 0.7 0.9 1 1.5
  35. 35. 28 Table 1.7: Overview of Events Across the Screening Profile Analogue IL-6 IL-10 IL-12 TNFa MHC II CD40 CD80 CD86 PHAG O* 0.1 ↓ ↓ ↓ ↓ 0.2 ↓ ↑ ↓ ↓ 0.3 - - ↓ ↓ 0.4 ↓ - ↓ ↓ 0.5 ↓ ↓ - - ↑ ↑ ↓ ↓ ↓ 0.6 ↓ ↓ ↑ ↑ 0.7 ↓ ↓ ↑ - ↓ ↓ ↑ ↑ ↓ 0.8 ↓ ↓ ↑ - 0.9 ↓ ↓ ↓ ↓ ↓ ↓ ↑ ↓ ↓ 1 ↓ ↓ ↓ ↓ ↓ ↓ ↑ ↓ ↓ 1.1 ↑ ↓ ↑ 1.2 ↓ ↑ ↑ 1.3 ↓ ↑ - 1.4 ↓ ↓ - 1.5 ↑ - ↓ ↑ ↓ ↓ ↓ ↑ ↓ ↓ = Suppression occurred - = level of secretion was level with internal control cells, +/- LPS ↑= level of expression/secretion was increased or maintained. *Phago = Phagocytosis.
  36. 36. 29 5.0 Discussion The purpose of this study was to screen of each of the 15 analogues on J774A.1 macrophage and by doing so generating data on the significant findings of these analogues in relation to the macrophage cell, a cell of the innate immune response. Throughout this experiment the theme of variance has been at the forefront of the majority of the analysis. The fifteen marine analogues were screened across an extensive immune response profile and five where selected for further testing in the hope of finding an ideal analogue that completely suppressed the pro-inflammatory responses and maintained or increased the level of anti- inflammatory responses. Unfortunately this was not the case in respect to any of the 15 analogues singly, however many did harbour very interesting effects across the screening profile which may prove significant for potential future research. The major findings of this study included the varied effect on cytokine secretion that the analogues possessed; both suppression and increased secretion of the cytokines was observed through the use of ELISAs (see section 2.3), the suppression and increased expression of cell receptors MHC II, CD40, CD80 and CD86 and the suppressive action the analogues had on phagocytosis. Over the screening profile the analogues have displayed strong evidence that individually they harbour many advantageous properties. With further studies these analogues could be used in therapeutics tailored to various inflammatory diseases in order to dampen specific targets. As these marine analogues all remain under patent none of the mechanisms of action are known. Each analogue had various affects across the screening profile, this may indicate the presence of different mechanisms in each analogue. It may also be possible that the analogues may carry out more than one mechanism. Each of the 15 analogues proved successful in dampening the immune response on some level. The hosts immune response is critical for survival. Without both arms of the immune response a common infection, often fought by the host’s immune system daily could wipe out entire communities of species. However a dysregulation of the immune response especially in the case of inflammatory diseases can often have devastating effects on the host. Pain caused by an overactive inflammatory response is seen in diseases such as RA and CD. The desired role of these analogues is in the dampening of this inflammatory response. In the case of most inflammatory diseases the causative pathway or agent is unknown however the dampening of various aspects of the immune
  37. 37. 30 response such as cytokine secretion has proved advantageous in suppressing the host’s response to the unknown stimulus. The marine analogues have shown competency in successful suppression of the immune response in many areas. The secretion of pro-inflammatory cytokines is often the driving force behind inflammatory diseases such as RA and CD. There has been clear evidence proposed by Zhang and An in 2007 to suggest that IL-6, TNFα and IL-1β are heavily involved in the development of pathological pain through inflammation. Pain in relation to the hallmark symptoms of inflammation is expected, this can be seen in common illnesses and is often a critical defence action. However inflammation in relation to a unknown cause can have devastating effects. For many years increased TNFα secretion has been associated with RA pathogenesis. RA is an extremely complex disease and the response of patients to anti-TNFα treatment has been seen to vary, this causes limitations for drug development (Vasanthi et al. 2007). TNFα is found in excess in the synovial fluid of RA sufferers. TNFα plays a pivotal role in inflammation and joint destruction, which are the hallmark symptoms seen in RA sufferers. In a study detailed by Vasanthi et al in 2007 RA sufferers showed a decrease in the level of pro- inflammatory cytokines present in the synovial fluid when treated with anti-TNFα treatment. This proved to alleviate the patient of the symptoms of RA. It has been highlighted that even with long term use therapeutics such as Etanercept, a recominbant TNF receptor, still achieves symptom alleviation in RA positive patients (Vasanthi et al 2007). Unfortunately the suppression of TNFα is not without adverse effects. TNFα is a crucial cytokine in the mounting of an immune response, acting through two separate pathways through its receptors TNFR1 and TNFR2. TNFα is responsible for the stimulation of many immune cells and regulates apopatosis (Zhang and An 2007). By suppressing the secretion of TNFα by the immune cells the immune response is impaired. Treating a patient with anti-TNFα therapeutics can increase the patients risk of infection with opportunistic infections such as pneumonia (Fiorino et al. 2012). For this reason an ideal therapeutic for the treatment of inflammatory diseases would have to be altered to meet the requirements of each disease. In inflammatory diseases in which TNFα is secreted in excess in comparison to other pro-inflammatory cytokines perhaps an analogue which showed strong suppression of TNFα would prove advantageous such as is seen in the case of analogue 0.1 which displayed a powerful level of TNFα suppression. There are a number of cytokines involved in the mounting of an immune response. This included the cytokines screened; IL-6, IL-10, IL-12p40, IL-23 and Il-27. Unfortunatly due to the 10% loss of protein upon freezing the supernatants, screening of the cytokines IL-23 and IL-27 had to be
  38. 38. 31 abandoned as the level of secretion was not high enough to be detected by ELISA. The levels of IL- 6, IL-10 and IL-12p40 were detectable. IL-6 is a pro-inflammatory cytokine which is closely linked with the pathogenesis of CD. Increased levels of IL-6 have been observed in both the serum and intestinal tissues of CD positive patients (Ito 2003). For this reason targeting the hyper-secretion of IL-6 whilst maintaining a level of beneficial TNFα may prove advantageous to the production of future therapeutics for CD patients. Many of the marine analogues showed strong suppression of IL- 6. This was a significant observation when the macrophage cells were treated with analogues 0.4 – 0.9. However only analogues 0.5, 0.7 and 0.8 maintained TNFα whilst suppressing IL-6 secretion in macrophage cells. IL-12p40 was the last of the pro-inflammatory cytokines of the screening profile to be assessed. IL- 12p40 is a subunit of IL-12. IL-12 is a heterodimeric cytokine made up of two subunits p40 and p35 (Peluso et al. 2006). IL-12 is secreted by macrophage, monocytes and dendritic cells, which are all pivotal cells in the innate immune response (Peluso et al. 2006). IL-12 has been associated with inflammatory disease for many years and its involvement is spread across the inflammatory disease network. Increased levels of IL-12 is seen in inflammatory diseases such as RA, atherosclerosis and CD (eBioscience). IL-12 is responsible for inducing the production of IFN-γ, Stimulation of NK cells and T-cells and by doing so, cell proliferation (eBioscience). The marine analogues showed varied effects on the secretion of IL-12p40. Analogues 0.1, 0.2, 0.3 and 0.4 showed a competent suppression of IL-12 along with analogues 0.9, 1 and 1.5. In terms of the anti-inflammatory cytokines included in the cytokine screen profile IL-10 was used. IL-10 is a member of a family, this family includes IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, IL- 28A, IL-28B and IL-29 (Ouyang et al 2011). This family of anti-inflammatory cytokines play a central role in the maintenance of the integrity and homeostasis of the tissue epithelial layers. In addition to this the IL-10 family are also involved in the healing process in injuries sustained during infection or inflammation (Ouyang et al 2011). For this reason in inflammatory diseases in which injury is being sustained a treatment inducing an increase in the level of the IL-10 family would be advantageous. Through treatment with the marine analogues many displayed a capability to increase the secretion of IL-10 or maintain its expected level of secretion. These analogues included 0.2, 0.3, 0.4, 1.2, 1.3 and 1.5. However only analogue 1.3 showed an ability to maintain the level of TNFα, increase secretion of IL-10 and suppress secretion of IL-6. Unfortunately a result was not recorded for this analogues effect on IL-12p40 due to time constraints and low IL-12p40 secretion levels due
  39. 39. 32 to the supernatants undergoing a freeze thaw. However this may be an analogue with potential for the treatment of inflammatory disease in which IL-6 is over-secreted such as is seen in CD. In addition to the analogues effect on cytokine secretion two additional events which take place in an immune response were studied and these were cell receptor expression and phagocytosis. Cell receptor expression has been shown to be increased in response to a stimulus such as LPS. However in the case of dysregulation of the inflammatory response the stimulant causing upregulation in cell receptor expression is unknown. The cell receptors included in the screening profile were MHC II, CD40, CD80 and CD86. All of the aforementioned cell receptors have a role in the mounting of an immune response. MHC II is a family of molecules found on the cell surface of professional APCs of the immune system. The main function of major histocompatibility complex (MHC) class II molecules is to present processed antigens (Holling et al. 2004). CD40 is a co-stimulatory protein found on the cell surface of APCs and is vital for their activation. CD40 is commonly associated with inflammatory diseases and is upregulated in the presence of TNFα which is a cytokine commonly seen in excess in inflammatory diseases. (Liang et al 2008). CD86 is a protein found expressed on the cell surface of APCs. CD86 is partially responsible for the activation and survival of T-cells in the adaptive immune response. The binding of CD86 to CD28 on the surface of T-cells acts as an activation mechanism (Woldai 2014). CD80, alike CD86 is a protein found on the cell surface on APCs. CD80 expression is upregulated in a mounting immune response in order to stimulate and maintain T-cells (Woldai 2014). Cell receptor expression was assessed using flow cytometry (see section 2.4). The marine analogues showed a varying effect on cell receptor expression. No one analogue suppressed all of the receptors of the screening profile however a significant finding was that analogue 0.9 showed successful suppression of receptor CD40, CD86 and MHC II in addition to this analogue 0.9 showed suppression of both and the pro and anti-inflammatory cytokines. Analogue 0.9 failed however to suppress CD80. Analogue 1.5 displayed a similar effect on cell receptor expression as 0.9 however analogue 1.5 failed to suppress IL-6 and spiked the level of TNFα being secreted. Suppression of the cell receptor expression on macrophage cells is another attempt at dampen the immune system. As analogue 0.9 showed successful suppression of all receptors except CD80 and significant suppression of the screening profile cytokines it may prove useful in therapeutics targeting an extreme case of severe dysregulation of the immune response such as systemic inflammatory response syndrome in which both the pro and anti-inflammatory responses are dysregulated (Kaplan and Pinsky 2015). Anti TNFα treatments have been used in systemic inflammatory response syndrome in the past and showed no efficacy (Kaplan and Pinsky 2015). However an analogue such as analogue 0.9 which displays powerful suppression across the
  40. 40. 33 screening profile may prove useful in the treatment of acute inflammation rather than chronic inflammation. The final assessment carried out in the screening profile was the effect the analogues displayed on the process of phagocytosis. Phagocytosis is the primary action of foreign agent destruction carried by macrophage cells. Phagocytosis is an important cell debris clearance mechanism. As figure 1.0 shows a non-activated macrophage cell resides in the host tissue until activation by a stimulus such as LPS. Once activated the macrophage will begin the process of phagocytosis. The effect the analogues had on phagocytosis was assessed using latex beads (see table 1.5) and flow cytometry (see section 2.5). Of the five significant analogues brought forward for further analysis all of the analogues suppressed the process of phagocytosis when compared to the negative control. This is a significant finding as phagocytosis is the first mode of defence carried out by macrophage in response to a stimulus. Overall the screening of the fifteen analogues over the cytokine profile showed varied results. Upon bringing five analogues forward for further testing the significance of the cytokine results became clear. In the case of analogue 0.9 a complete suppression of the screening profile was observed with the exception of CD80. As aforementioned analogue 0.9 would prove effective in the treatment of a severe dysregulation of the inflammatory response. Analogue 1.0 showed an identical effect to analogue 0.9 however with further testing and a broader screening profile it may be possible to isolate the differences that may arise in their powerful suppressive properties. No one analogue showed an ideal profile however each analogue harboured potential advantageous properties for drug development with specific targets such as a requirement for stronger suppression of IL-6 i.e in CD, or suppression of TNFα in RA. Inflammatory diseases differ in their requirement for treatment thus the use of a general immunosuppressive therapeutics to suppress all auto- inflammatory events will cause the patient to be immunocompromised and as a result expose them to a new level of susceptibility which may prove more fatal than the inflammatory disease itself. In this way it is useful to design drugs with a specific target however in the category of auto-inflammation in many cases the causative agent is unknown thus making the development of specific target therapeutics limited. This leaves researchers and medical professionals with the current issue of the level of immune-suppression needed to alleviate the symptoms of the inflammatory disease without exposing the patient to external infections without the defence provided by the immune system.
  41. 41. 34 6.0 Conclusion To conclude this report, the analogues were screened on macrophage cells. This was a novel study. As aforementioned variance was a trend seen throughout the analysis. The analogues have provided strong evidence that they harbour advantageous properties which may be used in potential future drug development. Each analogues exhibits anti-inflammatory properties whether these properties are ideal for the treatment of auto-inflammation or not. These analogues have shown competency in mediating the processes which occur in a mounting immune response. This competency is seen over the entire screening profile. Analogues have displayed the ability to suppress almost 90% of the events studied. Analogues which have shown the ability to elicit this level of suppression will prove useful in future studies even outside of the chronic inflammation seen in auto-inflammatory diseases. Each of the analogues studied have displayed many advantageous properties and isolation of these properties for each analogue is the next step in future research. By doing so, it may be possible to develop therapeutics in which specific events are targeted. This may not be in the category of auto- inflammation but perhaps in treating acute inflammation. Additionally it has been observed that the analogues harbour differentiation properties. Future studies may include the development of a model in which dual treatment with the analogues occurs in a bid to exploit the advantageous properties of analogues which may led to the creation of an ideal hybrid analogue.
  42. 42. 35 8.0 Appendices Appendix A: Recipes for Various Buffer and Other Solutions Used Throughout Analysis of Marine Analogues Buffer/Solution Materials Required and Corresponding Source 10X PBS buffer 160g NaCl (Sigma) 23.2g Na2HPO4 (Sigma) 4g KH2PO4 (Sigma) 4g KCl (Sigma) 2000mL DH2O p.H corrected to 7.4 1% formaldehyde fixing buffer 9mL FACs buffer 1mL of 37% formaldehyde ELISA wash buffer (5L) 500mL of 10X PBS buffer 2.5mL of 0.05% Tween (Sigma) 4500mL of DH2O Reagent Diluent 1XPBS 500ml (Gibco) 5g of bovine serum albumin. (Sigma) DMEM for macrophage 50mL foetal calf serum (Gibco) 15mL Penicillin Streptomycin Preparation of Lipopolysaccaride (100ng/mL) 495µL of DMEM media 5µL of LPS (Enzo Lifesciences)
  43. 43. 36

×