Enhanced Expression of Receptor Tyrosine Kinase Mer (MERTK) on SOCS3-Treated Polarized RAW 264.7 Anti-Inflammatory M2c Macrophages

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Enhanced expression of receptor tyrosine kinase Mer (MERTK) on SOCS3-treated polarized RAW 264.7 anti-inflammatory M2c macrophages

Sankhadip Bhadra Wright State University

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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

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-

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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.

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

RESULTS ...... 23

FIGURES ...... 26

DISCUSSION ...... 36

FUTURE RESEARCH ...... 39

REFERENCES ...... 41

SUPPLEMENTAL FIGURES …………………………………………………………. 44

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 expression of MERTK receptor between IL-13 treated M2a macrophage (left) and untreated M0 macrophage as control

(right) ...... 28

Figure 9: Dynamic nature of M2 macrophages exhibited during expression of MERTK receptor between IL-4 polarized M2a macrophages and IL-13 polarized M2a macrophages at 24 hours using flow cytometry ...... 29

Figure 10: Immunofluorescence staining using green fluorescence protein (GFP) filter for expressing MERTK receptor on SOCS3 treated polarized IL-10 M2c macrophage, SOCS3

vii treated M0 macrophage and IL-10 polarized M2c macrophage after 24 hours ...... 31

Figure 11: Immunofluorescence staining using green fluorescence protein (GFP) filter for expressing MERTK receptor on SOCS3 treated IL-10 polarized M2c macrophage, SOCS1 treated M0 macrophage and SOCS3 treated M0 macrophage after 24 hours ...... 32

Figure 12: Immunofluorescence staining using green fluorescence protein (GFP) filter for expressing MERTK receptor on IL-10 polarized M2c macrophage, SOCS1 treated M0 macrophage and SOCS3 treated M0 macrophage after 24 hours ...... 33

Figure 14: Immunofluorescence staining using green fluorescence protein (GFP) filter for expressing MERTK receptor on SOCS3 treated IL-10 polarized M2c macrophage, SOCS3 treated IL-13 polarized M2a phenotype and SOCS1 treated IL-10 polarized M2c macrophage after 24 hours ...... 35

Figure 15: Expression of MERTK receptor is enhanced by SOCS3 addition in IL-10 polarized M2c macrophage, but not when added to IL-13 polarized M2a macrophage .. 38

Supplemental Figure 1. Immunofluorescence staining using green fluorescence protein

(GFP) filter for comparing the expression of MERTK receptor on SOCS3 treated IL-10 polarized M2c macrophage (a), SOCS3 treated M0 macrophage (b), and IL-10 polarized

M2c macrophage (c) after 24 hours ...... 44

Supplemental Figure 2. Immunofluorescence staining using green fluorescence protein

(GFP) filter for comparing the expression of MERTK receptor on SOCS3 treated IL-10 polarized M2c macrophage (a), SOCS3 treated IL-13 polarized M2a macrophage (b), and

viii

SOCS1 treated IL-10 polarized M2c macrophage (c) after 24 hours ...... 45

Supplemental Figure 3. The dynamic nature of M2 macrophages were exhibited during immunofluorescence staining using green fluorescence protein (GFP) filter for comparing the expression of MERTK receptor on IL-13 polarized M2a macrophage (a) and IL-4 polarized M2a macrophage (b) after 24 hours ...... 46

LIST OF TABLES

Table 1: Cytokines and SOCS protein concentrations used for the current study ...... 15

Table 2: Primary antibody and isotype concentrations used for the current study ...... 17

Table 3: Corrected Total Cell Fluorescence (CTCF) values for different macrophage polarized treatments using immunofluorescence staining ...... 30

LIST OF ABBREVIATIONS

ATCC = American Type Culture Collection

CD = Cluster of differentiation

CRT = Calreticulin

CSB = Cell Staining Buffer

CTCF = Corrected Total Cell Fluorescence

DMEM = Dulbecco’s Modified Eagle Medium

EGF = Epidermal Growth Factor

FITC = Fluorescein isothiocyanate

FNIII = Fibronectin Type III

Gas6 = Growth arrest-specific 6

GFP = Green fluorescent protein

Gla = Gamma carboxyglutamic acid

GM-CSF = Granulocyte-macrophage colony-stimulating factor

IFN-g = Interferon gamma

IgL = Immunoglobulin lambda

IL = Interleukin iNOS = Inducible nitric oxide synthase

JAK = Janus kinase

KIR = Kinase Inhibitory Region

KLF-2 = Kruppel-like Factor 2

LG = Laminin G

LPS = Lipopolysaccharide

LTA = Lipoteichoic acid

M1 = Pro-inflammatory macrophage

M2 = Anti-inflammatory macrophage

M-CSF = Macrophage colony-stimulating factor

MERTK = Receptor tyrosine kinase Mer

NF-kb = Nuclear factor-kappa beta

PBS = Phosphate buffered saline

PPAR-g = Peroxisome proliferator-activated receptor gamma

Pro S = Protein S

PtdSer = Phosphatidylserine

RAW 264.7 = Murine macrophage cell line

SH2 = Src Homology 2

SOCS = Suppressor of cytokine signaling

SR-A = Scavenger receptor-A

STAT = Signal transducers and activators of transcription

TAM = Tyro3, Axl, Mer

TGF-b = Transforming growth factor-beta

Th1 / Th2 = Type I T helper / Type 2 T helper

TKD = Tyrosine Kinase Domain

TLR = Toll-like receptor

TNF-a = Tumor Necrosis Factor-alpha ng/mL = Nanograms per milliliter

µM = Micromolar

xii

HYPOTHESIS

Increased expression of MERTK receptor is seen on anti-inflammatory M2c macrophages polarized with IL-10 when compared to treatment with M2a macrophages using flow cytometry.

The addition of SOCS3 to IL-10 enhances the expression of MERTK receptor when compared to SOCS3 alone, whereas on the other hand SOCS1 does not affect the expression of MERTK receptor.

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ACKNOWLEDGEMENTS

Firstly, I would like to dedicate my work to Mom and Dad for their constant support, backing and trust on me and my field of interest. I wouldn’t have been able to love what I am doing without the freedom provided to me by them.

I am privileged to work under my advisor and guide Dr. Nancy J. Bigley, who opened her laboratory and played a significant role in shaping this study. Her vast knowledge in the field of immunology and her passion for research has motivated me throughout the time spent in working under this project. A big thank you to my committee members Dr. Dawn

P. Wooley and Dr. Marjorie M. Markopoulos for their suggestions and feedback on my report.

The study would not be completed without my current and former lab colleagues, who have trained me in learning new techniques, helped me when I got stuck in between experiments and provided me with valuable inputs while writing my thesis. A big high-five to all my friends who constantly encouraged and pushed me during difficult times and whenever laziness stepped in between.

I cannot end without thanking all my teachers from school and college who have always imparted knowledge, dealt patiently with all my doubts and queries and instilled confidence in me throughout.

I am grateful to Wright State University for giving me the golden opportunity to study abroad and providing an environment where I enjoyed conducting my research.

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INTRODUCTION

Capan (2017) showed an increase in phagocytosis of N2a tumor cells by the M1 phenotype of murine RAW 264.7 macrophages following treatment of the macrophages with SOCS1 peptide mimetic. There was no such enhancement in IL-10 polarized M2 macrophages. In contrast, SOCS3 treatment increased phagocytosis of the tumor cells by IL-10 polarized

M2 macrophages of the tumor target cells in which the calreticulin (CRT) “eat-me” signal was blocked by anti-CRT. CRT is an “eat me” signal present on the surface of apoptotic cells. Consequently, Madhkhali (2019) examined the effects of SOCS3 on two populations of M2a-polarized macrophages for phagocytosis of the N2a tumor cells treated with anti-

CRT. First, she noted insignificant increases in the phagocytic ability of SOCS1-treated

M2a and M2c macrophages for the anti-CRT-treated tumor cell N2a cells, while SOCS3 treatment of these same cells upregulated the phagocytic capacity of these macrophage phenotypes cells for N2a cells.

This current study is an extension of the observations of Capan (2017) and Madhkhali

(2019) and focused on the SOCS peptide mimetics effect on the expression of MERTK receptor on M2 macrophage phenotypes (Figure 4). IL-10 polarized M2c and IL-4/IL-13 polarized M2a macrophage phenotypes were used. Because Nishi et al. (2014) showed that the MERTK “eat-me” signal on macrophages was enhanced by phosphorylation upon contact with apoptotic cells, we wanted to determine whether SOCS3 peptide could also increase expression of MERTK by immunofluorescence. This seems likely because Zhang et al. (2016) showed that blocking MERTK expression on RAW 264.7 macrophages also

1 suppressed expression of SOCS3. Immunofluorescence led to comparison on the upregulation of MERTK receptor expression when treated with SOCS3 and SOCS1 along with the polarizing other cytokines IL-10, IL-4 and IL-13.

LITERATURE REVIEW

1.1 Macrophage subtypes

The fight of the immune system against bacterial and viral pathogens involve phagocytic cells which recognize, bind and engulf pathogens, leading to their clearance from the immune system. Innate immunity is the first line of defense against any pathogen- associated infection which leads to inflammation initiating healing process in the body against tissue damage and injuries.

It has been a century since the discovery of macrophages by Nobel Prize in Medicine winner Ilya Metchnikoff. Macrophages play a significant role in regulating the innate immune response (Saqib et al., 2018). They are able to differentiate between apoptotic, necrotic and viable cells without triggering inflammation and immune responses (Gordon et al., 2018). The apoptotic nature of the macrophages plays a major role in the development of organs, tissue remodeling, and post-injury cell replacement (Gordon et al.,

2018). The generation of specific tissue and microbial stimuli to phagocytose materials affects the heterogeneous nature of mononuclear phagocytes in tissues (Gordon et al.,

2018). They are a type of phagocytic cell which can be differentiated into proinflammatory macrophage (M1) and anti-inflammatory macrophage (M2) based on their reactions to different growth factors and cytokines (Xu et al., 2006).

1.2 Macrophage polarization

Arginine metabolism led to the differentiation between M1 and M2 macrophages in T helper type 1 (Th1) (C57BL/6) and T helper type 2 (Th2) (Balb/c) strains of mice (Mills et al., 2000). Classical M1 activation is stimulated by IFN-g and TLR ligands whereas alternative M2 macrophages are activated by IL-10, IL-4 and IL-13 (Mantovani et al.,

2012). Different stimuli and transcriptional factors led to the induction of different subtypes of M2 macrophages namely M2a, M2b, M2c and M2d (Mantovani et al., 2004). M2a subtype is triggered by IL-4, IL-13 and infections caused by fungus and helminth. IL-10,

IL-13, IL-4, dexamethasone and macrophage colony stimulating factor (M-CSF) stimulates M2 macrophages (Myers et al., 2019). IL-10, TGF-b and glucocorticoids triggers M2c whereas immune complexes and LPS generates M2b subtype.

Pro-inflammatory cytokines are produced by M1 macrophages and they have characteristics involving microbicidal properties, pathogenic resistance, and Th1 responses. M1 macrophages cause inflammation which is deleterious to health.

M2 macrophages involves enhanced phagocytic activity, tumor production, tissue remodeling, and parasitic control. The M2 macrophages are polarized into anti- inflammatory cytokines like interleukin-10 (IL-10), which assist M2 in engulfing and clearing the apoptotic cell (Xu et al., 2006). The interaction between apoptotic cells and phagocytes are based on three signals, “find me”, “eat me”, and “don’t eat me”. Previous analysis and reports suggested that phagocytosis displayed an anti-inflammatory response through upregulation of CD 163 and CD206 receptors (Gordon et al., 2018). The maintenance of immune system under normal physiological conditions is termed as

“immune homeostasis” which can be disrupted if the M1-M2 balance in the body is not

4 regulated. Beside homeostasis, macrophages control tissue remodeling and metabolism.

The damaged cells are termed as “apoptotic cells” which need to be cleared from the body to maintain immune homeostasis; the M2 macrophage plays a crucial role in engulfing these dead cells, preventing inflammation and formation of auto-immune diseases (Xu et al., 2006).

Xu et al., 2006 hypothesized that an anti-inflammatory phagocytic subset is responsible for recognizing and clearing the apoptotic cell, preventing autoimmunity and maintaining immune homeostasis. The macrophage colony stimulating factor (M-CSF) driven M2 has anti-inflammatory property producing IL-10 and granulocyte-macrophage CSF (GM)-CSF driven M1 has pro-inflammatory properties producing IL-6 and TNF-a. The authors suggest that IL-10 producing M2 macrophages are involved in the apoptotic cell recognition and clearance process.

The diverse and flexible nature of monocyte-macrophage lineage cells marks the authentic nature of these cells (Funes et al., 2018).

1.3 Anti-inflammatory cytokines

Parasitic infection and allergies trigger Type 2 immunity in tissues which leads to an initial response at mucosal barriers, which is then amplified and activated by effector cells followed by tissue repair mechanisms (Dyken et al., 2013). Type 2 immunity shifts from proinflammatory cytokine and nitric oxide production to anti-inflammatory molecules and tissue repair mechanisms through IL-4 and IL-13.

IL-13, a T-cell derived cytokine (Th2 type), along with IL-4, controls and operates the immune response (Elsner et al., 2011). The morphology, phenotype, and functioning of

5 monocytes are controlled in a similar way by both IL-13 and IL-4 (Lomo et al., 1997). It also plays a primary role in some chronic inflammatory diseases like asthma and ulcerative colitis (Elsner et al., 2011). Beside the similarities between these two cytokines, there are some differences. IL-13 is inactive on T cells due to the absence of receptors on these cells.

IL-4 is primarily generated by helper TH2 subset, whereas all three T helper subsets TH0,

TH1, and TH2 release IL-13 (Lomo et al., 1997). Janus kinases JAK1 and JAK3 proteins are activated by IL-4 but IL-13 can only activate JAK1 (Lomo et al., 1997).

Type II IL-4 receptor contains IL13Ra1, made up of dimers IL4Ra and IL13Ra1 chain to which both IL-4 and IL-13 binds (Elsner et al., 2011). IL-4 also binds to Type I receptor but IL-13 is solely dependent on IL13 Ra1 (Elsner et al., 2011). Hence, it has been proved that the binding sites of IL-13 and IL-4 shares a common signaling subunit.

1.4 Phagocytosis by macrophages

These Type II receptors phosphorylate and activates Janus tyrosine kinases (JAK) proteins which are bound to these receptors in unstimulated cells. Phosphorylated JAK proteins phosphorylates IL4Ra chain which binds, dimerizes, and activates STAT6 which translocate to the nucleus regulating specific transcription.

The action of CD103+CD11b+ and CD11b+ macrophages on apoptotic intraepithelial cells enhances the expression of growth arrest specific protein 6 (Gas6) and MERTK genes

(Gordon et al., 2018). The transcription factor Kruppel-like factor-2 (KLF2) regulates the expression of genes responsible for phagocytosis by macrophages (Gordon et al., 2018).

CX3CR1+CD64+MERTK+CD11c+ resident macrophages are the major efferocytic cells in the T zone of lymph nodes.

The cells on the verge of death trigger signals inducing mitosis and mutual interaction with macrophages (Gordon et al., 2018).

1.5 Receptor tyrosine kinase Mer (MERTK)

The family of receptor tyrosine kinase (TAM) removes apoptotic cells through efferocytosis. These receptors polarize macrophages to an alternatively-active form of M2 phenotype (Myers et al., 2019). The immunoregulatory functions of the tyrosine receptor kinase family (Tyro3, Axl and MERTK) in macrophages and dendritic cells assist in phagocytosis and clearance of apoptotic cells. The injured tissues could be repaired by macrophages through an apoptotic ligand detected by MERTK and Axl.

Mer receptor tyrosine kinase (MERTK) receptor was originally derived from v-ryk oncogene of avian retroviruses and was placed under the Axl receptor family (Myers et al.,

2019). MERTK is also mentioned as MER, RP38, c-Eyk, c-mer and Tyro12 in several contexts (Myers et al., 2019). MERTK receptor plays a major role in efficiently removing dead neutrophils and maintaining homeostasis during IL-10 production to prevent the formation of chronic inflammation and autoimmunity (Zizzo et. al, 2018). The receptor performs dual role in binding the apoptotic cells to the surface of the macrophages and engulfing the cells (Figure 1). There are two specific ligands Gas6 and Protein S which activates the receptors of the TAM family (Tyro3, Axl and MERTK). Phosphatidylserine

(PtdSer) located on the plasma membrane of the apoptotic cell acts as an “eat me” signal when the amino terminal Gla domain of Gas6 and Protein S binds to it (Dransfield et al.,

2015) (Figure 3). The “eat me” signal generated by the apoptotic cell is recognized and then internalized by macrophages (Dransfield et al., 2015). The autocrine production of

Gas6 ligand of MERTK receptor by M2c polarized cells stimulates the production of IL-

10. Studies on murine macrophages have observed a synergistic interaction of MERTK receptor with other receptors involved in phagocytosis like avb5 integrin, scavenger receptor A (SR-A) and C1q (Zizzo et. al, 2018).

Immunoglobulin-like repeats (IgL) External Fibronectin Type III (FNIII)

Cell Surface Plasma membrane

Tyrosine kinase domain (TKD) Internal

Structure of MerTK receptor

Figure 1: Structure of MERTK receptor. The receptor contains two immunoglobulin- like repeats domain (IgL) and two fibronectin type III repeats domain (FNIII) exposed on

Gla EGF-like LG-like the outer surface of the macrophage. The tyrosine kinase domain (TKD) is embedded in

the cytoplasm. Adapted from Myers,MerTK K. V., Amend, S. R., & Pienta, K. J. (2019).MerTK Targeting MerTK MerTK

Tyro3, Axl and MIL-10ERTK (TAM receptors): implications for macrophages in the tumor M2c SOCS3 M2c microenvironment. Molecular cancer, 18(1), 94.

MerTK MerTK

IL-13 M2a SOCS3 M2a

Immunoglobulin-like repeats (IgL) External Fibronectin Type III (FNIII)

Cell Surface Plasma membrane

Tyrosine kinase domain (TKD) Internal

Structure of MerTK receptor

Gla EGF-like LG-like

MerTK MerTK MerTK Figure 2: The ligands of the MERTK receptor Gas6 and Protein S contains a single MerTK

IL-10 domain of g-carboxyglutamicM2c acid (Gla), four domainsSOCS3 of epidermal growth factor repeatsM2c

(EGF) and two domains of laminin G (LG) like domains. Adapted from Myers, K. V.,

Amend, S. R., & Pienta, K. J. (2019).MerTK Targeting Tyro3, Axl and MERTK (TAM receptors): MerTK implications forIL-13 macrophages in the tumor microenvironment. Molecular cancer, 18(1), M2a SOCS3 M2a 94.

The “two bridging ligands” Gas6 and Pro S of MERTK receptor shares structural similarity. They comprise of an amino terminus γ-carboxyglutamic acid (Gla) domain followed by four epidermal growth factor (EGF) like domain repeats and two laminin G

(LG) like domain at the carboxy terminus end (Myers et al., 2019) (Figure 2).

Apoptotic cell PtdSer Gas6 Gas6

MerTK "bridging"ligands ProS ProS

Macrophage TKD TKD

Intracellular signals

Phagocytosis

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. The “eat me” signal is transported from the outer environment to the cytoplasm of the macrophage by the cytoplasmic domain tyrosine kinase (TKD) which leads to phagocytosis. Adapted from Myers, K. V., Amend, S. R., & Pienta, K. J. (2019). Targeting

Tyro3, Axl and MERTK (TAM receptors): implications for macrophages in the tumor microenvironment. Molecular cancer, 18(1), 94.

Phosphatidylserine (PtdSer) located in the lipid membrane of the apoptotic cell serves as an “eat me” signal to the macrophages through high affinity binding to the Gla domain of

Gas6 and Pro S ligand (Myers et al., 2019). The LG domain is bound to the TAM receptors.

The binding of the ligands to PtdSer is followed by receptor dimerization and the tyrosine kinase domain (TKD) is auto-phosphorylated (Myers et al., 2019). This triggers the intracellular signaling cascades of the macrophage initiating phagocytosis of apoptotic cells.

1.6 Suppressor of cytokine signaling (SOCS)

Polarization of M1 and M2 macrophages are controlled by a family of intracellular cytokine activated proteins termed SOCS. The SOCS family consists of eight proteins

(SOCS1-SOCS7 and CIS). Resting macrophages has lower expression of these proteins and are enhanced upon activation (Wilson, 2014). The SOCS family of proteins are structurally organized into three domains which includes a variable amino terminal domain, a carboxy-terminal box motif, and Src homology 2 (SH2) domain (Wilson, 2014).

SOCS proteins comprise a region of seventy amino acids termed as the SOCS box followed by a SH2 domain. SOCS1 and SOCS3 has an additional twelve amino acid extended SH2 domain called the Kinase Inhibitory Region (KIR) (Qasimi et al., 2006).

Cytokines, toll-like receptors (TLR), and immune complexes are common activators and inducers of both macrophages and SOCS protein. The interaction between phosphorylated

11 tyrosine residues with JAK proteins or cytokine receptors negatively regulates JAK/STAT signaling by SOCS proteins (Wilson, 2014). The kinase inhibitory region (KIR) present on

SOCS1 and SOCS3 proteins leads to direct suppression of JAK tyrosine kinase activity

(Wilson, 2014).

1.7 SOCS1 and SOCS3

The inhibition of IFN-g induced JAK2/STAT1 pathway and TLR/NF-kb signaling modulates M1 macrophage activation by SOCS1 (Wilson, 2014). Expression of SOCS1 is enhanced within a M2 polarizing habitat in vitro and in vivo whereas SOCS3 is upregulated in a M1 polarizing niche in vivo (Wilson, 2014). SOCS1 controls the iNOS/Arginase I expression ratio to counteract the immune responses during sudden changes within the microenvironment (Wilson, 2014).

SOCS3 expression is upregulated in pro-inflammatory M1 macrophage surroundings in vivo. They regulate TLR4 signaling and activates M1 macrophages by inhibiting STAT3 activation (Wilson, 2014). Treatment of macrophages with lipopolysaccharide (LPS) produces IL-10, IL-6, and TGF-b enhancing SOCS3 expression (White et al., 2011).

1.8 Effect of SOCS3 on macrophages

SOCS3 protein enhances the expression of receptor tyrosine kinase Mer (MERTK) which helps in increasing the phagocytic nature of M2 macrophages (Zhang et al., 2016). Reports suggest that lipoteichoic acid (LTA) induced activation of MERTK receptor through TLR2 signaling in RAW 264.7 macrophages upregulated the expression of SOCS3 (Zhang et al.,

2016).

IFN-y

20 ng/mL M1 M2

pro-inﬂammatory/ anti-inﬂammatory/ classically activated alternatively activated

IL-4

M2a M2b M2c M2d

20 ng/mL 20 ng/mL IL-10 IL-13

SOCS3 M2a SOCS3M2c

35 micromolar IL-10 IL-13

MerTK MerTK MerTK MerTK SOCS3 SOCS3 M2a M2c

Phagocytosis of apoptotic cells

Figure 4: Layout of the current study. M0 macrophages were polarized into cytokine treated M1 (IFN-g) and M2 (IL-10, IL-4 and IL-13) macrophages. Addition of SOCS3 leads to contrasting effects on the expression of MERTK receptors on IL-13 polarized M2a and IL-10 polarized M2c macrophages.

MATERIALS AND METHODS

2.1 Cell lines

RAW 264.7 macrophage transformed cell lines from Abelson leukemia viruses were used in this project obtained from American Type Culture Collection (ATCC). This particular cell line was obtained from an adult male BALB/c mouse. These adherent cells were grown overnight in HyClone Dulbecco’s Modified Eagle Medium (DMEM) (Fisher Scientific) supplemented with 10% HyClone Fetal Clone II Serum (Fisher Scientific) on 25 cm2 and

75 cm2 sterile BioLite Cell Culture treated flasks (Fisher Scientific). The cultured flasks

0 were incubated in a humid environment with 5% carbon dioxide (CO2) at 37 C. The cells undergo multiple passages before they are ready for treatment and are split once they reach

70% confluency by scraping the surface of the flask using cell scrapers (Fisher Scientific).

2.2 Macrophage polarization treatment

The M0 RAW 264.7 macrophage cells were grown in 75 cm2 cell culture flasks and incubated overnight to approximately 70% growth confluency. The cells adhere to the surface of the culture flasks and were removed via cell scraper (Fisher Scientific), transferred to a 15 mL centrifuge tube, and centrifuged at 1700 revolutions per minute

(40C) for five minutes. The supernatant was removed using a vacuum tube and the pellets were re-suspended in 1 mL of 10% DMEM growth medium. The cells were counted using hemocytometer and were aliquoted in 25 cm2 culture flasks at a seeding density of 1.0 x

106 cells per flask. These M0 macrophage cells were polarized into M2c macrophages by treating them separately with IL-10, IL-4, & IL-13 (BioLegend) at a concentration of 20 ng/mL (Table 1). SOCS1-KIR (GenScript) and SOCS3-KIR (GenScript) peptide mimetics

14 were also used in the presence and absence of IL-4, IL-10, and IL-13 to measure the expression of MERTK receptor at a concentration of 35 µM (Table 1). The cells were incubated for 24 hours post-cytokine and SOCS1/3 treatment.

Table 1: Cytokines and SOCS protein concentrations used for the current study.

Treatment Concentration IL-10 20 ng/mL IL-13 20 ng/mL IL-4 20 ng/mL IFN-g 20 ng/mL SOCS1-KIR 35 µM SOCS3-KIR 35 µM

2.3 Cell counting

Untreated cells and treated cells with cytokines and SOCS1/3 were scraped from the culture flasks 24 hours post incubation, transferred to a 15 mL centrifuge tube and centrifuged at

1700 revolutions per minute (40C) for five minutes. The supernatant was removed by using a vacuum tube and the pellets were re-suspended in 1 mL of 10% DMEM growth medium.

The re-suspended cells were stained with trypan blue (Fisher Scientific) at a 1:2 ratio and added to a hemocytometer for cell counting and viability. The viable cells appeared translucent whereas dead cells were stained blue when observed under phase contrast microscope.

The following equation was used for cell count and cell viability:

Cell confluency = [(Number of cells in hemocytometer (5 boxes) / 5) x 250 x 2] / 1000

Cell viability = [Viable cells (translucent)/Total number of cells (dead and alive)] x 100

2.4 Flow cytometry (Cell Surface Staining)

Both treated (polarized) and non-treated (control) RAW 264.7 macrophage cells were scraped, centrifuged and checked for cell count and viability. Around two million cells were added to 1.5 mL centrifuge tubes and centrifuged at 11,000 revolutions per minute at

40C for six minutes. The supernatants were removed, and pellets were treated with 1 mL of Cell Staining Buffer (BioLegend). The cells were centrifuged at 2100 revolutions per minute at 40C for six minutes.

FITC anti-mouse MERTK (Mer) Antibody (BioLegend) and FITC Rat IgG2a, k Isotype

Control Antibody (BioLegend) were diluted at a ratio of 1:100 with Cell Staining Buffer

(BioLegend) (Table 2). These conjugated antibodies are light sensitive, so all further steps were performed in the dark. The pellets were treated with 100 µL of the diluted antibody and its isotype. The antibody treated cells were incubated in ice for fifteen minutes in the dark. The cells were centrifuged at 1100 revolutions per minute at 40C for six minutes. The supernatant was removed, and the cells were washed thrice with 1 mL of Cell Staining

Buffer (CSB) (BioLegend). After every wash, the cells were centrifuged at 2100 revolutions per minute at 40C for six minutes. The final pellets were resuspended in 500

µL of Cell Staining Buffer (CSB) and incubated for five minutes at room temperature. The cells were filtered through Falcon tubes and transferred to a fresh 1.5 mL centrifuge tube.

The samples were analyzed on Accuri C6 Flow Cytometer (BD Biosciences) and raw data was transformed into flow histogram in FCS Express 6 Flow Cytometry Software (De

Novo Software).

Table 2: Primary antibody and isotype concentrations used for the current study.

Antibody Concentration (Stock) Company

FITC anti-mouse MERTK 0.5 mg/mL BioLegend (Mer) FITC Rat IgG2a, k Isotype 0.5 mg/mL BioLegend Control

2.5 Immunofluorescence staining

20,000 cells were seeded per well, in a 12 well chamber slide along with the required treatment of IL-10, IL-4, IL-13, and SOCS1/3 on the cells. The total volume per well of the chamber slide was filled with 200 µL with 10% DMEM growth medium. The chamber slides were incubated for 24 hours in a humid environment with 5% carbon dioxide (CO2) at 370C. The cells adhered to the base and the media was aspirated the following day and washed thrice with 200 µL of chilled 1X HyClone Phosphate Buffer Saline (PBS) (GE Life

Sciences). The cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature and washed thrice with 200 µL of chilled 1X PBS. Cell Staining Buffer (CSB) was used as a blocking agent and 200 µL was added to the cells for 30 minutes and then washed thrice with 200 µL of chilled 1X PBS. FITC anti-mouse MERTK (Mer) Antibody

(BioLegend) were diluted at a ratio of 1:100 in PBS with 5% Fetal Bovine Serum (FBS).

The diluted antibody was added to each well and incubated in dark for an hour at 40C.

2.6 Cell mounting for viewing

The antibody solution was removed and washed twice with cold 1X PBS. The PBS was aspirated completely, the silicone wells were removed, and the slide was left to dry. Several

17 drops of VECTASHIELD Antifade Mounting Medium (Vector Laboratories) was added along the center of the slide. The coverslip was placed on top of the VECTASHIELD and allowed to harden for twenty minutes. The edges of the coverslip were sealed using nail polish and the slides were visualized using ACCU-SCOPE EXC-350 microscope with scope LED fluorescence illuminator.

2.7 Slide observation under fluorescence microscope

The slide is placed on the stage, fluorescent cells were viewed, and the field of interest was set under 10X objective lens. Once the cells are clearly visible, the objective lens was changed to 50X for higher resolution and better image under oil immersion. The turret must be set to 20 for better image. The wavelength must be changed based on the type of filter being used (DAPI=1, GFP=2 and Texas Red=3).

2.8 Selecting pictures using Infinity Capture

The selection of the best field view of fluorescent cells will transfer the picture from the eyepiece to the camera. The camera settings must be optimized along with the exposure time suitable for the cell sample and must be kept constant throughout all treatments. The picture will be saved using the option called “Still Image” in the Infinity Capture program.

2.9 Measuring corrected total cell fluorescence (CTCF)

Fiji (ImageJ) software was used for measuring fluorescence. Individual distinct cells or stacked cells in groups post immunofluorescence were selected using freehand selection tool (Figure 5a). The area mean gray value and integrated density were selected in “Set measurements” under the “Analyze” menu in ImageJ.

The values were displayed once we select “Measure”. The background noise has been negated by selecting four areas on an average around each cell of interest in the field of view and measuring the background fluorescence for accumulating accurate intensities

(Figure 5b).

Corrected Total Cell Fluorescence (CTCF) was calculated after recording all the values from our total field of view using the following formula (Table 3)-

CTCF = Integrated density – (Area of selected cell x Mean fluorescence of

background readings)

A graph is plotted and compared with different treatment groups.

(a)

Figure 5: (a) Measuring fluorescence using ImageJ. Individual cells (green) were selected using the freehand selection tool (yellow) and analyzed. Several parameters like area, mean and integrated density were measured. Stacked cells were selected all at once and the parameters measured.

(b)

(b) Measuring background noise by selecting four areas around each cell. Four areas

(yellow) around each cell were selected along with the parameters (area, mean and integrated density). In case of stacked cells, based on the number of individual cells present in the stack, the corresponding background areas were selected.

2.10 Statistical Significance

The statistical significance was calculated using One-Way ANOVA Sigma Plot 13.0. All experiments were performed in triplicates.

RESULTS

M2c macrophages and IL-13 polarized M2a macrophages shift towards an increase in the expression of MERTK receptor in comparison to IL-4 treated M2a macrophages in flow cytometry

Polarization of RAW 264.7 M0 macrophages after 24 hours with IL-10 and IL-13 followed by treatment with FITC anti-mouse MERTK (Mer) antibody (BioLegend) and FITC Rat

IgG2a, k Isotype Control Antibody (BioLegend) showed an increasing trend in the expression of MERTK receptor in polarized M2c macrophages (Figure 7) when compared to polarized IL-4 treated M2a macrophage (Figure 6). The increase was statistically non- significant.

Overnight incubation of both polarized M2c macrophage and polarized M2a (treated with

IL-13) macrophage expressed similar increase in the expression of MERTK receptors post treatment with FITC anti-mouse MERTK (Mer) antibody (BioLegend) and FITC Rat

IgG2a, k Isotype Control Antibody (BioLegend) (Figure 6).

IL-13 polarized M2a macrophages shift towards an increase of MERTK receptor expression in comparison to IL-4 polarized M2a macrophages in flow cytometry displaying the dynamic nature of macrophages

Following 24-hour treatment of M0 macrophages with IL-13 and IL-4 and addition of FITC anti-mouse MERTK (Mer) antibody (BioLegend) and FITC Rat IgG2a, k Isotype Control

Antibody (BioLegend), IL-13 polarized M2a macrophages showed an increasing trend in the expression of MERTK receptor (Figure 8 and Figure 9) than IL-4 polarized M2a macrophages . The increase was statistically non-significant.

Immunofluorescence green fluorescent protein (GFP) staining of M2c macrophages with SOCS3 significantly enhances the expression of MERTK receptor

SOCS3 treated IL-10 polarized M2c macrophages significantly enhanced the expression of MERTK receptor when compared to SOCS3 treated M0 macrophages and IL-10 polarized M2c macrophages (Figure 10, Supplemental Figure 1). The enhancement in the

MERTK expression increases the phagocytic nature of SOCS3 treated M2c macrophages.

There was a non-significant increase in the expression of MERTK receptor in SOCS3 treated M2c macrophages when compared to M2c macrophages.

SOCS3 treated M2c macrophages enhances the expression of MERTK receptor when compared to SOCS1 and SOCS3 treated M0 macrophages using immunofluorescence green fluorescent protein (GFP) staining

Immunofluorescence staining after 24-hour polarization treatment of M0 macrophages with SOCS3+IL-10, SOCS1 and SOCS3 treated M0 macrophages, enhanced an increase in MERTK receptor expression in SOCS3 treated M2c macrophages (Figure 11). SOCS1 negatively regulates the phagocytic nature of macrophages by decreasing the expression of

MERTK receptor (Figure 11).

GFP-Immunofluorescence stained M2c macrophages increases the expression of

MERTK receptor when compared to SOCS1 and SOCS3 treated M0 macrophages

The phagocytic ability of M2c macrophages are higher than SOCS1 and SOCS3 treated

M0 macrophages due to the higher expression of MERTK receptor in IL-10 treated M2c macrophages (Figure 12).

IL-13 polarized M2a macrophage significantly increased the expression of MERTK receptor when compared to IFN-g polarized M1 phenotype and IL-4 polarized M2a macrophages using immunofluorescence staining

Previous studies have observed an increase in Axl receptor with IL-13, which is structurally and functionally similar to MERTK receptor and belongs to the same family of TAM receptors. The current immunofluorescence study also displays a significant increase in the expression of MERTK receptor with IL-13 polarized M2a macrophages when compared to IFN-g treated M1 macrophages and IL-4 treated M2a macrophages (Figure 13,

Supplemental Figure 3).

SOCS3 negatively regulates the expression of IL-13 polarized M2a macrophages but significantly enhances the expression of IL-10 polarized M2c macrophages

Immunofluorescence staining of 24-hour polarized SOCS3 treated IL-10 M2c macrophages significantly enhanced the expression of MERTK receptor whereas SOCS3 treated IL-13 decreased the expression confirming the fact that SOCS3 only enhances the phagocytic capacity of IL-10 polarized M2c macrophages by increasing the expression of the phagocytic receptor MERTK (Figure 14, Supplemental Figure 2). SOCS1 treated IL-

10 polarized M2c macrophages also reduced MERTK expression acting as a negative regulator (Figure 14, Supplemental Figure 2).

FIGURES

Comparison of MERTK receptor expression between IL-10 treated M2c cells, IL-13 treated M2a cells and IL-4 treated M2a cells

10 Percentage of MERTK expression (%)

IL-4 M2a / IgG2a IL-13 M2a / IgG2a

IL-4 M2a / anti-MERTK Ab IL-10 M2c / anti-MERTKIL-10 AbM2c / anti-MERTKIL-13 AbM2a / anti-MERTK Ab

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. The black bar represents IL-10 polarized

M2c macrophages, IL-13 polarized M2a macrophages and IL-4 polarized M2a macrophages with FITC anti-mouse MERTK (Mer) primary antibody (BioLegend) and grey bars depicts treatment with FITC Rat IgG2a, k Isotype Control Antibody

(BioLegend). IL-10 polarized M2c macrophages show an increasing trend in the expression of MERTK receptor. However, both IL-10 polarized M2c macrophages and IL-

13 polarized M2a macrophages show similar expression of MERTK receptor.

FITC-IL-10-03.28.2018.c6 (sample a01.fcs) compensated 6 FITC-IL-10-03.28.2018.c6 (sample a02.fcs) compensated 1.1 x10 1.1 x106

5 8.2 x10 P1 68.53% 8.2 x105 P1 64.74% 5 5.5 x10 5

SSC-A 5.5 x10 SSC-A

5 2.7 x10 2.7 x105

0 3 4 10 10 0 FL1-A 103 104 FL1-A

FITC-IL-10-03.28.2018.c6 (sample a01.fcs) compensated FITC-IL-10-03.28.2018.c6 (sample a02.fcs) compensated 1910 2010 26.10% 14.76 %

1433 1508

955 1005 Count Count

478 503

0 0 1 2 3 4 5 6 7 1 2 3 4 5 6 7 10 10 10 10 10 10 10 10 10 10 10 10 10 10 FL1-A FL1-A

b a

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). (a) IL-10 treated M2c macrophages (left) and (b) untreated M0 macrophages (right) with FITC anti-mouse MERTK (Mer) primary antibody

(BioLegend) is shown in black whereas the same set of cells with FITC Rat IgG2a, k

Isotype Control Antibody (BioLegend) were shown in red.

FITC-IL-13-08.26.2018.c6 (sample a01.fcs) compensated FITC-IL-13-08.26.2018.c6 (sample a02.fcs) compensated 6 1.8 x106 1.8 x10

6 1.3 x106 1.3 x10

P1 P1 P2 P2 73.81% 5 49.77% 9 x105 78.28% 9 x10 69.14% SSC-A SSC-A

5 4.5 x105 4.5 x10

0 0 3 4 103 104 10 10 FL1-A FL1-A

FITC-IL-13-08.26.2018.c6 (sample a01.fcs) compensated FITC-IL-13-08.26.2018.c6 (sample a02.fcs) compensated 1689 Ungated- 34.85% 868 Ungated- 14.17% P2- 30.10% P2- 12.55%

1267 651

434

845 Count Count

217

422

0 1 2 3 4 5 6 7 10 10 10 10 10 10 10 0 FL1-A 1 2 3 4 5 6 7 10 10 10 10 10 10 10 FL1-A

b a

Figure 8: Flow histogram displaying a positive shift in the expression of MERTK receptor between IL-13 treated M2a macrophage (left) and untreated M0 macrophage as control (right). (a) IL-13 treated M2a macrophages (left) and (b) untreated M0 macrophages (right) with FITC anti-mouse MERTK (Mer) primary antibody

(BioLegend) is shown in black whereas the same set of cells with FITC Rat IgG2a, k

Isotype Control Antibody (BioLegend) were shown in red.

Comparison of MERTK receptor expression between IL-4 treated M2a cells vs IL-13 treated M2a cells

10 Percentage of MERTK expression (%)

IL-4 M2a / IgG2a IL-13 M2a / IgG2a

IL-4 M2a / anti-MERTK Ab IL-13 M2a / anti-MERTK Ab

Figure 9: Dynamic nature of M2 macrophages exhibited during expression of

MERTK receptor between IL-4 polarized M2a macrophages and IL-13 polarized

M2a macrophages at 24 hours using flow cytometry. The black bar represents IL-4 polarized M2a macrophages and IL-13 polarized M2a macrophages with FITC anti-mouse

MERTK (Mer) primary antibody (BioLegend) and grey bars depicts treatment with FITC

Rat IgG2a, k Isotype Control Antibody (BioLegend). IL-13 polarized M2a macrophages exhibit an increasing trend in MERTK expression when compared to IL-4 polarized M2a macrophages.

Table 3: Corrected Total Cell Fluorescence (CTCF) values for different macrophage polarized treatments using immunofluorescence staining. The CTCF values of the polarized macrophages were shown with respect to their binding with FITC anti-mouse

MERTK (Mer) primary antibody (BioLegend).

Macrophage treatments Total number of cells Corrected Total Cell (n) Fluorescence (CTCF) SOCS3+IL-10 polarized 40 106796.826 M2c IL-10 polarized M2c 43 73114.6853

SOCS3 treated M0 40 65930.5357

SOCS3+IL-13 polarized 51 38023.3569 M2a

SOCS1 treated M0 37 30440.079

IL-13 polarized M2a 60 166100.899

IL-4 polarized M2a 97 71250.1304

SOCS1+IL-10 polarized 65 45655.3833 M2c IFN-g polarized M1 68 59611.1139

Comparison of MERTK receptor expression between SOCS3+IL-10 treated M2c cells, SOCS3 treated M0 cells SOCS3+IL-10 M2c/anti-MERTK Ab and IL-10 treated M2c cells +++ , ++ , +

1.2e+5 * * SOCS3+M0/anti-MERTK Ab 1.0e+5 +++ , ++ , + 8.0e+4 * *

IL-10 M2c/anti-MERTK Ab 6.0e+4 +++ , ++ , +

4.0e+4

SOCS3+IL-10 M2c/IgG2a 2.0e+4 +++ , ++ , + Corrected Total Cell Fluorescence (CTCF) 0.0

SOCS3+M0/IgG2a +++ , ++ , +

S3+M0 / IgG2a (n=24)

IL-10 M2c / IgG2a (n=36) IL-10 M2c/IgG2a +++ , ++ , + S3+IL-10 M2c / IgG2a (n=41) S3+M0 / anti-MERTK Ab (n=40)

IL-10 M2c / anti-MERTK Ab (n=43) S3+IL-10 M2c / anti-MERTK Ab (n=40)

Figure 10: Immunofluorescence staining using green fluorescence protein (GFP) filter for expressing MERTK receptor on SOCS3 treated polarized IL-10 M2c macrophage, SOCS3 treated M0 macrophage and IL-10 polarized M2c macrophage after 24 hours. The black bar represents SOCS3+IL-10, SOCS3 and IL-10 polarized macrophages treated with FITC anti-mouse MERTK (Mer) primary antibody (BioLegend) and grey bars depicts treatment with FITC Rat IgG2a, k Isotype Control Antibody

(BioLegend). The stars on top of the bar graph shows significant increase in the expression of MERTK receptor. (All images above were captured at 500X oil magnification in fluorescent microscope, scale bar = 100 µm). (P<0.05 *) (n=total number of cells counted for fluorescence).

Comparison of MERTK receptor expression between SOCS3+IL-10 treated M2c cells, SOCS1 treated M0 cells SOCS3+IL-10 M2c/anti-MERTK Ab and SOCS3 treated M0 cells +++ , ++ , +

1.2e+5 * * * 1.0e+5 SOCS1+M0/anti-MERTK Ab +++ , ++ , + 8.0e+4 *

SOCS3+M0/anti-MERTK Ab 6.0e+4 +++ , ++ , +

4.0e+4

2.0e+4 SOCS3+IL-10 M2c/IgG2a

+++ , ++ , + Corrected Total Cell Fluorescence (CTCF) 0.0

SOCS1+M0/IgG2a +++ , ++ , +

S1+M0 / IgG2a (n=40) S3+M0 / IgG2a (n=24)

SOCS3+M0/IgG2a S3+IL-10 M2c / IgG2a (n=41) S1+M0 / anti-MERTK Ab (n=37) S3+M0 / anti-MERTK Ab (n=40) +++ , ++ , +

S3+IL-10 M2c / anti-MERTK Ab (n=40)

SOCS1 treated M0 macrophages with FITC anti-mouse MERTK (Mer) primary antibody

(BioLegend) and grey bars depicts treatment with FITC Rat IgG2a, k Isotype Control

Antibody (BioLegend). The stars on top of the bar graph shows significant increase in the expression of MERTK receptor. (All images above were captured at 500X oil magnification in fluorescent microscope, scale bar = 100 µm). (P<0.05 *) (n=total number of cells counted for fluorescence).

Comparison of MERTK receptor expression between IL-10 treated M2c cells, SOCS1 treated M0 cells IL-10 M2c/anti-MERTK Ab and SOCS3 treated M0 cells +++ , ++ , + 80000 * * * SOCS1+M0/anti-MERTK Ab +++ , ++ , + 60000

SOCS3+M0/anti-MERTK Ab 40000 +++ , ++ , +

20000

IL-10 M2c/IgG2a +++ , ++ , + Corrected Total Cell Fluorescence (CTCF) 0

SOCS1+M0/IgG2a +++ , ++ , +

S1+M0 / IgG2a (n=40) S3+M0 / IgG2a (n=24)

IL-10 M2c / IgG2a (n=36)

SOCS3+M0/IgG2a +++ , ++ , + S1+M0 / anti-MERTK Ab (n=37) S3+M0 / anti-MERTK Ab (n=40) IL-10 M2c / anti-MERTK Ab (n=43)

FITC anti-mouse MERTK (Mer) primary antibody (BioLegend) and grey bars depicts treatment with FITC Rat IgG2a, k Isotype Control Antibody (BioLegend). The stars on top of the bar graph shows significant increase in the expression of MERTK receptor. (All images above were captured at 500X oil magnification in fluorescent microscope, scale bar

= 100 µm). (P<0.05 *) (n=total number of cells counted for fluorescence).

Comparison of MerTK receptor expression between IL-13 treated M2a cells, IL-4 treated M2a cells IL-13 M2a/anti-MERTK Ab and IFN treated M1 cells +++ , ++ , +

1.8e+5 * 1.6e+5 IL-4 M2a/anti-MERTK Ab +++ , ++ , + 1.4e+5

1.2e+5

1.0e+5 IFN-y M1/anti-MERTK Ab +++ , ++ , + 8.0e+4

6.0e+4

4.0e+4

IL-13 M2a/IgG2a 2.0e+4 +++ , ++ , + Corrected Total Cell Fluorescence (CTCF) 0.0

IL-4 M2a/IgG2a +++ , ++ , +

IL-4 M2a / IgG2a (n=41) IFN-y M1 / IgG2a (n=49) IL-13 M2a / IgG2a (n=45) IFN-y M1/IgG2a +++ , ++ , + IL-4 M2a / anti-MERTK Ab (n=97) IFN-y M1 / anti-MERTK Ab (n=68) IL-13 M2a / anti-MERTK Ab (n=60)

Figure 13: Immunofluorescence staining using green fluorescence protein (GFP) filter for expressing MERTK receptor on IL-13 polarized M2a macrophage, IL-4 polarized M2a macrophage and IFN-g polarized M1 macrophage after 24 hours. The black bar represents IL-13 polarized M2a macrophage, IL-4 polarized M2a macrophage and IFN-g polarized M1 macrophage with FITC anti-mouse MERTK (Mer) primary antibody (BioLegend) and grey bars depicts treatment with FITC Rat IgG2a, k Isotype

Control Antibody (BioLegend). The stars on top of the bar graph shows significant increase in the expression of MERTK receptor. (All images above were captured at 500X oil magnification in fluorescent microscope, scale bar = 100 µm). (P<0.05 *) (n=total number of cells counted for fluorescence).

Comparison of MERTK receptor expression between SOCS3+IL-10 treated M2c, SOCS3+IL-13 treated M2a cells SOCS3+IL-10 M2c/anti-MERTK Ab and SOCS1+IL-10 treated M2c cells +++ , ++ , +

1.2e+5 * * SOCS3+IL-13 M2a/anti-MERTK Ab * +++ , ++ , + 1.0e+5

8.0e+4

SOCS1+IL-10 M2c/anti-MERTK Ab 6.0e+4 +++ , ++ , + *

4.0e+4

SOCS3+IL-10 M2c/IgG2a 2.0e+4 +++ , ++ , + Corrected Total Cell Fluorescence (CTCF) 0.0

SOCS3+IL-13 M2a/IgG2a +++ , ++ , +

SOCS1+IL-10 M2c/IgG2a S3+IL-10 M2c / IgG2a (n=41) S3+ IL-13 M2a/ IgG2a (n=23) S1+IL-10 M2c / IgG2a (n=87) +++ , ++ , +

S3+IL-10 M2c / anti-MERTK Ab (n=40) S1+IL-10 M2c / anti-MERTK Ab (n=65) S3+ IL-13 M2a / anti-MERTK Ab (n=51)

10 polarized M2c macrophage after 24 hours. The black bar represents SOCS3+IL-10 polarized M2c macrophage, SOCS3+IL-13 polarized M2a macrophage and SOCS1+IL-10 polarized M2c macrophage with FITC anti-mouse MERTK (Mer) primary antibody

(BioLegend) and grey bars depicts treatment with FITC Rat IgG2a, k Isotype Control

DISCUSSION

The trans-membrane receptor tyrosine kinase Mer (MERTK) present on the outer surface of macrophages is responsible for binding to the “eat me” signal, in this case phosphatidylserine (PtdSer) which are released by the apoptotic cell followed by engulfment of the cell. The current study focuses on the expression of the MERTK receptor on IL-10 treated M2c RAW 264.7 macrophages, IL-13 and IL-4 polarized M2a macrophages. IL-10 polarized M2c macrophages shows an increasing trend towards

MERTK expression. The results also demonstrate an enhancement in the expression of the receptor by addition of KIR-SOCS3 to M2c macrophages when compared with

SOCS3+IL-13 M2a macrophages (Figure 15). There is no increase observed in the

MERTK receptor expression on SOCS3 and SOCS1 treated M0 macrophages which suggest that SOCS3 protein acts as a negative regulator of the MERTK receptor unless in combination with IL-10 polarized M2c phenotype. SOCS1 on the other hand acts as a negative regulator of MERTK receptor expression when treated with M0 macrophage and with IL-10 polarized M2c phenotype.

Reports based on the current study showcase the dynamic, diverse, and plastic nature of macrophages as they readily switch from one phenotype to another when there are changes within the cytokine environment. The increase in the expression of MERTK receptor on

IL-13 polarized M2a macrophages but not when compared to IL-4 polarized M2a macrophage exhibit the fact that two distinct cytokines which are responsible for polarizing

RAW 264.7 macrophage into M2a phenotype have contrasting effects on the expression of

MERTK receptor exhibit the flexible nature of macrophages. The increase of MERTK receptor expression on IL-13 polarized M2a macrophage in the current study aligns with

36 reports involving murine bone marrow derived macrophages (BMDMs) showing an increase in the expression of Axl receptor which belongs to the same family as MERTK, in IL-13 and IL-4 polarized M2a phenotype when compared to LPS and IFN-g polarized

M1 macrophage (Myers et al., 2019). The MERTK-mRNA transcript levels also increases in IL-13 polarized M2a subtype in comparison to M1 phenotype (Myers et al., 2019).

In cancer immunotherapy, not much progress has been made with M2 macrophage polarization. M2 macrophages polarized by IL-10 helps in tumor progression as there is a tendency to polarize macrophages into the anti-inflammatory phenotype in a tumor environment (Weagel et al., 2015). The current study with SOCS3 enhancing the expression of the phagocytic receptor MERTK on IL-10 polarized M2c macrophage might act as the perfect combination of factors required for SOCS3+ MERTK+ M2c macrophages to phagocytose cancer cells and exhibit anti-cancer properties in vivo.

The lack of a significant increase in MERTK receptor expression statistically in flow cytometry is because of the asynchronous culture used during the study. The cell confluency on separate treatments had slight variations and the exact morphology and nature of the respective macrophage phenotype in culture based on their treatments were not finite. A small portion of the MERTK receptor called the tyrosine kinase domain (TKD) was present in the cytoplasmic region of the macrophage, hence the antibody in use could only detect the exposed region of the receptor.

The current study enhancing the expression of phagocytic receptor MERTK on

SOCS3+IL-10 M2c phenotype supports the data of Capan (2017) and Madhkhali (2019) on the positive impact of SOCS3 on IL-10 polarized M2c macrophages, increasing the

Immunoglobulin-like repeats (IgL) External Fibronectin Type III (FNIII)

Cell Surface Plasma membrane

Tyrosine kinase domain (TKD) Internal

Structure of MerTK receptor

phagocytosis of tumor cells following blockage of CRT “eat me” signal (Capan, 2017) and upregulating theGla phagocytic capacityEGF-like of macrophageLG-like for N2a cells (Madhkhali, 2019).

MerTK MerTK MerTK MerTK

IL-10 M2c SOCS3 M2c

MerTK MerTK

IL-13 M2a SOCS3 M2a

Figure 15: Expression of MERTK receptor is enhanced by SOCS3 addition in IL-10 polarized M2c macrophage, but not when added to IL-13 polarized M2a macrophage.

Hence, SOCS3 negatively regulates MERTK receptor expression in IL-13 polarized M2a macrophage, although IL-13 increases the receptor expression when treated with M0 macrophages.

FUTURE RESEARCH

Previous studies have suggested that macrophage receptor expression and cytokine secretion are affected in an apoptotic environment. Expression of the phagocytic receptor

MERTK on macrophages enhances uptake (phagocytosis) of nearby apoptotic cells releasing “eat-me” signals at their cell surfaces. Briefly, these “eat-me” signals increase the function of the MERTK ligands Gas6 and ProS on the macrophage to bridge with

PtdSer present on the apoptotic cell, leading to MERTK-receptor mediated phagocytosis.

Based on the results of this present study, in a future study, SOCS3 treated IL-10-polarized

MERTK-positive M2c macrophages should exhibit a higher phagocytic rate of tumor cells such as N2a neuroblastoma cells, when compared to macrophages treated with IL-10 or

IL-4 or IL-13 alone. The higher phagocytic rate of SOCS3+IL-10 polarized MERTK- positive M2c macrophages would help in cancer diagnostics, where the combination of

SOCS3+IL-10 might lead to engulfment of cancerous cells, which was not possible with

IL-10 treatment alone.

Although IL-10 polarized M2c macrophages shows a non-significant increase in MERTK expression by flow cytometry, IL-10 requires other factors like M-CSF and PPAR-g antagonists to enhance the MERTK expression than IL-10 alone. IL-10 along with M-CSF or PPAR-g would not only increase MERTK expression but might also increase the phagocytic rate around apoptotic cells. It would be interesting to observe the impact of

SOCS3 when treated with a combination of IL-10+MCSF or IL-10+PPARg in an apoptotic environment.

Another factor to consider is that the TKD portion of the MERTK receptor is embedded in the cytoplasm and not exposed on the macrophage cell surface and hence is not readily identified by flow cytometry. In the present study, immunofluorescent cytochemistry was able to differentiate the increase in expression of MERTK following cytokine treatments of IL-10, IL-13 and by SOCS3+IL-10 but not by SOCS3+IL-13. Since IL-13 induces expression of the Axl receptor similar in structure to MERTK, future studies should be directed to determining if Axl expression is not enhanced by SOCS3 treatment of the M2a phenotype whereas MERTK expression is enhanced by S0CS 3 treatment of M2 phenotype macrophages as shown in the present study.

M2a macrophages polarized with IL-13 might also exhibit have a phagocytic increase of

N2a cells and this increase could be reversed by treatment of these IL-13 polarized macrophages with SOCS3 because SOCS3 did not enhance expression of MERTK on IL-

13-treated M2a macrophages while it did on IL-10-treated M2c macrophages.

REFERENCES

Capan, C. (2017). Effects of SOCS1 and SOCS3 Peptide Mimetics on Macrophage

Phagocytosis of Malignant Cells. Wright State University.

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SUPPLEMENTAL FIGURES SOCS 3 + IL-10 M2c + FITC anti-MerTK Ab

+++ ++ +

SOCS 3 M0 + FITC anti-MerTK Ab CTCF: 320654.98 CTCF: 208509.77 CTCF: 185370.56 a +++ ++ +

IL-10 M2c + FITC anti-MerTK CTCF: 342102.158 CTCF: 257937.88 CTCF: 71293.746 b +++ ++ +

CTCF: 273001.57 CTCF: 197063.69 CTCF: 191519.48 c

Supplemental Figure 1. Immunofluorescence staining using green fluorescence protein (GFP) filter for comparing the expression of MERTK receptor on SOCS3 treated IL-10 polarized M2c macrophage (a), SOCS3 treated M0 macrophage (b), and IL-10 polarized M2c macrophage (c) after 24 hours. SOCS3 treated IL-10 polarized

M2c macrophages enhances the expression of MERTK receptor. SOCS3 alone negatively regulates MERTK receptor expression on M0 macrophages. (All images above were captured at 500X oil magnification in fluorescent microscope, scale bar = 100 µm).

(+++, ++, + = decreasing order of fluorescence). SOCS 3 + IL-10 M2c + FITC anti-MerTK Ab

+++ ++ +

CTCF: 320654.98SOCS 3 + IL-13 M2aCTCF: + 208509.77 FITC anti-MerTK Ab CTCF: 185370.56 a +++ ++ +

SOCS 1 + IL-10 M2c + FITC anti-MerTK Ab CTCF: 460082.011 CTCF: 44431.42 CTCF: 24038.439 b +++ ++ +

CTCF: 76420.8333 CTCF: 64225.9398 CTCF: 20730.612 c

Supplemental Figure 2. Immunofluorescence staining using green fluorescence protein (GFP) filter for comparing the expression of MERTK receptor on SOCS3 treated IL-10 polarized M2c macrophage (a), SOCS3 treated IL-13 polarized M2a macrophage (b), and SOCS1 treated IL-10 polarized M2c macrophage (c) after 24 hours. SOCS3 only enhances the MERTK receptor expression when combined with IL-10 polarized M2c phenotype. SOCS3 reduces the receptor expression when combined with

IL-13. SOCS1 negatively regulated MERTK receptor expression on IL-10 polarized M2c phenotype. (All images above were captured at 500X oil magnification in fluorescent microscope, scale bar = 100 µm). (+++, ++, + = decreasing order of fluorescence).

IL-13 M2a + FITC anti-MerTK

+++ ++ +

CTCF: 1533416.33 CTCF: 1122313.67 CTCF: 251364.354 IL-4 M2a + FITCa anti-MerTK

+++ ++ +

CTCF: 330320.281 CTCF: 305758.127 CTCF: 194068.01 b 46

(a) and IL-4 polarized M2a macrophage (b) after 24 hours. Both IL-13 and IL-4 polarizes M2a macrophage, but only IL-13 increases MERTK receptor expression and IL-

4 has no significant increase. This expresses the flexible nature of M2c macrophage phenotype. (All images above were captured at 500X oil magnification in fluorescent microscope, scale bar = 100 µm). (+++, ++, + = decreasing order of fluorescence).