Synergistic induction of macrophage tumoricidal activity

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Synergistic induction of macrophage tumoricidal activity

by

Sanjib Halder

Thesis for Master’s Degree in Molecular Bioscience 60 study points

Department of Molecular Biosciences

The Faculty of Mathematics and Natural Sciences

University of Oslo

April 2016

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Synergistic induction of macrophage tumoricidal activity

by

Sanjib Halder

Master Program in Molecular Biology Department of Molecular Biosciences

The Faculty of Mathematics and Natural Sciences University of Oslo

Master Thesis, 60 study points Supervised by Alexandre Corthay

Performed at the Department of Pathology Oslo University Hospital, Rikshospitalet

April 2016

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April 2016

Synergistic induction of macrophage tumoricidal activity.

Sanjib Halder

http://www.duo.uio.no/

Trykk: Reprosentralen, Universitetet i Oslo

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Acknowledgements

The work presented in this thesis was performed within the Tumor Immunology group, at the Department of Pathology, Oslo University Hospital (Rikshospitalet), during the period July 2014 to January 2016.

I would like to thank Alexandre Corthay for welcoming me to his group and being a fabulous supervisor. Thank you for your great guidance and advice which helped me to keep going with the work, even in time of frustration and stress. Thank you for your feedback during the lab work and writing process. You taught me how to work under pressure and think like a researcher.

Many thanks to Elisabeth Müller for helping me with the techniques of my work, for sharing her knowledge, tips and tricks. Also thanks to Kahsai Beraki, the senior research technician, for all the help and the technical experience he shared during my work and taking care of the cells and other matters.

I am grateful to Inger Øynebraten and Elisabeth Müller for critically reading this thesis even on their busy schedule. Thanks to Branislava Stankovic and Baiba Ķūrēna for their help to give feedback on my writings.

My gratitude goes to all the great members of the Tumor Immunology group to help me while I needed the help, thank you for being there to share thoughts and frustrations, for giving feedback on presentations. I thank you all for the support you gave me during all this time.

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Abbreviations

AP1 Activator protein 1

Ci Curie

CD Cluster of Differentiation CpG ODN CpG oligodeoxynucleotide CPM Counts per minute

CXCL C-X-C motif chemokines ligand DC Dendritic cell

DNA Deoxyribonucleic acid

h Hour

IFNγ Interferon gamma

Ig Immunoglobulin

IL Interleukin

iNOS Inducible nitric oxide synthase IRF Interferon regulatory factor LBP LPS –binding protein LLC Lewis lung carcinoma LPS Lipopolysaccharides LTA Lipoteichoic acid

MD-2 Myeloid differentiation protein 2 MHC Major histocompatibility complex MOPC Mineral oil induced plasmacytoma

MyD88 Myeloid differentiation primary response 88 MΦ Macrophage

NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells NK Natural killer

NO Nitric Oxide

NO2- Nitrite

PAMP Pathogen associated molecular pattern Poly(I:C) Polyinosinic-polycytidylic acid

PRR Pattern recognition receptor TAM Tumor associated macrophages TCR T cell receptor

TGF Transforming growth factor

TH T helper cell

TLR Toll-like receptor

TNFα Tumor necrosis factor alpha Treg T regulatory cell

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Abstract

Background: An important function of the innate immune system is immune surveillance of cancer which consists of detecting and eliminating transformed cells. Macrophages and dendritic cells (DCs) are two main types of innate immune cells involved in anti-tumor immunity.

Method: We used an in vitro growth inhibition assay to analyze the inhibited growth of cancer cells by activated macrophages or DCs. Nitric oxide (NO) production was quantified by Griess assay. Combination of cytokine interferon (IFN)-γ and various Toll-like receptor (TLR) ligands were tested as activators. The macrophage cell line J774.A1 and the DC cell line D2SC/1 were used as effector cells. Target cells were the MOPC315 plasmacytoma and the Lewis Lung carcinoma (LLC). The TLR4 ligand lipopolysaccharides (LPS) was used as a positive control.

Results: IFNγ or a TLR ligand alone was not sufficient to render macrophages or DCs cytotoxic to cancer cells. Macrophages and DCs were shown to require two signals to become tumoricidal and secrete NO. TLR ligands Pam3, Poly(I:C), flagellin, CL264, and CpG ODN were identified as molecules being able to render macrophages and DCs tumoricidal, when given in combination with IFN . Cancer cell killing may be mediated by NO secretion.

Conclusions: Induction of tumoricidal activity in macrophages and DCs requires two signals.

Five TLR ligands other than LPS were identified as being able to synergize with IFNγ to render macrophages and DCs cytotoxic to cancer cells. These molecular combinations may serve as a basis for the development of novel immunotherapy protocols for cancer.

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1 Introduction

1.1 The immune system

Defense against pathogens like viruses, bacteria and parasitic worms is a core function in all living organisms. A set of strategies, collectively called innate immunity, evolved in early life forms, designed to eliminate the invading microorganisms as a starting line of defense. The strategies differ among species, but all share several key features^{1, 2}. Sophisticated and more robust defense mechanisms are found in vertebrates which possess both innate and adaptive immune systems³.

Innate and adaptive immunity are interacting with each other in a fine-tuned manner to mediate effective defense mechanisms against infections. As the first line of defense, the innate immune system is activated by a small set of invariant pattern-recognition receptors (PRRs) which recognize pathogen-associated molecular patterns(PAMPs) from the invading microorganisms¹. PRR associates with the innate immune system to discriminate and recognize different classes of pathogens through PAMPs⁴. Components involved in the innate immune system are epithelial barriers (skin), phagocytic macrophages, dendritic cells, specialized lymphocytes like natural killer (NK) cells and several plasma proteins including the complement system^{2, 4, 5}(Fig. 1). Some cells of the innate immune system are good at processing and presenting the encountered pathogens (i.e. antigens) to other components of the immune system to initiate a strong response or help others to memorize the pathogens for future attacks⁶.

While the innate immune system provides a rapid response to the invading pathogens, it lacks specificity and memory potentials. The adaptive immune system comes to help when a pathogen reoccurs in the host, to defend these reoccurring pathogens the adaptive immune systems developed specific recognition and memory potentiality^{2, 6}. The adaptive immune system requires being educated before it can raise the immune response and eradicate the invaders. The innate immune system helps adaptive immune system to recognize and orchestrate a strong immune response to the reoccurring pathogens. This whole process of educating adaptive immune system by innate immune system provides a safeguard against unwanted activation of strong adaptive response⁴ (Fig. 1). The adaptive system does not response in a rapid way like the innate system do.

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Figure 1. Principal mechanisms of innate and adaptive immunity. The mechanism of innate immunity provides the initial defense against infections. Adaptive immune responses develop later and are mediated by lymphocytes and their products. The kinetics of innate and adaptive immune responses are approximations and may vary in different infections. Figure adapted from Abbas et al.²

1.2 Cancer immunosurveillance

Several mutations in oncogenes and tumor suppressor genes could lead normal cells to develop into cancerous cells by disrupting their homeostasis⁷. These mutated cells may express some danger signal which could activate the immune system. Paul Ehrlich was the first to form the concept that the immune system could repress the frequency of carcinomas, this was in 1909⁸. More than four decades later, Lewis Thomas and Macfarlane Brunet re- established the immunosurveillance concept^{9, 10} and Macfarlane proposed that long-lived animals have some mechanisms that prevent genetic changes leading to cancer development¹¹ Opposing concepts have also been proposed, such as the concept is given by Balkwill and Mantovani, suggesting that the inflammatory immune cells and cytokines present in the tumor may promote cancer cell growth¹². Recent works regarding this concept have shown that the surveillance by immune system may select the emergence of primary tumors with reduced immunogenicity capable of escaping immune recognition and destruction¹³.

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3 Many findings now point towards the host-protective and tumor-sculpting function of the immune system throughout the tumor development, and this forms the basis of the cancer immunoediting hypothesis¹⁴.

Cancer immunoediting is a dynamic process designed upon three distinct phases: elimination, equilibrium, and escape¹⁵. In the elimination phase (which is similar to immunosurveillance), the immune system is, able to detect genetic changes in the tumor and to eliminate tumor from the body. If tumor cells remain after the elimination phase, they can mutate into new variations which resist the attacks from the immune system. These dynamic changes in the tumor cells form new populations with less immunogenicity and stay in dormant condition due to the immune pressure. In the escape phase, the balance between the immune response and the tumor bias towards tumor growth, resulting from new variation of tumor or exhausted immunity helps tumor cells to avoid the immune pressure. Tumors gained progressive growth capability and can be detected clinically in the escape phase^{15, 16} (Fig. 2).

Recent reports also bring to light that the immune system applies various strategies to recognize and eliminate newly transformed cells. Different lymphocyte subsets like the cluster of differentiation 4⁺ (CD4⁺) and CD8⁺ T cells, γδ T cells, NK cells, NKT cells, and effector mechanisms such as IFNγ, perforin, and TNF-related apoptosis-inducing ligand (TRAIL) have been found to be critical for various types of malignancies^{13, 17}.

Corthay et al. (2005) showed that the tumor-specific CD4⁺ T cells could provide a primary antitumor immune response with the help of professional antigen presenting cells. Moreover, their findings also support the importance of T cell-derived IFNγ, which has a significant role in activating macrophages to become cytotoxic and to inhibit the tumor cells¹⁷.

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Figure 2. Cancer immunoediting. The process of cancer immunoediting is envisaged as three phases:

elimination, equilibrium and escape. The first phase, the elimination phase (formerly known as cancer immunosurveillance), consists of the recognition of transformed cells by the innate and the adaptive immune system, leading to the killing of these cells. Recognition by innate and adaptive immune mechanisms leads to direct killing of tumour cells, as well as production of chemokines and other cytokines that facilitate killing of tumour cells. If some tumour cells are not killed in the elimination phase, then the process can progress to the second phase: the equilibrium phase, a subclinical phase in which the tumour persists but is prevented from expanding by immune pressure. The third phase, escape, begins when the balance between the immune response and the tumour tilts towards tumour growth as a result of immune exhaustion or inhibition or as a result of the emergence of tumour-cell variants (shown in purple) that enable the tumour to evade immune pressure. Non- immunogenic transformed cells directly enter the escape phase. This phase concludes with the appearance of clinically detectable, progressively growing tumours. Figure adapted from Dunn et al.¹⁶.

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1.3 Cancer-related inflammation

Inflammation refers to a complex process initiated by the immune system to eliminate invading pathogens and allow for wound healing, and it is considered to be the usual response of the body to harmful stimuli¹⁸. In such cases, acute inflammation by innate immunity may lead to the development of protective immunity. Whereas in the case of chronic inflammation, which has been shown to contribute to tumorigenesis¹⁹. Inflammation may or can contribute to cancer initiation, cancer promotion, and cancer progression by inducing genotoxic stress, or by promoting cellular proliferative agents, or by enhancing angiogenesis and tissue invasion²⁰.

Pro-inflammatory cytokines such as interleukin (IL)-1β, IL-23 and tumor necrosis factor (TNF)-α, and signaling molecule myeloid differentiation primary response 88 (MyD88) may participate both in tumor induction and tumor immunity, depending at which time point they are recruited to the cancer development process¹⁹. Reports showed MyD88 dependent signaling and induction of IL-1β induce tumor progression in mouse models, where the wild- type mice develop progressive tumor compare to MyD88 knockout mice^{21, 22}, the same signaling molecules and the products it induces have opposite functions at the later stages of tumorigenesis^{23, 24}. Tumor cells that went through immunogenic death during tumorigenesis release molecules which could be recognized by the immune system and immune system promotes the development of protective immune response due to the production of IFNγ, IFN-α/β, TNF-α, and IL-12, or generation of T cells against established tumor cells. In a study with mouse myeloma model showed that the antigen secreted by the cancer cells is required to induce the immune response against tumors²⁵. This dual role of the inflammatory cytokines supports the observation that the immune system has both tumor-promoting and antitumor activity. Recent studies performed on mouse model showed that the IFNγ and TNF-α both have the ability of tumor formation and immune rejection of tumors^{26, 27}.

Haabeth et al. (2011) argued for a cancer-suppressive nature of inflammation when they showed that particularly inflammation driven by tumor-specificT helper (TH)-1 cells may repress cancer proliferation²⁸. In an idiotype (Id)-specific T cell receptor (TCR) transgenic mice injected with MOPC315 plasmacytoma in Matrigel, they observed that an increase in nine core cytokines (IL-1α, IL-1β, IL-2, IL-3, IL-6, IL12p70, IFNγ, CXCL9, and CXCL10) was consistently associated with tumor rejection (Fig. 3a). Eight days after tumor injection

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they also found CD11b⁺ macrophages in the Matrigel plugs, which relate to M1 activated macrophages characterized by having a high number of major histocompatibility complex (MHC) class II molecules on their cell surface. These macrophages were activated in vivo by Id-specific TH1 driven cytokine IFNγ and showed tumoricidal activities. The authors propose that IFNγ activated macrophages use two strategies simultaneously, (i) direct killing of cancer cells, and (ii) indirect inhibition of tumor growth through secretion of angiostatic chemokines such as CXCL9 and CXCL10.

Figure 3: A model for how inflammation may either suppress or promote cancer. (a) Tumor suppressive inflammation. Successful cancer immunosurveillance is mediated by a tumor-specific Th1-driven inflammation.

In this process, tumor-specific Th1 cells collaborate with tumor-infiltrating macrophages and nine cytokines synergize to prevent cancer. Proinflammatory cytokines (IL-1α, IL-1β, and IL-6) participate in tumor eradication by recruiting immune cells from the circulation and by stimulating T-cell functions. (b) Tumour-promoting inflammation. In the absence of sufficient numbers of tumour-specific Th1 cells, IL-1α, IL-1β, and IL-6 may participate in tumor progression by stimulating angiogenesis, vascular permeability and tumor invasiveness.

MΦ, macrophage; CTL, cytotoxic CD8⁺ T cells. Figure adapted from Haabeth et al²⁸.

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1.4 Macrophages

Macrophages are innate immune cells and are part of the mononuclear phagocytic system which is generated from the haematopoietic stem cells in the bone marrow. Their precursors, monocytes, are released into the circulation from where they can extravasate through the endothelium into the tissues, where monocytes are differentiated into macrophages²⁹.

Élie Metchnikoff was the first person to identify the macrophage based on it phagocytic nature³⁰. Macrophages are ancient cells in metazoan phylogeny and are highly professional phagocytic cells, which participate in tissue maintenance and homeostasis³¹.

Macrophages also detect microbes and endogenous danger signals with their dedicated PRRs, which can serve different purposes. PRRs are transmembrane receptors and are located either on the cell surface or inside the phagosomes and endosomes. Microbial pathogens associated PAMPs and endogenous damage signals released during cell death or damaged cells associated damage-associated molecular patterns (DAMPs) can a induce the dynamic range of responses through their respective PPR³². Toll-like Receptors (TLRs) are an example of PRRs and are common to most of the mononuclear phagocytic system^{1, 33, 34}.

Macrophages have influential activity in every biological aspect of an organism, from hosts steady state to hosts protective defense²⁹. Upon recognition of PAMPs or DAMPs macrophages in response produce different effector molecules such as cytokines, chemokines, or nitric oxide. Also, the activation status of the macrophages is changed, as they show increased phagocytosis, or increased antigen presentation to activate the adaptive immune system to provide the protective response to the cause. The macrophages could be separated in different classes and subtypes regarding their developmental origin and their functions³⁵. Charles Mills et al. (2000) distinguished macrophages in two different phenotypes based on their response to arginine metabolism, M1/kill, and M2/heal. This was based on a study with TH1 and TH2 strains of mouse models and the responses of the mouse models to immune stimulants such as LPS³⁶. Mills et al. showed that the macrophages from the TH1 mouse strain metabolized arginine to produce nitric oxide (NO) in response to LPS, whereas the TH2 mouse strain increased the production of ornithine by metabolizing arginine with the similar LPS stimuli. Other studies regarding the macrophage activation in vitro have stated M1

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macrophage as classically activated with IFNγ and LPS, and M2 macrophages as alternatively activated in response to IL-4 or IL-13³⁷.

These two subtypes of activated macrophages have different functions depending on host steady state or defense mechanism. In the sterile wound, the M2 heal macrophages produce ornithine to provide healing features to the wound, while M1 macrophages produce NO for few days until the wound is free from pathogens. The M1 macrophage responses relate to IL- 12 and IL-8 production, surface marker CD80/86 expression, or stimulate TH1 responses.

Where the M2 macrophages produce transforming growth factor (TGF)-β, vascular endothelial growth factor (VEGF) or epidermal growth factor (EGF)-like growth factors, surface expression of CD163 or 206 and stimulate TH2 response to humoral immunity³⁸.

1.4.1 M1 macrophage

The effector macrophages that are produced during a T cell-mediated immune response are designated as M1 macrophages. Originally, activated characterization of macrophages is dependent on a combination of two signals IFNγ and TNF resulted in a population of macrophage that shows enhanced microbicidal or tumoricidal capacity³⁹ (Fig. 4). Typically, a TLR ligand activates the MyD88-dependent pathway will induce the transcription of TNF.

TNF that induced in this TLR-MyD88 pathway cooperates with IFNγ as a positive feedback to activate the macrophage population⁴⁰. These M1 macrophages secrete increased level of inflammatory cytokines, chemokines, and metabolize arginine to produce NO^{38, 39, 41}.

The NK cells produce the cytokine IFNγ in response to PAMPs or DAPMs; IFNγ acts as a booster to activate and maintain the M1 macrophages fucnctionaliy^{40, 42}. Because of transient production of IFNγ by NK cells, the adaptive immune response is necessary to support the activated macrophages, which is provided by TH1cells producing IFNγ for the rest of the inflammation period. TLR ligands in association with MyD88 and Toll/Interleukin (TIR)-1 Receptor-domain-containing adaptor protein inducing IFNβ (TRIF)-dependant pathway induce the transcription of TNF and IFNβ respectively^{40, 43}. TNF, which is produced by MyD88 signalling, activates the macrophages in an autocrine manner, and IFNβ produced by the TRIF dependent pathway able to replace IFNγ⁴⁰.

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9 During the activation, M1 macrophages secrete cytokines, chemokines, and other effector molecules that may contribute to the immunity and wound clearing. Such cytokines are IL-1α, IL-1β, IL-12, IL-23, IL-6, and TNF; the chemokines are CCL2, CXCL10, or CXCL9, and they attract other immune cells; the effector molecules are superoxide anions, oxygen, and nitrogen. M1 activated macrophages express an increased number of MHC class II molecules on the surface, and also express CD80/86 molecules which stimulate the TH1 response28, 38, 40, 43, 44. That allow macrophages to provide a better host defense against pathogens or self- damaged cells. Moreover, M1 macrophages have been observed to provide antitumor protection, which correlates with increased survival in some cancer types (non-small cell lung cancer, myeloma)^{45, 46}.

Figure 4. Cytokines produced by immune cells can give rise to macrophage with distinct physiology.

Classically activated macrophages arise in response to interferon-gamma (IFNγ), which can be produced during an adaptive immune response by T helper 1 (TH1) cells or CD8⁺ T cells (not shown) or during an innate immune response by natural killer (NK) cells, and tumour-necrosis factor (TNF), which is produced by antigen- presenting cells (APCs). Wound-healing (alternatively activated) macrophages arise in response to interleukin-4 (IL-4), which can be produced during an adaptive immune response by Th2 cells or during an innate immune response by granulocytes. Regulatory macrophages are generated in response to various stimuli, including immune complexes, prostaglandins, G-protein coupled receptor (GPCR) ligands, glucocorticoids, apoptotic cells or IL-10. Each of these three populations has a distinct physiology. Classically activated macrophages have microbicidal activity, whereas regulatory macrophages produce high levels of IL-10 to suppress immune responses. Wound-healing macrophages are similar to the previously described alternatively activated macrophages and have a role in tissue repair. TLR, Toll-like receptor. Figure adapted from Mosser et al.⁴⁰.

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1.4.2 M2 macrophages

The macrophages can differentiate to a different phenotypic state rather than M1 macrophage and this phenotype is known as the M2 macrophage. This activation does not depend on a two signal system as required for M1 macrophages. The M2 phenotype can be induced by IL-4 or IL-13 in the absence of TLR signals (Fig. 4).

Cells of the immune system, such as eosinophils, macrophages or TH2 cells produce IL-4. IL- 4 activated macrophage decrease its phagocytic property and secret low level of IL-12 and high level of IL-10, IL-1Ra, and TGF-β⁴⁴. The M2 macrophages participate in TH2 polarization to help parasite clearance, dampen inflammation, tumor progression, wound healing, and have immunoregulatory functions^{47, 48}.

M2 macrophages can be further divided into three classes, M2a, M2b, and M2c according to theirs activation signal source. Among these, M2a represent the alternative activation, activated with the association of TH2 cells and IL-4 or IL-13 and gives resistance to allergens and parasites⁴⁹. M2b is the type II activation of M2 macrophages that requires a combined signalling similar to M1 activation, but instead of IFNγ, the M2b macrophages are generated in the response to ligands of FcγRs and TLRs. These M2b macrophages turn off IL-12 and induce IL-10 production⁵⁰. Moreover, M2c activation stimulated by glucocorticoids, inhibit the transcription of inflammatory cytokine genes and decrease mRNA stability⁵¹. M2c macrophages influence the production of TGF-β to contribute in immunoregulatory functions^{40, 52} (Fig. 5).

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Figure 5. Inducers and selected functional properties of different polarized M2 macrophage population.

Macrophages polarize and acquire different functional properties in response to environment-derived signals. M2 macrophages are in general more prone to immunoregulatory and protumoral activities. In particular, M2a (induced by exposure to IL-4 and IL-13) and M2b (induced by combined exposure to immune complexes and TLR or IL-1R agonists) exert immunoregulatory functions and drive type II responses, whereas M2c macrophages (induced by IL-10) are more related to suppression of immune responses and tissue remodelling.

Abbreviations: DTH, delayed-type hypersensitivity; IC, immune complexes; MR, mannose receptor; PTX3, the long pentraxin PTX3; SLAM, signaling lymphocytic activation molecule; SRs, scavenger receptors; TLR, Toll- like receptor. Figure adapted from Mantovani et al. ³⁷

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1.4.3 Tumor-associated macrophages (TAM)

Chemokines and growth factors produced by tumor cells can recruit monocytes and differentiate into macrophages^{53, 54}. Macrophages accumulating in the tumor mass termed as tumor-associated macrophages (TAM). Compared to the M1 macrophage TAMs show a poor antigen presentation, produce IL-10, TGF-β, and several growth factors to support tumor progression. TAMs inhibit the proliferation and activation of T-cells by releasing immunosuppressive cytokines⁵⁵. Besides their tumor-promoting characteristics, evidence of tumoricidal properties of TAM has been reported when they are activated as the M1 macrophage^{18, 56, 57}.

1.5 Dendritic cells (DCs)

To activate specific and effective immune responses against invading pathogens, the body requires a system that can survey, decipher and response quickly. The DCs are specialized in antigen processing and presentation, show high phagocytic activity and high cytokine producing capability within their different maturation stage and subtypes29, 58, 59. Different cytokines (GM-CSF, IL-4, IFN-γ, TNF-α) produced by various cells including NK cells, natural killer T cells (NKT), γδ T cells during steady state and immune response possibly affect the maturation, function, migration and antigen presenting capability of DCs60, 61, 62, 63. DCs are efficient in processing and presenting engulfed antigens, to naive T lymphocytes in association with MHC class MHC class I and class II proteins⁶⁴ (Fig. 6).

The DCs receptor repertoire is comprised of several families of receptors focusing on recognizing foreign, self-components or tumor antigens. The receptor families include TLRs, C-type lectin receptors, NOD-like receptors and RIG-I-like helicases. Dendritic cells go through a full maturation process to educate the adaptive immune system. This can be initiated by PAMPs or DAMPs of innate receptors^{65, 66}. Upon TLR-mediated maturation, DCs display a transient increase in their endocytic and phagocytic activity. Changes occur in DCs cytoskeleton rearrangement and gain rapid migration capabilities⁶⁷.

In addition to immature and mature DCs, there is a different phenotype of mature DCs observed, which is called regulatory DCs (DCreg). Regulatory DCs could induce Foxp3⁺ regulatory T cells (Treg) from CD4⁺ naive precursors. Regulatory DCs induce the production of co-stimulatory molecules and low level of inflammatory cytokines (IL-6, IL-12, and

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13 TNFα). DCs with both stimulatory and regulatory functions can be found in the tumor microenvironment. Depending on the cytokines and other immunomodulatory factors expressed by tumor cells, DCreg can be recruited to the tumor environment⁶⁸. Reports proposed that the DCs involvement to tumor environment could be essential to promote and provide the anti-tumor response⁶⁹. In particular, induction of the anti-tumor immunity mediated by the DCs requires IFNγ stimulation⁷⁰.

Figure 6. Stimulatory and regulatory dendritic cells in health and disease. DCs are a plastic lineage able to process and integrate signals from the microenvironment. Under pro-inflammatory conditions stimulatory DCs promote an effective immune response by stimulating T cell proliferation and shaping T cell responses toward TH1, TH2, or TH17 phenotypes. This crucial role allows the immune system to clear pathogens and keep transformed cells in check. Nevertheless, uncontrolled DC activation can lead to tolerance ablation, fostering the development of autoimmune diseases like rheumatoid arthritis. Under a tolerogenic environment DCs acquire regulatory functions suppressing T cell activation and proliferation and providing signals that enable Treg and Tr1 differentiation and expansion. This function maintains tolerance in organs like the gut which are exposed to a variety of harmless antigens. However, DCreg function can be exploited by tumors and pathogens leading to tumor progression and chronic infection. Figure adapted from Schmidt et al. ⁶⁸

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1.6 The Toll-like receptors (TLRs)

The Toll was first identified as a gene controlling the correct dorsoventral patterning embryo in Drosophila by Nüsslein-Volhard in 1985. A homologue of Toll proteins called Toll-like receptors were later found in mammals and other vertebrates, associated with resistance to microbes. A homologue of the Toll protein domains are also involved in the production of antimicrobial peptides in plants, indicating these domains association with the host defense since the evolution of life^{6, 71}.

The TLRs belong to the family of PRRs which are able to recognize different microbial PAMPs. PPRs recognize a set of specific patterns of microbial components that are conserved among microorganisms, but not found in mammals⁷². TLRs share a significant homology in their cytoplasmic regions with the interlukin-1 receptor super family, collectively named Toll/IL-1R (TIR) domain. TLR proteins also contain a leucine-rich repeat (LRR) domain in their extracellular region⁷³.

The TLR family consists of ten receptors in humans (TLR1-TLR10) and twelve receptors in mice (TLR1 to TLR9, TLR11 to TLR13)⁷⁴. There are multiple ligands for the corresponding TLRs, but the best known microbial TLR ligands are; LPS from the gram-negative bacteria which stimulates TLR4; lipoproteins, lipoteichoic acid, and fungal zymosan stimulate TLR1, TLR2, and TLR6; bacterial flagellin stimulates TLR5; and profilin-like molecules from protozoan stimulate TLR11, these TLRs are located on the surface of the immune cells.

Double-stranded RNA stimulates TLR3; single-stranded RNA stimulates TLR7 and TLR8, and un-methylated CpG motifs present in bacterial DNA stimulate TLR9, these TLRs are active within the endosomal compartment ^{75, 76} (Fig. 7).

Recognition of PAMPs by TLRs enhances phagocytosis, leads to the induction of inducible nitrogen oxide synthetase (iNOS), plays a role in host protective adaptive immune responses such as T cell activation, leads to up-regulation of the costimulatory molecules CD80/CD86, and to the production of factors that inhibit regulatory T-cell activity⁷⁵.

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Figure 7. Overview of TLR signalling pathway. Toll-like receptors (TLRs) are present on the cell surface and in endosomes, where they detect microbial cell-wall components, non-self nucleic acids or danger-associated self molecules. Upon stimulation, TLRs activate two types of pathway that involve myeloid differentiation primary response protein 88 (MYD88) and/or TIR-domain-containing adaptor protein inducing IFNβ (TRIF). Crosstalk with other signalling pathways ensures that the TLR signal is properly regulated and leads to either apoptosis or cell survival, and the transcription of pro-inflammatory cytokines and chemokines, and type I interferons (IFNs).

AP-1, activator protein 1; CREB, cAMP-responsive element-binding protein; dsDNA, double-stranded DNA;

dsRNA, double-stranded RNA; ERK, extracellular signal-regulated kinase; FADD, FAS-associated death domain; IκBα, inhibitor of NF-κBα; IKK, inhibitor of NF-κB kinase; IRAK, interleukin-1 receptor-associated kinase; IRF, IFN-regulatory factor; ISRE, IFN-stimulated response element; JNK, JUN N-terminal kinase; LBP, LPS-binding protein; LPS, lipopolysaccharide; MAL, MYD88 adaptor-like protein; MAP3K, mitogen-activated protein kinase kinase kinase 8; MD2, myeloid differentiation factor 2; MEK, mitogen-activated protein kinase/ERK kinase; MKK, mitogen-activated protein kinase kinase; NEMO, NF-κB essential modulator; NF-κB, nuclear factor-κB; PI3K, phosphoinositide 3-kinase; PKCε, protein kinase Cε; RIP1, receptor-interacting protein 1; ssRNA, single-stranded RNA; TAB, TAK1-binding protein; TAK1, TGFβ-activated kinase 1 (also known as MAP3K7); TBK1, TANK-binding kinase 1; TRAF, tumour necrosis factor receptor-associated factor; TRAM, TRIF-related adaptor molecule. Figure adapted from Nicholas et al. ⁷⁷

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A major number of the TLRs share a common signaling pathway upon activation with PAMPs. This common signaling pathway is initiated via the adaptor molecule MyD88, which has a TIR domain in the C-terminus and a death domain (DD) in the N-terminus⁷³. Besides the MyD88 associated signaling pathway, TLRs can also signal through other pathways: TIR- domain-containing adaptor protein (TIRAP)/MyD88-adaptor-like (MAL) protein, TIR domain-containing adaptor inducing IFNβ (TRIF)/TIR domain-containing adaptor molecule (TICAM-1)⁷⁵ (Fig. 7).

MyD88 is the central adaptor required for the downstream signalling by all TLRs except TLR3; a different adaptor protein TRIF comes into assistance at this point. TRIF signaling pathway is also used by TLR4 at the late endosomes following phagocytosis⁷⁸.

When TLRs become bound to their specific ligands, they induce a signaling cascade that activates Mitogen-activated protein kinases (MAPKs), c-Jun N-terminal kinases (JNKs), the transcription factors; nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and interferon regulatory factors (IRF3, IRF5 and IRF7) and leads to the induction of inflammatory cytokines and chemokines^{75, 79}.

Among the TLRs, TLR4 was the first to be described⁸⁰ and the one which has been studied in the most detail. A long list of studies suggests that several PAMPs can stimulate TLR4, including LPS, fusion protein from respiratory syncytial virus⁸¹, envelope protein from mouse mammary tumor virus⁸², as well as endogenous molecules like heat-shock protein⁸³, hyaluronic acid⁸⁴, and β-defensin2^{85, 86}. LPS is a highly potent activator of the innate immune system, and TLR4 recognizes the LPS with a set of proteins including LPS-binding protein (LBP), CD14, MD-2⁸⁷, where TLR4 acts as core receptor for LPS.

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1.7 Inducible nitric oxide synthase (iNOS/NOS2)

Nitric oxide (NO) is a free radical, has an inorganic gaseous property and has the important functions as a messenger and effector molecule. A family of enzymes named nitric oxide synthase (NOS) helps to synthesis the NO in biological system⁸⁸. Three isoforms of nitric oxide synthase are found, and among them is the inducible nitric oxide synthase (iNOS/NOS2) which allow macrophages to produce a micromolar concentration of NO for longer periods of time in their active state⁸⁹. The breakdown of L-arginine in the presence of oxygen to L-citrulline and NO is a complex oxidoreductase reaction which can be catalyzed by all three NOS in a calcium dependent (nNOS & eNOS) or independent (iNOS) manner⁹⁰ (Fig. 8).

Figure 8. Biosynthesis of NO. NO is produced by three nitric oxide synthase (NOS) isoforms neural, endothelial and inducible NOS (nNOS, eNOS, and iNOS). They catalyze the oxidation reaction of L-arginine to L-citrulline. Figure adapted from Vannini et al.⁸⁹.

Resting immune systems do not produce iNOS, but a variety of extracellular stimuli can trigger different pathways which lead to initiate the iNOS expression and NO production.

Microbial pathogens or their PAMPs and cytokines released from infected host cells are

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18

responsible for triggering the downstream pathway to activate iNOS expression. TLR4 in association with CD14-LPS complex and cytokines like TNFα and, IL-1β can activate the intracellular signaling cascades, leading to the transcription of iNOS and result in NO production. IFNγ is also able to activate the NO production⁹¹. Microbial PAMPs and cytokines through their associated receptors can induce transcription factors NF-κB or IRF-1, which in result leads to the expression of iNOS⁹⁰. An upsurge of LPS induced iNOS expression can be achieved with a synergistic effect of IFNγ, resulting in the synthesis of two transcription factors NF-κB and IRF-1⁹². The high-mobility group (HMG)-I(Y) protein interacts with IRF-1 and NF-κB, forming a multi-subunit complex that increases the transcription of iNOS⁹³ (Fig. 9). Regulation of the iNOS expression is controlled by the cytokine TGF-β through transcriptional, post-transcriptional and post-translational mechanisms⁹⁴. A variety of signaling molecules, such as IL-4, IL-10, IL-13 and macrophage deactivation factors are included in the inhibition of iNOS expression⁹¹.

Figure 9. Expression of iNOS. iNOS expression through the activation of different signal transduction pathway by various stimulators. TLRs induce transcription factor NF-κB and IFNγ to induce transcriptional factor IRF-1.

HMG-I(Y) protein interacts with NF-κB and IRF-1 to increase the iNOS expression. The homodimers form of iNOS produces NO and citrulline by catalyzing L-arginine in the presence of oxygen. Expression of iNOS is regulated by TGF-β. Figure adapted from Lowenstine et.al. ⁹¹.

.

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19 A long list of immune cells such as DCs, NK cells, macrophages, monocytes, and eosinophil generate NO using iNOS in response to cytokine the IFNγ, and microbial pathogens or their products⁹⁵.

NO produced by iNOS expressing tumor cells or by iNOS-positive tumor infiltrated myeloid cells have a dual role in tumor immunology, depending on the concentration of NO in the tumor environment⁹⁶. NO might regulate tumor activity by one of the following mechanisms:

(i) inhibition of enzymes essential for tumor growth⁹⁷, (ii) inhibition of tumor growth via iNOS-dependent depletion of arginine, (iii) TNF-induced cytotoxicity⁹⁵, (iv) cell cycle arrest by down-regulating cyclin D1⁹⁸, or (v) apoptosis by accumulating p53⁹⁹.

1.8 MOPC315 murine myeloma

Multiple myeloma is a malignant plasma cell tumor, located at multiple sites in the bone marrow compartment and high level of monoclonal protein is associated with the blood and serum¹⁰⁰. In our study, one of the tumor cell lines is MOPC315 (MOPC: Mineral Oil induced Plasmacytoma), induced in BALB/c mouse strain by intraperitoneal injection of mineral oil.

MOPC315 cell produces an immunoglobulin (Ig)-A myeloma protein named M315¹⁰¹. This cell line was used as a mouse model of plasmacytoma in this study.

1.9 Lewis Lung Carcinoma (LLC)

The Lewis Lung carcinoma was established by Dr. Margaret R. Lewis in 1951, from a spontaneously originated carcinoma of the lung of C57BL mouse and has been extensively applied as a transplantable tumor model. The LLC-derived cell line LLC1 was established in C57BL mouse by Bertram & Janik¹⁰². The cell line is highly tumorigenic, with a doubling time of 21 h. LLC cell line was used as a mouse model for carcinoma in our study.

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2 Aims of the study

Primary Objective: To identify TLR ligands which synergize with IFNγ to render macrophage tumoricidal.

Sub-goal:

1. To test combination of IFNγ with TLR ligands for induction of tumoricidal activity by macrophages or DCs.

2. To measure NO production by the activated macrophages or DCs.

3. To test tumoricidal activity towards plasmacytoma (MOPC315) and carcinoma (LLC).

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3 Materials and methods

3.1 Mitomycin C

Mitomycin C is a naturally occurring antibiotic, isolated from the gram-negative bacteria Streptomyces caespitosus. Mitomycin C is a DNA cross-linker that binds 5’-CpG-3’

nucleotide sequence, inhibiting DNA synthesis by covalently reacting with DNA and forming a crosslink between the complementary DNA sequences¹⁰³. Mytomicin C was obtained from Sigma-Aldrich.

In our experiment, we used 5µg/ml of Mitomycin C to inhibit the proliferation of macrophage and dendritic cells¹⁰⁴. The cells were exposed to Mitomycin C for two and a half hours followed by a three washing steps with Dulbecco’s phosphate buffered saline (PBS) from Sigma-Aldrich.

3.2 Ligands for Toll-Like Receptors (TLRs)

Here in this study, we have done experiment with eight Toll-Like Receptor (TLRs) ligands, to activate mouse macrophage and dendritic cell lines. All the TLR ligands were resuspended in endotoxin-free water, except LPS and LTA, which were resuspended in sterile water.

Resuspended ligands were aliquoted before applying in our experiments.

3.2.1 Lipopolysaccharides (LPS)

LPS is a potent activator of the immunity, major component of the outer membrane of gram- negative bacteria. TLR4 with CD14 and MD2 receptor complex has been identified as the receptor for LPS⁸⁷. LPS of Escherichia coli was obtained from Sigma-Aldrich.

3.2.2 Lipoteichoic Acid (LTA)

Lipoteichoic acid is a major constituent of gram-positive bacteria’s cell wall. LTA used in this experiment was from Staphylococcus aureus, obtained from InvivoGen. LTA can be recognized by the innate immune system via LPS Binding Protein (LBP), CD14 and TLR2 receptor complex¹⁰⁵ in association with TLR6¹⁰⁶.

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3.2.3 Pam3CSK4

Pam3CSK4 is a synthetic lipoprotein mimics the triacylated bacterial lipopeptide, was obtained from InvivoGen. Pam3CSK4 can be recognized by the innate immune system via LBP, CD14, and TLR2 receptor complex in association with TLR1¹⁰⁷ able to induce pro- inflammatory transcription factor NF-κB¹⁰⁸.

3.2.4 Polyinosinic: polycytidylic acid; Poly (I:C)

Poly (I:C) is a synthetic analog of double-stranded RNA. It represents itself as a molecular pattern associated with viral infection and can be recognized by TLR3¹⁰⁹. In our study, Poly (I:C) was obtained from InvivoGen.

3.2.5 Flagellin

Flagellin ST Ultrapure from Salmonella typhimurium was obtained from InvivoGen. Flagellin is the main component of bacterial flagellum filament and can be recognized by TLR5¹¹⁰.

3.2.6 CL264

CL264 is a novel 9-benzyl-8-hydroxyadenine derivative and an analog to adenine, obtained from InvivoGen. CL264 specifically recognizes by TLR7 of the immune systems¹¹¹ to induce immune stimulatory effects.

3.2.7 CpG ODN

CpG ODNs are synthetic oligodeoxynucleotide containing unmethylated CpG deoxynucleotide. These CpG ODNs can be recognized by TLR9 of the immune system and induce immune stimulatory effects¹¹². In our study, Class-C CpG ODNs were used, obtained from InvivoGen, has a 22 oligomer sequence (5’-tcgtcgttttcggcgc:gcgccg-3’) with a CpG- containing palindromic motif (underlined).

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3.3 Interferon gamma (IFN-γ)

Mouse recombinant interferon gamma (IFNγ) was obtained from PeproTech, with the endotoxin level less than 0.1 ng/µg. Sterile water was used to resuspend the IFNγ. In the experiments concentration of IFN-γ was 200 unit/ml.

3.4 Cell lines

In these experiments, we have used two different target cell lines and two different effector cell lines. Cells were cultured in RPMI 1640 + GlutaMAX-I (Life Technology) supplemented with 10% heat-inactivated foetal bovine serum (Sigma), 0.1 µM non-essential amino acids, 1mM sodium pyruvate (both from Lonza), 50 µM Monothioglycerol (Sigma) and 40 mg/ml Gentamycin (Sanofi-Aventis Norge As) (hereafter denoted culture medium).

For co-culturing effector and target cells, RPMI 1640+GlutaMAX-I (Life Technology) supplemented with 10% heat-inactivated FBS (Biochrom AG), 0.1 µM non-essential amino acids, 1mM sodium pyruvate (both from Lonza), 50 µM Monothioglycerol (Sigma) and 40 mg/ml Gentamycin (Sanofi-avensis Norge As) were used (hereafter denoted biochrom medium).

3.4.1 MOPC315 cell line

Cell line MOPC315 is a IgA and λ2³¹⁵secreting, MHC class II negative transplantable BALB/c plasmacytoma¹⁰¹ obtained from the ATCC and propagated as in vitro growing cells.

Cells were grown in the culture medium as in suspension, using a 175 cm² Nunc Cell Culture Treated EasYFlask (Thermo Scientific).

3.4.2 Lewis Lung Carcinoma (LLC) cell line

The cell line LLC was collected from CLS and grown in RPMI 1640+GlutaMAX-I (Life Technology) with 5% heat-inactivated FBS (Sigma). LLC was established from a mouse strain C57BL lung tissue with spontaneously formed lung cancer¹⁰². Cells were grown in the culture medium as adherent and suspension, using a 75cm² Nunc Cell Culture Treated EasYFlask (Thermo Scientific).

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3.4.3 J774.A1 macrophage cell line

The J774.A1 cells have the typical macrophage phenomena, obtained from adult female BALB/c mouse with reticulum cell sarcoma, derived from mouse ascites tissue¹¹³, kindly given by Ander Ø. Grammelsrud at the Norwegian Veterinary Institute. Cells were grown in the culture medium as adherent and suspension, using a 75cm² Nunc Cell Culture Treated EasYFlask (Thermo Scientific). Cells were prepared by scraping and centrifugation for subculture.

3.4.4 D2SC/1 dendritic cell line

D2SC/1 cell lines are the oncogene-immortalized mouse dendritic cell line of BALB/c origin and propagated as in vitro growing cells¹¹⁴. D2SC/1 cells were established from mouse spleen tissue, shows immature dendritic characteristics¹¹⁵. Activated D2SC/1 cells induce increased expression of DC markers CD11c, B7, and MHC I and MHC II on their surface. D2SC/1 cells also induce NO in response to IFNγ and LPS treatment¹¹⁴. The cell line D2SC/1 was kindly provided by Francesca Granucci, University of Milan.

Cells were grown in the culture medium as adherent and suspension, using a 75cm² Nunc Cell Culture Treated EasYFlask (Thermo Scientific). Subcultures of the cells were prepared by scraping and centrifugation.

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3.5 Growth Inhibition Assay (GIA)

Inhibition of the tumor cell growth was mediated by macrophages and DCs in vitro. The rate of the cell growth was measured by the incorporation of [³H]-radio labeled thymidine by the cells during co-culture with a different effector: target ratios. The assay protocol has been developed based on the experience of Corthay et al.¹⁷.

J774.A1 and D2SC/1 cells (effector cells) were harvested from the culturing flask using scraper followed by centrifugation at 300g for 6 min. Culture medium was removed by suction, and fresh biochrom medium was added, and cells were prepared for counting. The required numbers of cells were taken and incubated in 5% CO2 at 37⁰C for two and a half hours with Mitomycin C, followed by a three-step washing procedure with PBS. The washed cells were re-suspended in biochrom medium and counted to prepare cell concentrations. The concentrations of the effector cells were varied depending on the target cells (MOPC315 or LLC). The effector cells were seeded in 96 well cell culture cluster flat bottom plates (Costar) in four concentrations. The effector cells concentration for MOPC315 cells were 7.5x10⁵ cells/ml, 5x10⁵ cells/ml, 2.5x10⁵ cells/ml and 2.5x10⁴ cells/ml, and for LLC cells were 3.75x10⁵ cells/ml, 2.5x10⁵ cells/ml, 1.25x10⁵ cells/ml and 1.25x10⁴ cells/ml. Effector cell suspensions were applied in triplicates, in a volume of 200 µl to the wells.

Macrophage or DC cells were primed with IFN-γ (200 U/ml, 10 µl/well), with negative controls left untreated and incubated at 37⁰C in 5% CO2 for 18 h. LPS or other co-stimulators (TLR ligands) for effector cells were added in different period (for 18 h together with IFNγ or for 2 h after 18 h primed with IFNγ). The medium was removed after the activation, and the supernatant was used for NO assay. Fresh biochrom medium containing 2.5x10⁴ cells/ml (MOPC315) or 1.25x10⁴ cells/ml (LLC) in a volume of 200 µl/well was added to each well giving 30:1, 20:1, 10:1 and 1:1effector.target ratios. The fresh biochrom medium was added to the wells with effector cells only and considered as a control for effector cell growth. This plate was incubated at 37⁰C in 5% CO2 for another 18 h (see Fig. 10 for an example set-up).

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After 18 h incubation of co-cultured cells, [³H]-Thymidine (Hartman analytic) equivalent of 1 µCi/ml in a volume of 10 µl/well was added to the cultures and incubated for 24 h. Later the cells went through a freeze-thaw step to rupture the cell barrier to analyzing the [³H]

thymidine incorporation by the dividing cells, using a 1450 MicroBeta Trilux Microplate Scintillation counter (Fig. 11). The results were expressed in counts per minutes (CPM) and represent the incorporation of radioactive thymidine into the growing cells.

The results were plotted and analyzed with GraphPad, followed by a layout design with Adobe Illustrator.

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Figure 10. Example setup for growth inhibition assay. The effector cells were primed with IFN-γ and negative controls left untreated. LPS was added to the wells together with IFN-γ or in separate time. “LPS alone 2 h or 20 h” rows used for control of IFN-γ independent activation and “No activation” row used for MOPC315 baseline growth. MOPC315 cells were added to all the rows, except the “MΦ alone” rows. No macrophages were seeded into “MOPC alone” row.

Figure 11. The schematic presentation of growth inhibition assay. The effector cells were allowed to grow in 96-well plate for 18 h with activators. After activation, cell medium was replaced with cancer cell suspension with fresh biochrom medium. After 18 h of incubation, [³H]-Thymidine was added to the co-cultures, and cells were harvested 24 h later to measure [³H]-Thymidine uptake by the dividing cells.

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3.6 Nitric Oxide (NO) assay

As its nature of a free radical NO reacts with oxygen, superoxide radical or hydrogen peroxide yielding NO2, peroxynitrite and NO2- respectively, which depends on the redox environment¹¹⁶.

Griess assay was applied to measure the NO indirectly as nitrite (NO2-), which is a product of auto-oxidation of NO. NO2- reacts with sulfanilamide to form a diazonium salt intermediates which reacts with N-(1-napthyl) ethylenediamine to form the final compound, an azo dye¹¹⁷ (Fig. 12).

Figure 12. Griess Assay. The reaction of NO2- with Griess reagents form an azo dye that is easily detected spectrophotometrically to extrapolate NO concentrations released from the sample. Figure adapted from Coneski et al.¹¹⁷.

Two different types of Griess reagents were prepared, reagent-A containing 1% sulfanilamide and 2.5% of phosphoric acid in a 5 ml total volume of MilliQ water. Where reagent-B containing 0.1% N-(1-napthyl) ethylenediamine (NED) in 5 ml MilliQ water.

A dilution of NaNO2 was prepared for the standard curve. 100 µM of NaNO2 was prepared from a stock of 100 mM NaNO2 and diluted with MilliQ water to get 100, 50, 25, 12.5, 6.25, 3.13, 1.56 μM concentration of NaNO2.

The cell supernatant was collected as described above in the growth inhibition assay. The supernatant was centrifuged for two minutes at 300g to discard any cells. 50 µl of the cell supernatant was transferred to 96 well flat bottom cluster plate. Three different cell concentrations were used in triplicates to measure the level of NO. Fig. 13 showing a schematic diagram of set-up for the NO assay.

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29 50 µl of the Griess reagent-A was added to each well and kept in dark at room temperature for 10 minutes. 50 µl of Griess reagent-B was added after the incubation, and the level of NO was measured with the Gen5-microplate reader from BioTek, at 540 nm wavelength.

Figure 13. Example setup for NO assay. 50 µl of the effector cell supernatant from the growth inhibition assay was transferred to the plate as same order except the lowest cell concentration. The standard dilution was set-up in a triplicate manner followed by a half of the concentration of 100 µM of NaNO2.

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4 Results

4.1 Growth inhibition and NO assay

A growth inhibition assay was used to evaluate the effectiveness of the antitumor agent candidates on the proliferation of cancer cells. For these experiments, we used the murine macrophage cell line J774 or the DC cell line D2SC/1 as effector cells. Two cancer cell lines, MOPC315 and the LLC, were used as target cells. In previous experiments, it was shown that a sufficient number of effector cells are needed to hinder cancer cells growth, and the ratio between the number of effector cells and target cells is of importance³. Therefore, in this study, the following ratios for the number of effector cells:target cells were used: 30:1, 20:1, 10:1, and 1:1. The number of macrophages or DCs per well was 5x10³ when MOPC315 was the target cell. Because of the high growth rate of LLC, the number of macrophages or DCs was reduced to 2.5x10³ cells when LLC was the target cell line.

The J774 cells were primed with IFNγ (200 U/ml) for 18 h before co-activation with LPS (750 ng/ml) for 2 h (Fig. 14A). Alternatively, the J774 cells were activated in a combination of IFNγ and LPS together for 20 h. Some J774 cells were left untreated or treated with IFNγ or LPS alone to be used as controls. The cell supernatant was removed after activation and MOPC315 cells (5x10³cells/well) were added to the culture. After 18 h, the co-culture of effector and cancer cells were pulsed with [³H]-Thymidine and left for another 24 h in culture before harvesting the cells. Incorporation of [³H]-Thymidine by the dividing cells was measured with a scintillation counter. The optimal concentration and culturing time with [³H]- Thymidine applied in this study were 1µCi/ml and 24 h respectively, were found in a previous study³.

Growth inhibition of MOPC315 was seen at 30:1 and 20:1 effector:target cell ratio by 18 h IFNγ primed J774 cells stimulated with LPS for 2 h (Fig.14B). Less or no inhibition was observed at 10:1 and 1:1 effector:target ratio respectively. No inhibition of MOPC315 growth was seen by untreated J774 cells or those only treated with either of activators.

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31 Production of NO by the activated J774 cells was measured by the Griess method.

Supernatant from the activated J774 cells were analyzed for NO production (Fig. 14C). J774 cells were activated in two different ways in regards to the length of LPS activation. Data showed that effector cells activated with IFNγ and LPS together (20 h) produced more NO compared to IFNγ and LPS (2h) in separate activation protocol. The cell line J774 stimulated with LPS alone for 20 h produced higher level of NO compared to LPS alone for 2 h. Cells treated with IFNγ alone did not produce any notable amount of NO in the medium from different cell concentrations.

The LLC cells result showed inhibited growth when co-cultured with activated J774 cells at 30:1 and 20:1 effector:target cell ratio (Fig. 14D). 18 h of IFNγ primed J774 cells co- stimulated with LPS for 2 h was the best activation treatment to inhibit the LLC cell growth.

However, the highest level of NO was produced by J774 cells treated with IFNγ in combination with LPS for 20 h (Fig. 14C). No inhibition of LLC was seen with untreated J774 cells or those treated with IFNγ or LPS alone.

Synergistic induction of macrophage tumoricidal activity

Synergistic induction of macrophage tumoricidal activity

Sanjib Halder

Thesis for Master’s Degree in Molecular Bioscience 60 study points

Department of Molecular Biosciences

The Faculty of Mathematics and Natural Sciences

University of Oslo

Synergistic induction of macrophage tumoricidal activity

Sanjib Halder

Master Program in Molecular Biology Department of Molecular Biosciences

The Faculty of Mathematics and Natural Sciences University of Oslo

Master Thesis, 60 study points Supervised by Alexandre Corthay

Performed at the Department of Pathology Oslo University Hospital, Rikshospitalet

April 2016

Table of Contents

Acknowledgements

Abbreviations

Abstract

1 Introduction

1.1 The immune system

1.2 Cancer immunosurveillance

1.3 Cancer-related inflammation

1.4 Macrophages

1.5 Dendritic cells (DCs)

1.6 The Toll-like receptors (TLRs)

1.7 Inducible nitric oxide synthase (iNOS/NOS2)

1.8 MOPC315 murine myeloma

1.9 Lewis Lung Carcinoma (LLC)

2 Aims of the study

3 Materials and methods

3.1 Mitomycin C

3.2 Ligands for Toll-Like Receptors (TLRs)

3.3 Interferon gamma (IFN-γ)

3.4 Cell lines

3.5 Growth Inhibition Assay (GIA)

3.6 Nitric Oxide (NO) assay

4 Results

4.1 Growth inhibition and NO assay