Wright State University CORE Scholar Browse all Theses and Dissertations Theses and Dissertations 2019 Enhanced expression of receptor tyrosine kinase Mer (MERTK) on SOCS3-treated polarized RAW 264.7 anti-inflammatory M2c macrophages Sankhadip Bhadra Wright State University Follow this and additional works at: https://corescholar.libraries.wright.edu/etd_all Part of the Immunology and Infectious Disease Commons, and the Microbiology Commons Repository Citation Bhadra, Sankhadip, "Enhanced expression of receptor tyrosine kinase Mer (MERTK) on SOCS3-treated polarized RAW 264.7 anti-inflammatory M2c macrophages" (2019). Browse all Theses and Dissertations. 2105. https://corescholar.libraries.wright.edu/etd_all/2105 This Thesis is brought to you for free and open access by the Theses and Dissertations at CORE Scholar. It has been accepted for inclusion in Browse all Theses and Dissertations by an authorized administrator of CORE Scholar. For more information, please contact [email protected]. Enhanced expression of receptor tyrosine kinase Mer (MERTK) on SOCS3-treated polarized RAW 264.7 anti-inflammatory M2c macrophages A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science By SANKHADIP BHADRA M.Sc., Microbiology, GITAM University, INDIA, 2015 B.Sc., Microbiology, University of Calcutta, INDIA, 2013 2019 Wright State University WRIGHT STATE UNIVERSITY GRADUATE SCHOOL July 31, 2019 I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY Sankhadip Bhadra ENTITLED Enhanced expression of receptor tyrosine kinase Mer (MERTK) on SOCS3-treated polarized RAW 264.7 anti-inflammatory M2c macrophages BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science. ----------------------------- Nancy J. Bigley, Ph.D. Thesis Director ------------------------------ Dawn P. Wooley, Ph.D. Chair, Microbiology and Immunology Program, College of Science and Mathematics Committee on Final Examination: -------------------------------------------- Nancy J. Bigley, Ph.D. Professor, Microbiology and Immunology Program ---------------------------------------------- Marjorie Markopoulos, Ph.D. Director, Department of Environmental Health and Safety ------------------------------------------------ Dawn P. Wooley, Ph.D. Professor, Department of Neuroscience, Cell Biology and Physiology ------------------------------------------------- Barry Milligan, Ph.D. Interim Dean of the Graduate School ABSTRACT Bhadra, Sankhadip M.S. Microbiology and Immunology Graduate Program, Wright State University, 2019. Enhanced expression of receptor tyrosine kinase Mer (MERTK) on SOCS3-treated polarized RAW 264.7 anti-inflammatory M2c macrophages. Macrophages are phagocytic cells located in tissues, organs and even circulated within our body as white blood cells. They are critical in detecting tissue damage and infection. Resident tissue macrophages initiate the signals for inflammation recruiting neutrophils and blood monocytes which mature into macrophages at sites of infection and in the resolution of inflammation. Based on the local cytokine milieu in tissue sites, macrophages may be polarized into pro-inflammatory M1 or anti-inflammatory M2 phenotypes. Receptor tyrosine kinase Mer (MERTK) helps in clearing dead neutrophils and other apoptotic cells from damaged tissue sites preventing chronic inflammation and autoimmune disorders. MERTK aids in the maintenance of tissue homeostasis and wound healing. Phosphatidylserine (PtdSer) present on the surface of apoptotic cells release “eat me” signals which are recognized by the two “bridging ligands” of MERTK receptor, Gas6 and ProS. The binding of the ligands to PtdSer initiates intracellular signals leading to phagocytosis of the cell. MERTK receptor is expressed mostly on M2c macrophages. The current study explores the expression rate of the phagocytic receptor MERTK, on macrophages polarized with either IL-10 (M2c ells) or IL-4 or IL-13 (M2a macrophages) following treatment with the suppressor of cytokine signaling SOCS3 in comparison with macrophage polarization with only IL-10 or IL-4 or IL-13 . The current study exhibits an enhancement in the expression of the phagocytic MERTK receptor on the surface of IL-10 polarized M2c macrophage when treated with SOCS3 in comparison to IL-10 polarized M2c macrophage, IL-4 polarized M2a macrophage and IL- iii 13 treated M2a macrophage. IL-13 polarized M2a macrophage also shows an increase in the expression of MERTK receptor which is similar to a previous study where a similar receptor to MERTK termed “Axl receptor” is enhanced by IL-13 treatment on bone- marrow derived macrophage. SOCS3 when treated with IL-13 polarized M2a macrophage acts as a negative regulator of MERTK receptor by decreasing the expression contrasting to the effect of SOCS3 on IL-10 which enhances the expression. Based on these observations, future research will involve co-culturing SOCS3 polarized M2 macrophages with apoptotic cells such as N2a neuroblastoma cells. iv TABLE OF CONTENTS INTRODUCTION .............................................................................................................. 1 LITERATURE REVIEW ................................................................................................... 3 1.1 Macrophage subtypes ................................................................................................ 3 1.2 Macrophage polarization .......................................................................................... 4 1.3 Anti-inflammatory cytokines .................................................................................... 5 1.4 Phagocytosis by macrophages .................................................................................. 6 1.5 Receptor tyrosine kinase Mer (MERTK) .................................................................. 7 1.6 Suppressor of cytokine signaling (SOCS) .............................................................. 11 1.7 SOCS1 and SOCS3 ................................................................................................. 12 1.8 Effect of SOCS3 on macrophages .......................................................................... 12 MATERIALS AND METHODS ...................................................................................... 14 2.1 Cell lines ................................................................................................................. 14 2.2 Macrophage polarization treatment ........................................................................ 14 2.3 Cell counting ........................................................................................................... 15 2.4 Flow cytometry (Cell Surface Staining) ................................................................. 16 2.5 Immunofluorescence staining ................................................................................. 17 2.6 Cell mounting for viewing………………………………………………………...17 2.7 Slide observation under fluorescence microscope .................................................. 18 2.8 Selecting pictures using Infinity Capture ................................................................ 18 2.9 Measuring corrected total cell fluorescence (CTCF) .............................................. 18 2.10 Statistical Significance .......................................................................................... 22 v RESULTS ......................................................................................................................... 23 FIGURES .......................................................................................................................... 26 DISCUSSION ................................................................................................................... 36 FUTURE RESEARCH ..................................................................................................... 39 REFERENCES ................................................................................................................. 41 SUPPLEMENTAL FIGURES …………………………………………………………. 44 vi LIST OF FIGURES Figure 1: Structure of MERTK receptor ............................................................................. 8 Figure 2: The ligands of the MERTK receptor Gas6 and Protein S ................................... 9 Figure 3: Phosphatidyl serine (PtdSer) on the surface of apoptotic cell acts as an “eat me” signal which binds to the “bridging ligands” of MERTK receptor Gas6 and Protein S ... 10 Figure 4: Layout of the current study ............................................................................... 13 Figure 5: (a) Measuring fluorescence using ImageJ. ........................................................ 20 (b) Measuring background noise by selecting four areas around each cell ...................... 21 Figure 6: Expression of MERTK receptor between IL-10 polarized M2c macrophages, IL- 13 polarized M2a macrophages and IL-4 polarized M2a macrophages at 24 hours using flow cytometry .................................................................................................................. 26 Figure 7: Flow histogram displaying a positive shift in the expression of MERTK receptor between IL-10 treated M2c macrophage (left) and untreated M0 macrophage as control (right) ................................................................................................................................ 27 Figure 8: Flow histogram displaying a positive shift in the