ABSTRACT Title of Dissertation: THE ENDOGENOUS REGULATION OF THE HUMAN MACROPHAGE ACTIVATION RESPONSE Kajal Hamidzadeh, 2020 Dissertation directed by: Dr. David M. Mosser, Professor Department of Cell Biology & Molecular Genetics Macrophages are innate immune cells that participate in host defense to invading pathogens. They are powerful producers of cytokines and inflammatory mediators due to their efficient recognition of pathogen associated molecular patterns (PAMPs) via toll like receptors (TLRs). We and others have shown that the activation response to PAMPs is transient. In the present work, we demonstrate that stimulated macrophages produce adenosine and prostaglandin E2, which function as regulators of the macrophage activation response. Macrophages also upregulate receptors for these regulators to terminate inflammation and promote wound healing. We performed high throughput RNA sequencing to characterize the transcriptomes of human monocyte-derived macrophages in response to stimulation with LPS + Adenosine or LPS + PGE2. These cells exhibited a decrease in inflammatory transcripts and an increase in transcripts associated with cell growth and repair when compared to cells stimulated in the absence of these regulators. Macrophages can be generated from precursor cells in response to two different growth factors; M-CSF (macrophage colony stimulating factor) and GM-CSF (granulocyte-macrophage colony stimulating factor). M-CSF is expressed constitutively in a variety of tissues, while GM-CSF is expressed primarily in the lung, but can be induced in other tissues under inflammatory conditions. We demonstrate that human macrophages differentiated in M-CSF readily adopt an anti-inflammatory, growth promoting phenotype in response to LPS + Adenosine or LPS + PGE2, while macrophages differentiated in GM-CSF do not. This observation suggests that M-CSF derived human macrophages may be better able to alter their activation state in response to surrounding signals in order to maintain homeostasis. GM-CSF derived macrophages, in contrast, may undergo a more prominent activation response that is associated with inflammation and tissue destruction due to their inability to efficiently respond to resolving molecules. THE ENDOGENOUS REGULATION OF THE HUMAN MACROPHAGE ACTIVATION RESPONSE by Kajal Hamidzadeh Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, College Park in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2020 Advisory Committee: Professor David M. Mosser, Chair Professor Kevin McIver Professor Wenxia Song Professor Volker Briken Professor Xiaoping Zhu ? Copyright by Kajal Hamidzadeh 2020 Dedication I would like to dedicate this dissertation to my parents, Babak and Stacey Hamidzadeh. ii Acknowledgements First, I would like to thank my advisor, Dr. Mosser. His encouragement and excitement for my project truly helped me through the graduate school process. He taught me invaluable lessons about how to approach scientific problems, how to ask questions, and how to communicate ideas, which I will carry with me for the rest of my career. I would also like to thank my committee members, Dr. McIver, Dr. Song, Dr. Briken, and Dr. Zhu for their insightful advice and time dedicated to the development of my project. I would like to thank my collaborators Dr. El-Sayed and Dr. Belew for sharing their scientific expertise with me and for their kindness. It was a privilege to work with them. I would like to thank former members of the Mosser Lab: Dr. Christensen, Dr. Chandrasekaran, and Dr. Costa da Silva. I couldn?t have picked a better fellow graduate student to sit next to for 6 years than Steve. Prabha not only taught me lab techniques, but also life lessons. Carol motivated and cheered me on since day one. Mostly, I am thankful to all three for their friendship. Lastly, I would like to thank my parents for their unconditional love and support throughout my graduate school journey and for all the opportunities they have given me in my lifetime. I would also like to thank my sister for her support and enthusiastic spirit. iii Table of Contents Dedication .......................................................................................................................... ii Acknowledgements .......................................................................................................... iii Table of Contents ?.......................................................................................................... iv List of Tables .................................................................................................................... vi List of Figures .................................................................................................................. vii List of Illustrations ............................................................................................................ ix List of Abbreviations ......................................................................................................... x Chapter 1: Introduction ...................................................................................................... 1 1.1 Macrophages and innate immunity .................................................................. 1 1.2 Macrophage polarization ................................................................................. 2 M1 macrophages ........................................................................................ 3 M2 macrophages ........................................................................................ 3 Regulatory macrophages ............................................................................ 4 1.3 Toll like receptors ............................................................................................ 5 TLR signaling ............................................................................................ 5 Lipopolysaccharide and TLR4 ................................................................... 6 1.4 Adenosine in the immune system .................................................................... 6 ATP release and hydrolysis ....................................................................... 8 Adenosine receptor signaling ..................................................................... 8 Adenosine in disease .................................................................................. 9 1.5 Prostaglandin E2 in the immune system ........................................................ 10 PGE2 synthesis and secretion ................................................................... 10 PGE2 receptor signaling ........................................................................... 11 PGE2 and the immune response ............................................................... 11 1.6 Interferons and innate immunity .................................................................... 12 Type I interferon ...................................................................................... 12 Type II interferon ..................................................................................... 13 1.7 Colony stimulating factors ............................................................................. 14 M-CSF ..................................................................................................... 14 GM-CSF ................................................................................................... 15 1.8 Macrophage activation during disease or injury............................................. 16 Sepsis ....................................................................................................... 16 Tumor Associated Macrophages .............................................................. 17 Intracellular parasitic infections ............................................................... 18 Wound healing ......................................................................................... 19 1.9 Scope and Limitations .................................................................................... 20 Chapter 2: Materials and Methods ................................................................................... 21 2.1 Mouse BMDM preparation ............................................................................ 21 2.2 Mouse BMDM stimulation ............................................................................ 21 2.3 Human macrophage differentiation ............................................................... 21 iv 2.4 Human macrophage stimulation .................................................................... 22 2.5 RNA-sequencing sample and library preparation .......................................... 22 2.6 RNA-sequencing data assessment, visualization and differential expression analysis ........................................................................................................... 23 2.7 Single cell RNA-sequencing sample and library preparation ........................ 23 2.8 Single Cell RNA-sequencing analysis ........................................................... 24 2.9 ELISA ............................................................................................................ 24 2.10 Quantitative real-time PCR .......................................................................... 25 2.11 Measurement of PGE2 production ............................................................... 28 2.12 Measurement of ATP degradation ............................................................... 28 2.13 HUVEC tube formation assay ..................................................................... 28 2.14 Flow cytometry ............................................................................................ 29 Chapter 3: The effects of interferons on purinergic signaling in mouse macrophages ... 30 3.1 Introduction .................................................................................................... 30 3.2 Results ............................................................................................................ 31 Type II interferon modulation of macrophage activation ........................ 31 Type I interferon modulation of macrophage activation ......................... 35 3.3 Discussion ...................................................................................................... 40 Chapter 4: Characterization of the transcriptome of human M-CSF derived macrophages stimulated with LPS + Adenosine and LPS + PGE2 ........................................................ 42 4.1 Introduction .................................................................................................... 42 4.2 Results ............................................................................................................ 42 RNA-sequencing analysis ........................................................................ 42 Pathway analysis ...................................................................................... 47 Single cell RNA-sequencing analysis ...................................................... 52 4.3 Discussion ...................................................................................................... 56 Chapter 5: Comparison of human GM-CSF derived macrophages to M-CSF derived macrophages both stimulated with LPS + Adenosine and LPS + PGE2 .......................... 60 5.1 Introduction .................................................................................................... 60 5.2 Results ............................................................................................................ 60 Adenosine and PGE2 sensing in human macrophages ............................. 62 Transcriptome comparison of M-CSF and GM-CSF macrophages ......... 65 Functional assays of human macrophages ............................................... 73 Kinetics and modulation of cytokine production ..................................... 79 5.3 Discussion ...................................................................................................... 86 Chapter 6: Conclusions and future directions .................................................................. 93 References ........................................................................................................................ 97 v List of Tables Table I. Primer Sequences Table II. Single cell RNA-sequencing markers for LPS + PGE2 versus LPS Table III. Single cell RNA-sequencing markers for LPS + Ado versus LPS vi List of Figures Figure 1. Adenosine receptor gene expression following IFN? priming and stimulation with various TLR ligands Figure 2. Adenosine receptor expression in STAT1 knockout BMDMs Figure 3. Cytokine levels in WT and STAT1 knockout BMDMs Figure 4. Cytokine levels in WT and A2B receptor knockout BMDMs Figure 5. Adenosine receptor expression following priming with Type I, II and III IFNs Figure 6. Adenosine receptor expression in IFNAR knockout BMDMs Figure 7. Cytokine levels in WT and A2A receptor knockout BMDMs Figure 8. Most highly upregulated and downregulated genes by adenosine and PGE2 relative to LPS stimulated macrophages alone. Figure 9. Degree of transcriptional similarity between LPS + Adenosine and LPS+PGE2 stimulated macrophages Figure 10. Cluster visualization of M-CSF macrophage single cell transcriptomes Figure 11. Functional characteristics of shared DEGs between LPS + Adenosine and LPS + PGE2 relative to LPS stimulation Figure 12. Functional interaction groups in shared DEGs between LPS + Adenosine and LPS + PGE2 relative to LPS stimulation Figure 13. Violin plots of candidate RNA markers for LPS + Adenosine and LPS + PGE2 stimulated M-CSF macrophages Figure 14. Feature plots of candidate RNA markers for LPS + Adenosine and LPS + PGE2 stimulated M-CSF macrophages Figure 15. M1 genes in M-CSF macrophages Figure 16. Flow cytometry validation of M-CSF and GM-CSF derived macrophages Figure 17. ATP degradation and PGE2 production by human macrophages Figure 18. mRNA expression of purinergic pathway and PGE2 receptor genes in M-CSF and GM-CSF macrophages vii Figure 19. mRNA expression of PGE2 synthesis pathway genes Figure 20. Purinergic and PGE2 pathway gene expression following stimulation with various TLR ligands Figure 21. PCA plot of stimulated human macrophage samples Figure 22. Volcano plot visualization of DEGs from LPS + Ado and LPS + PGE2 stimulated macrophages relative to LPS alone Figure 23. Comparison of expression of DEGs by LPS + Adenosine and LPS + PGE2 relative to LPS alone in M-CSF and GM-CSF macrophages Figure 24. mRNA expression of genes of interest in M-CSF and GM-CSF macrophages Figure 25. HUVEC cell tube formation in the presence of macrophage conditioned media Figure 26. Flow cytometry of CD300E surface expression Figure 27. Flow cytometry of PLAUR surface expression Figure 28. Growth promoting, anti-inflammatory cytokine secretion by M-CSF and GM- CSF macrophages Figure 29. Inflammatory cytokine secretion by M-CSF and GM-CSF macrophages Figure 30. Modulation of macrophage IL-12p40 secretion by increasing concentrations of adenosine and PGE2 Figure 31. Priming in M-CSF and GM-CSF macrophages Figure 32. Kinetics of inflammatory TNF and IL-12p40 cytokine secretion by M-CSF and GM-CSF macrophages Figure 33. Pharmacological inhibition of adenosine and PGE2 receptors Figure 34. Proposed model of M-CSF and GM-CSF macrophage activation viii List of Illustrations Illustration 1. ATP catabolism at the macrophage surface ix List of Abbreviations Ado adenosine ATP adenosine triphosphate BMDM bone marrow derived macrophage cAMP cyclic adenosine monophosphate CD cluster of differentiation COX cyclooxygenase DEG differentially expressed gene ELISA enzyme-linked immunosorbent assay Epac exchange protein activated by cAMP GM-CSF granulocyte macrophage colony stimulating factor GO gene ontology GPCR g-protein coupled receptor HUVEC human umbilical vein endothelial cell IFN interferon IFNAR interferon alpha receptor IRF interferon regulatory factor IL-6 interleukin-6 IL-10 interleukin-10 IL-12p40 interleukin-12p40 JAK Janus kinase KO knockout Log2FC log2 fold change x LPS lipopolysaccharide M-CSF macrophage colony stimulating factor mRNA messenger ribonucleic acid NK natural killer PAMP pathogen associated molecular pattern PAP pulmonary alveolar proteinosis PGE2 prostaglandin E2 PKA protein kinase A PCA principal component analysis PLAUR plasminogen activator, urokinase receptor RT-PCR reverse transcriptase polymerase chain reaction RNA-seq RNA sequencing SLE systemic lupus erythematosus STAT signal transducing THBS1 thrombospondin-1 TGF? transforming growth factor beta TLR toll like receptor TNF tumor necrosis factor TYK tyrosine kinase UMAP uniform manifold approximation and projection VEGFa vascular endothelial growth factor alpha WT wild type xi 1 Introduction Parts of this chapter are adapted from published works: Hamidzadeh, K., S.M. Christensen, E. Dalby, P. Chandrasekaran, and D.M. Mosser. 2017. Macrophages and the Recovery from Acute and Chronic Inflammation. Annu. Rev. Physiol. 79:567?592. doi:10.1146/annurev-physiol-022516-034348. Hamidzadeh, K., and D.M. Mosser. 2016. Purinergic Signaling to Terminate TLR Responses in Macrophages. Front. Immunol. 7:74. doi:10.3389/fimmu.2016.00074. 1.1 Macrophages and Innate Immunity Macrophages reside in almost all tissues of the body and engage in inflammatory processes in order to protect the host from invading pathogens and to fight infection. They are part of the innate immune system, which is the host?s first line of defense once an immunogen has bypassed external barriers such as the skin. Macrophages are efficient phagocytes and are able to rapidly clear debris, dead cells, and microbes at the site of infection. In addition to phagocytosis, macrophages release important inflammatory mediators including cytokines in order to initiate systemic immune responses. These inflammatory macrophages are a vital component of host defense. However, the transition to an anti-inflammatory state is crucial during the resolution of infection in order to prevent damage to host tissue. The failure to resolve inflammation can result in autoimmunity. Macrophages originate from two different sources: the embryonic yolk sac, and hematopoietic stem cells in the bone marrow1. During inflammation, tissue macrophages can be derived from circulating blood monocytes. These cells migrate to the tissue, or 1 sites of inflammation, where they encounter signals such as macrophage-colony stimulating factor (M-CSF) and granulocyte macrophage-colony stimulating factor (GM- CSF) that promote their differentiation into macrophages. It has been demonstrated that during chronic infection, macrophages from embryonic origin largely disappear, and are replaced with macrophages of hematopoietic origin in the lung, liver, and peritoneum among other tissues2,3. Macrophages residing in close proximity to a variety of signals exhibit dramatically different phenotypes. 1.2 Macrophage polarization Macrophages represent a heterogeneous population of immune cells. Because they are present in so many tissues, they are exposed to a wide variety of microenvironments and must be able to respond to a wide range of stimuli. Macrophages express the family of surface receptors known as Toll-like receptors (TLRs), which are able to recognize pathogen associated molecular patterns (PAMPs). These are conserved patterns present on pathogens that the cells have evolved to recognize efficiently to mount an inflammatory immune response. In addition to PAMP signals, macrophages also recognize small molecules in their surroundings that allow them to alter their phenotype. This is termed macrophage ?plasticity? and it is important that macrophage phenotypes can be reversed. Due to the plasticity of these cells, macrophages with an infinite number of diverse phenotypes may exist4. However, it is generally accepted that 3 main populations of macrophages may represent the polar extremes of macrophage phenotypes. These include classically activated (M1), alternatively activated (M2), and ?regulatory? macrophages. While these 3 groups have been initially defined in the 2 mouse5, it is proposed that macrophage activation falls along a spectrum as they exist within diverse environments within the human host. M1 Macrophages M1 macrophages are defined by stimulation with interferon gamma (IFN?) + lipopolysaccharide (LPS). Early on in infection the type II interferon, IFN?, is produced by innate and adaptive immune cells. Macrophages exposed to IFN? are primed for secretion of large amounts of inflammatory cytokines, and reactive oxygen and nitrogen species6. This secretion is typically triggered by a second stimulus. Classically activated macrophages receive a secondary signal through TNF receptor or TLR stimulation. Stimulation of TLRs results in the activation of NF?B transcription factors, which induce the transcription of pro-inflammatory cytokines such as IL-12 and TNF, and many cytokines and mediators through interferon response elements. Our lab has contributed to the body of knowledge surrounding classically activated mouse macrophages by demonstrating that IFN? primed bone marrow-derived macrophages (BMDMs) do not downregulate their own activation state7. This occurs due to the failure to upregulate the A2br receptor, which renders these macrophages insensitive to the endogenous anti- inflammatory effects of adenosine. M2 Macrophages M2 macrophages are typically induced in response to IL-4 or IL-13. These macrophages produce precursors important for collagen production. These same precursors may lead to tissue fibrosis and Th2 pathology. While IL-4 alone does not induce cytokine production in these macrophages, it does promote the induction of arginase (Arg1)6. IL-4 receptor stimulation also results in the activation of STAT68. 3 Alternatively activated macrophages are considered to be more susceptible to microbial infection and intracellular pathogens and this is thought to be through the metabolism of L-arginine to polyamines9. In this way, arginase induction diverts arginine away from NO production and provides microorganisms with necessary nutrients for survival. Surprisingly, M2 or alternatively activated macrophages have not sufficiently been characterized in humans. Preliminary, unpublished, transcriptomic data from our lab indicates that IL-4 treatment of monocyte-derived macrophages has a more limited effect on their phenotype, with little overlap with the effects of IL-4 treatment of mouse macrophages. Regulatory Macrophages Regulatory macrophages (R-M?) were first defined in our laboratory in mouse bone marrow derived macrophages10,11. They require a combination of an inflammatory signal, such as TLR stimulation and a secondary signal, which can include immune complexes, adenosine or prostaglandin E 5,122 . The hallmark of regulatory macrophages is the reciprocal change of IL-10 and IL-12 cytokine production. In mouse, R-M? produce high levels of IL-10 compared to LPS stimulation alone. IL-10 is an anti-inflammatory cytokine, which can act to suppress macrophage activation and it is critical for the resolution of inflammation. R-M? stimulated with adenosine and PGE2 produce decreased amounts of IL-12 despite the inflammatory TLR signal. IL-12 is involved in the initiation of cell-mediated immune responses. Our lab has identified a number of other highly upregulated genes in regulatory macrophages, including growth and angiogenic factors, and we were able to demonstrate that these macrophages are distinct from classically and alternatively activated macrophages. From RNA sequencing data, it 4 is hypothesized that regulatory macrophages are not only involved in decreasing inflammation but also involved in promoting homeostasis. 1.3 Toll like receptors: The innate immune system is our first line of defense against foreign antigens. It is critical that the innate immune response acts quickly and efficiently in order to destroy invading pathogens. Through the evolutionary process, mammalian innate immune cells became equipped with a number of cell surface and intracellular receptors, known as Toll-like receptors (TLRs), that are able to recognize highly conserved and repetitive sequences known as pathogen associated molecular patterns (PAMPs)13. In this way, TLRs can rapidly trigger an immune response to different pathogens. TLR signaling TLRs are type I transmembrane glycoproteins. The extracellular portion of TLRs is composed of between 16 and 28 leucine rich repeats that contain conserved amino acid motifs14. TLR signaling originates with dimerization of the receptor and subsequent recruitment of adaptor proteins to its intracellular TIR domain15. There are five adaptors that can initiate TLR signaling: MyD88, MAL, TRIF, TRAM, and SARM16. The adaptor MyD88 is critical for the production of inflammatory cytokines by all TLRs. MyD88 recruits IRAK-4 to the receptors, which then activates IRAK-1 and IRAK217. Activated IRAKs associate with TRAF6, and this complex can then activate two pathways18. The first is the MAPK pathway leading to AP-1 transcription factor activation. The second is the phosphorylation of I?B kinase, which dissociates from the IKK complex in order to activate the NF-?B transcription factor. These transcription factors lead to the production of many inflammatory genes. 5 Lipopolysaccharide and TLR4 One of the most widely used tools to activate macrophages is lipopolysaccharide (LPS), a TLR ligand. LPS is a glycolipid present in the outer membrane of gram- negative bacteria including E. coli. The structure of LPS consists of a hydrophobic lipid A component, hydrophilic core of polysaccharides, and O-antigen19. Trace amounts of LPS in certain tissues, such as blood, can lead to a fatal disease called sepsis due to uncontrollable amounts of inflammation and this toxicity is primarily due to the lipid A component20. LPS is recognized by TLR4, which forms a heterodimer with myeloid differentiation factor 2 (MD-2) that aids in LPS binding21,22. Two additional proteins are also required for LPS recognition, LPS binding protein (LBP) and CD1423. LBP facilitates the association of LPS with CD1423. CD14 is expressed on myeloid cells and binds to LPS, subsequently presenting it to the TLR4/MD-2 complex24. CD14 is also required for endocytosis of TLR4, which is part of the signal transduction process25. TLR4 and its interaction with LPS is one of the most studied and well-characterized processes in innate immunity. 1.4 Adenosine in the Immune System: Adenosine is a purine nucleoside circulating at low levels in the blood and in tissue. Adenosine concentrations surrounding cells can increase via nucleoside transport proteins in the cell membrane, or via ATP catabolism (Illustration 1). ATP and adenosine can be released in local environments by platelets, dead and dying cells, tumors, and from endothelial cells, among other sources. It is proposed that all immune cells can contribute to adenosine concentrations due to the fact that ATP is produced by 6 Illustration 1. ATP catabolism at the macrophage surface. ATP is converted to adenosine via the action of two ecto-enzymes, CD39 and CD73. Adenosine signals through the A2a and A2b receptors which are coupled to G?s proteins. This leads to an increase in intracellular cAMP levels. Figure from: Hamidzadeh, K., and D.M. Mosser. 2016. Purinergic Signaling to Terminate TLR Responses in Macrophages. Front. Immunol. 7:74. doi:10.3389/fimmu.2016.00074. 7 glycolysis and immune cells express the ecto-enzymes required to convert this ATP to adenosine. ATP Release and Hydrolysis ATP release from resting macrophages is low, but ATP release is significantly increased following TLR activation26. Some of the cytosolic ATP generated following TLR stimulation is released into the extracellular milieu through pannexin-1 channels. This ATP is catabolized by macrophages in a coordinated two-step process. ATP is first hydrolyzed to AMP by the surface ecto-enzyme CD39 (E-NTPDase1) in a Ca2+ and Mg2+ dependent process27. Next, AMP is rapidly converted to adenosine by CD73 (Ecto5?NTase)28. These two enzymes and their expression level on the macrophage surface can greatly affect the concentration of adenosine in the extracellular environment directly adjacent to the cell. Adenosine Receptor Signaling Macrophages respond to adenosine via signaling through the P1 class of seven transmembrane G-protein coupled receptors (GPCR), which includes: A1R, A2aR, A2bR, and A3R29. The A1 and A3 receptors are coupled to the Gi family of proteins, which act to decrease cAMP levels. A2a receptors are G?s-coupled receptors which act to increase intracellular cAMP, and they are high affinity for adenosine30,31. Similarly, A2b receptors can signal through G?s or Gq proteins, also leading to increased cAMP, but these receptors are low affinity for adenosine30,32. In combination with TLR stimulation, adenosine drives the transition from a pro-inflammatory to a regulatory macrophage4. Adenosine is immuno-suppressive in mouse macrophages as it leads to increased IL-10 release and decreased TNF and IL-12 release26. High-throughput RNA-sequencing data 8 from our lab indicated that macrophages stimulated with LPS in combination with adenosine upregulated 501 transcripts and downregulated 610 transcripts relative to LPS exposure alone5. Furthermore a number of the upregulated transcripts were involved in cell growth, while many of the downregulated transcripts were involved in inflammation33. While signaling through GPCRs controls the levels of intracellular cAMP, the role of the cAMP/PKA pathway in terms of inflammatory cytokine inhibition by adenosine receptor signaling is not fully understood. Some researchers have proposed that the inhibition of macrophage TNF production by adenosine is due to a cAMP/PKA- independent pathway, and is rather a pathway dependent on phosphatases34. However, others have shown that cAMP/PKA levels are linked to TNF production in an inverse manner35. It has also been shown that the A2bR interacts with and inhibits NF?B, and that A2bR knockout macrophages produce less IL-10 and more IL-12 and TNF36. Thus, adenosine may regulate macrophage phenotypes by mechanisms that have not yet been fully elucidated. Adenosine in disease Adenosine receptors have been associated with a variety of diseases. These receptors are expressed in many tissues including the brain, heart, spleen, muscle and lung37,38. This ubiquitous pattern of expression is one of the obstacles of developing therapeutics that are able to specifically target the receptors. Studies have been done that reveal a role for A2aR and A2bR in diabetes due to the fact that they are involved in the regulation of glucose as a result of increased cAMP39?41. There is potential for A2ar agonists to be anti-inflammatory in ischemia reperfusion injury42. A2aR and A2bR have 9 both been implicated in reducing foam cell formation, which is a feature of atherosclerosis43,44. However, it has been demonstrated that knocking out the A2ar has a protective outcome in a mouse model of hypercholesterolemia largely due to the fact that macrophages in these mice are pro-inflammatory and therefore reduce atherosclerotic lesions45. Furthermore, adenosine receptors contribute to wound healing and modulate cytokine production by macrophages of patients with chronic obstructive pulmonary disease29,46. 1.5 Prostaglandin in the immune system: Prostaglandins are bioactive lipids present in many tissues that are implicated in processes including proliferation, angiogenesis, and inflammation. They are part of the prostanoid family of lipids, which are synthesized step-wise from fatty acids. There are numerous prostaglandins that can be synthesized, including PGI2, PGD2, and PGF2, but the most studied and widely acting prostaglandin is PGE2. PGE2 is typically seen as a perpetuator of inflammation, which is why it is the target of non-steroidal anti- inflammatory drugs (NSAIDs). This is true for many cell types including T and B cells. However, the opposite is true for macrophages, as PGE2 promotes the production of anti- inflammatory molecules and the shutting off of cytokine production. PGE2 synthesis and secretion First, phospholipases hydrolyze membrane phospholipids, releasing arachidonic acid47. Next, arachidonic acid is oxidized into PGG2 and reduced to PGH2 by the cyclooxygenase enzymes COX-1 and COX-247. These two enzymes are highly upregulated throughout the immune system in response to pro-inflammatory signals48,49. Lastly, PGH2 is converted into PGE2 via three synthases: mPGES-1, mPGES-2, and 10 cPGES49. PGE2 levels are regulated by both its synthesis and by its degradation. 15- PGDH and 13-PGR are catabolic enzymes that rapidly remove PGE2 from the cellular environment50. PGE2 receptor signaling Macrophages respond to PGE2 via four transmembrane G-protein coupled receptors: EP1-4. EP2 and EP4 are coupled to G?s proteins, which stimulate intracellular cAMP production51. In combination with TLR stimulation, PGE2 inhibits IL-12 and TNF and partially decreases IL-6 production by macrophages52?54. At the same time, PGE2 enhances IL-10 release from mouse macrophages in a PKA-dependent manner55. PGE2 inhibits inflammasome activation in macrophages via signaling through the EP4 receptor, dampening the production of IL-1?56. Additionally, IL-17 production is increased in the presence of PGE2 therefore promoting M2 macrophage microenvironments57. Our lab previously showed that mouse macrophages stimulated with LPS and PGE2 exhibit an immuno-regulatory phenotype58. PGE2 and the immune response PGE2 helps to regulate the activation of many cells of the innate immune system59. The involvement of PGE2 in acute inflammation has been well-documented60 but, in contrast, PGE2 has also been demonstrated to also play a significant role in immunosuppression60,61. Inflammation in the lung in response to allergens and pollutants, as well as colonic inflammation, is dampened by PGE2 through the EP4 receptor signaling on macrophages62,63. PGE2 also downregulates MHC class II expression on dendritic in lymphoid organs in order to decrease antigen presentation64. It has been demonstrated that PGE2 inhibits the phagocytosis of bacterial pathogens and 11 bacterial killing by alveolar macrophages in a dose-dependent manner65,66. Along with this, many bacteria and intracellular parasites, including L. donovani, have developed mechanisms to stimulate PGE2 production by macrophages in order to suppress inflammation and promote survival inside the host59,67,68. Cyclooxygenase (COX) inhibitors are commonly used NSAID drugs that inhibit inflammatory responses. However, it has been shown that chronic inhibition of the COX enzymes in macrophages drives them towards a pro-inflammatory phenotype, partly due to lower synthesis of PGE 692 . Furthermore, it has been proposed that these classic inhibitors of prostaglandin synthesis may act to prolong chronic inflammation when taken during the resolving phase70. Macrophages are well-known producers of PGE 712 . Exposure of macrophages to LPS increases arachidonic acid metabolism, leading to greater PGE2 secretion72. Since macrophages synthesize endogenous PGE2, we recognize the profound effects of this molecule on the regulation of macrophage activation. 1.6 Interferons and innate immunity: Type I Interferon Interferons (IFNs) are cytokines that are best known for their involvement in the immune response to viral pathogens. However, IFNs also affect the response to microbial pathogens, the stimulation of antigen presentation, cell proliferation and apoptosis73. Type I IFNs include IFN? and IFN? and signal through the heterodimeric IFN? receptor (IFNAR). Signaling through IFNAR activates Janus kinase 1 (JAK1) and tyrosine kinase 2 (TYK2), followed by activation of signal transducer and activator of transcription (STAT) transcription factors, STAT1 and STAT2 as well as IFN regulatory factor 9 (IRF9)74,75. Type I IFNs can be produced by many cells and in macrophages they 12 can be produced following stimulation of TLR4, TLR3, TLR7 and TLR918. The effects of Type I IFN in response to bacterial infection are complicated and not fully understood. Often times they can contribute to host resistance to bacteria but sometimes they can promote bacterial survival via suppression of the innate immune response76. In mouse models of sepsis, Type I IFN deficiency led to a reduction in endotoxin lethality despite no change in inflammatory cytokine levels77. Type I IFNs also have contradicting roles in autoimmune disorders. Patients with systemic lupus erythematosus (SLE) have increased type I interferon in circulation that is thought to promote disease78. On the other hand, IFN? administration is an effective treatment for multiple sclerosis79. The activity of Type I IFNs appears to be highly context dependent. Type II Interferon The Type II IFN family consists solely of IFN?, which signals through a heterodimeric IFN? receptor to activate JAK1 and JAK2, which subsequently activate STAT180. Further downstream, IRF1 is strongly induced by IFN? which promotes Th1 responses73,81. IFN? is highly efficient at priming macrophages for enhanced bacterial killing and inflammation82. Its ability to enhance bacterial killing is largely mediated through nitric oxide and superoxide production83,84. IFN? is used as effective treatment for patients with chronic granulomatous disease, in which phagocytes are defective in superoxide and hydrogen peroxide production85. Priming also acts in coordination with TLR signaling to stabilize mRNAs that encode inflammatory genes leading to more efficiency in both transcription and translation of these proteins86,87. Type II IFN is mainly produced by natural killer (NK) cells and Th1 cells in response to infection88. While it is highly effective at stimulating innate immune cells to control pathogens, it can 13 have negative consequences for the host. For example, IFN? contributes to the pathogenesis of rheumatoid arthritis and SLE89,90. Overall, IFN? is effective at potentiating inflammatory immune responses. 1.7 Colony-stimulating factors: M-CSF and GM-CSF are both colony-stimulating factors that are important in myeloid cell differentiation and immune modulation. Many studies have addressed the role of these two growth factors in terms of cell survival, as they prolong the life of macrophages and are regulators of hematopoiesis. A few gene expression studies have been done comparing macrophages differentiated in each growth factor in both mouse and human33,91?93. However, extensive research has not been done on the inflammatory response of both M-CSF and GM-CSF macrophage populations and their sensitivity to endogenous mediators. M-CSF M-CSF signals through the C-FMS receptor or CSF-1R. This receptor is a single pass type I membrane protein and has a tyrosine kinase domain94. Upon binding of M- CSF, receptors dimerize. Downstream of this receptor is PI3K, Src family kinases, Ras, ERK1/2, Akt, and PLC?2 signaling94?96. SHIP2 tyrosine phosphorylation following M- CSF stimulation leads to reduced Akt activation and inhibition of NF?B gene transcription independently of a functional SH2 domain, unlike SHIP197. Macrophages have been shown to regulate the levels of M-CSF in circulation by CSF-1R mediated endocytosis98. M-CSF is produced ubiquitously by many cells types including endothelial cells, lymphocytes, fibroblasts and monocytes99. CSF1R is critical for the maintenance of 14 monocyte and macrophage populations. Mice lacking CSF1R are deficient in several tissue macrophage populations including Kupffer cells, microglia and skin macrophages100. The addition of recombinant human M-CSF in mice resulted in a significant increase in blood monocytes in circulation and an expansion of resident macrophage populations101. Increased levels of M-CSF have been reported in numerous diseases including arthritis, pulmonary fibrosis, inflammatory bowel disease, and cancer102. A number of monoclonal antibodies and small molecules targeting the CSF- 1R have been in clinical trials for targeting solid tumors102. GM-CSF GM-CSF signals via the ?c receptor Type I cytokine receptor family, similarly to IL-3 and IL-5. A ternary complex between the ?c receptor, the GM-CSF receptor specific alpha chain and the GM-CSF molecule is required for signaling103. Downstream of the GM-CSF receptor complex is JAK2/STAT5 activation, MAPK, and PI3 kinase/Akt pathway activation104,105. GM-CSF also activates NF?B by interaction of the alpha chain of the GM-CSF receptor and I?B kinase106. Further downstream, GM-CSF activates IRF5 transcription factor, which shapes macrophage polarization107. It has recently been shown to also activate IRF4 in order to drive the CCL17. GM-CSF, like M-CSF, can also be produced by endothelial cells, epithelial cells, and fibroblasts that are activated, but is mainly produced by TH17 T cells and innate lymphoid cells in response to infection or trauma99. The inflammatory cytokine, IL-1 appears to be particularly important in the induction of GM-CSF from a number of cell types108?111. There are a multitude of diseases in which the circulating levels of GM-CSF are increased including encephalomyelitis, rheumatoid arthritis, systemic 15 inflammation112, and even allergic responses113. Multiple clinical trials for monoclonal antibodies to GM-CSF have been undertaken in the context of rheumatoid arthritis, asthma and multiple sclerosis102. The main tissue in which GM-CSF is constitutively expressed is the lung. Humans with a point mutation in the common beta chain of the GM-CSF receptor develop a condition called pulmonary alveolar proteinosis (PAP)114. Mice lacking GM-CSF develop lung abnormalities and also have symptoms mirroring PAP115. Additionally, these GM-CSF deficient mice are more susceptible to local infections, in the lung particularly115. For example, neutralization of GM-CSF in mice led to a reduction in protective immunity to histoplasma infection, indicating that this cytokine plays a role in host defense116. 1.8 Macrophage activation during disease or injury Sepsis Macrophages are critical drivers of inflammation due to their potent cytokine producing capabilities. In the case of sepsis, this high level of cytokine production, known as a ?cytokine storm?, by macrophages in the early phases of infection is what leads to a high mortality rate for the host. Sepsis is a serious condition that affects over 30 million people per year, and results in over 5 million deaths. The blockade of inflammatory cytokines such as TNF and IL-1 have proven to be inefficacious in the reversal of sepsis. However, the anti-inflammatory cytokine IL-10 contributes significantly to the progression of the disease. Cecal ligation and puncture model (CLP) in IL-10 knockout mice was associated with 15-fold higher serum levels of TNF, but treatment with recombinant IL-10 led to improved survival and a longer therapeutic window for rescue surgery117. Regulatory macrophages are partly defined by their ability 16 to secrete increased amounts of IL-10. A previous graduate student in our lab demonstrated in mice, that macrophages stimulated with LPS + Adenosine and LPS + PGE2 had a protective effect on mouse survival when injected into the peritoneum of the mice in an endotoxemia model118. Additionally, earlier work in our lab demonstrated that Fc?R ligation promotes IL-10 production in mice, leading to protection in sepsis models11. Therefore, multiple methods of generating these regulatory macrophages can have a potentially therapeutic effect in sepsis. Tumor associated macrophages Macrophages are one of the main immune cells residing in the tumor microenvironment. These macrophages are termed tumor associated macrophages (TAMs) and generally are not activated against the tumor. The production of pro- inflammatory cytokines by macrophages in the tumor environment is critical for the activation of cytotoxic T cell responses119. A few treatment strategies such as checkpoint inhibitors and CAR T cell therapies targeting tumor antigens have proven successful in many cancer patients. However, it has been demonstrated that in those patients in which these immunotherapies are unsuccessful, there can be an abundance of tumor associated myeloid cell infiltrates, including macrophages120. These macrophages display an immunosuppressive, and pro-angiogenic phenotype. Tumors secrete immunosuppressive molecules, including both adenosine and PGE2, which we believe shapes the macrophage phenotype to one that is anti-inflammatory and angiogenic in order to support the tumor?s growth121,122. Therefore, methods of reversing or preventing this macrophage phenotype could prove beneficial in cancer patients. 17 Intracellular Parasitic Infections Macrophages are host to numerous parasitic and bacterial pathogens. These pathogens include Leishmania, Mycobacteria, Toxoplasma, and Trypanosoma, among others123. These pathogens reside intracellularly and have evolved numerous mechanisms to evade the intense macrophage anti-pathogen response. The anti-pathogen response includes the formation of reactive nitrogen and oxygen species, protease activation, programmed cell death, and cytokine production124. The phenotype of macrophages largely affects the survival of these pathogens within them. For example, many studies have demonstrated that L. major have decreased cell proliferation and increased death in macrophages that produce nitric oxide and super oxide in mice125?128. A number of molecules can stimulate macrophages to produce these oxygen radicals including zymosan, LPS and IFN?. GM-CSF also changes the macrophage phenotype leading to poor L. major and L. tropicana survival129,130. Additionally, GM-CSF has been shown to increase hydrogen peroxide release by mouse peritoneal macrophages and GM-CSF cultured microglia restricted the intracellular multiplication of T. gondii via the synthesis of reactive nitrogen intermediates131,132. GM-CSF also leads to greater control of T. cruzi by inhibiting its replication via increased IL-12, NO and IFN? production133,134. Conversely, M. tuberculosis seem to have increased survival in GM- CSF macrophages compared to M-CSF macrophages135. One study showed that adding M-CSF to restore homeostatic levels in the lungs of M. tuberculosis infected mice, led to greater activation of the adaptive immune response to the pathogen and led to decreased survival136. Overall, manipulation of the macrophage phenotype has clear consequences for the survival of intracellular pathogens. 18 Wound Healing Macrophages play a critical role in the response to injury in muscle, skeleton, skin and other organs. While macrophages initially mount an inflammatory immune response following injury, they also are active in the return to homeostasis137. An early study in mice found that the depletion of macrophages in the wound sites led to delayed debridement and also slowed fibroblast recruitment and proliferation affecting the wound closure time138. The wound healing process is largely orchestrated by the macrophage secretome. The secretome includes molecules like TGF?, VEGF and EGF, which have been shown to be essential for angiogenesis. One early study indicated that recombinant human M-CSF accelerated wound healing in non-ischemic wounds in rabbits by significantly increasing the levels of TGF? 139. The timing of the conversion of inflammatory, TNF producing macrophages to growth promoting, TGF? secreting macrophages is particularly important in muscle regeneration140,141. Other molecules secreted by macrophages, such as anti-inflammatory cytokines IL-10, IL-4 and IL-13 along with lipid mediators like lipoxins and resolvins can contribute to the initiation of tissue repair. In muscle, infiltrating blood monocytes that differentiate into macrophages convert to pro-regenerative macrophages that promote the growth of new myofibers once phagocytosis of debris is complete142. Macrophages also indirectly contribute to wound healing by recruiting other cell types such as fibroblasts, mesenchymal cells and mesoangioblasts143. Subsequently, macrophages promote the proliferation of these cells at the wound site in order to instruct the tissue repair mechanism and initiate angiogenesis144. If macrophages do not function properly during wound healing, this can lead to fibrosis. 19 1.9 Scope and Limitations The research in this dissertation was performed on human monocyte-derived macrophages. These cells represent a subset of macrophages in the host that develop once monocytes migrate into different tissues in response to inflammatory signals, and subsequently encounter colony-stimulating factors. We studied these macrophages due to the feasibility of their collection from human blood. Although these macrophages are separate in origin from yolk sac or fetal liver-derived tissue macrophages, we believe that all macrophages exhibit plasticity and can respond to molecules in their microenvironment in order to alter their phenotype. 20 2 Materials and Methods 2.1 Mouse BMDM preparation Bone marrow hematopoietic stem cells were flushed from the femurs of 6-8 week old C57/bl6J mice (Cat# 000664, Jackson Laboratory, Bar Harbor, ME) in saline containing 1% penicillin/streptomycin145. Cells were plated on petri dishes in DMEM/F-12 + Glutamax media (Cat# 10565018, Life Technologies, Grand Island, NY) containing 10% heat-inactivated fetal bovine serum (Cat# S11550, Atlanta Biologicals, Flowery Branch, GA) supplemented with 20% conditioned media obtained from the culture of the L-929 mouse fibroblast cell line (Cat# ATCC). New media was added on day 4. On day 7, differentiated macrophages were removed from petri dishes using Cell stripper (Corning). Other strains of mouse macrophages were also used including A2br-/-146, A2ar-/- and STAT1-/- mice (Cat#s 010685, 012606, Jackson Laboratory). 2.2 Mouse BMDM stimulation Mouse macrophages were stimulated with the following reagents: 10 ng/mL ultra pure LPS from Escherichia coli K12, IFN? 10000 U/mL, IFN? 10000 U/mL, and IFN? 10000 U/mL. They were also stimulated with various TLR-ligands (Cat# tlrl-kit1mw, Invivogen). 2.3 Human macrophage differentiation Whole blood was isolated from healthy donors under University of Maryland, IRB approved protocols. Human monocytes were isolated via density gradient centrifugation followed by negative isolation using immunomagnetic beads (Cat# 130-096-537, Miltenyi Biotec, San Diego, CA). Monocytes were cultured for 7 days in X-VIVO 15 serum-free media (Cat# 04-744Q, Lonza, Walkersville, MD) containing 1% penicillin- 21 streptomycin, 1% L-glutamine (Cat# 25-005-CI, Gibco, Gaithersburg, MD), and supplemented with either 30 ng/mL recombinant human M-CSF or 20 ng/mL recombinant human GM-CSF (Cat# AF-300-25, Cat# 300-03 respectively, Peprotech, Rocky Hill, NJ). Media containing either growth factor was replenished on day 4 following initial culture. Prior to stimulation on day 7, media containing growth factor was replaced with X-VIVO 15 media containing 5% heat-inactivated fetal bovine serum (Cat # S11550, Atlanta Biologicals, Flowery Branch, GA). 2.4 Human macrophage stimulation LPS stimulated macrophages were generated by the addition of 10ng/mL ultra pure LPS from Escherichia coli K12 (Invivogen, San Diego, CA). LPS + Adenosine macrophages were generated by the addition of 10 ng/mL LPS and 50 ?M adenosine (Cat# A4036, Sigma-Aldrich, St. Louis, MO). LPS + PGE2 macrophages were generated by the addition of 10 ng/mL LPS and 50 nM PGE2 (Cat# 2296, Tocris, Bristol, UK). Pharmacological inhibitors, ONO AE3 208, PF 04418948, SCH 442416, were used to inhibit EP4, EP2 and A2aR, respectively (Cat#s 3565, 4818, 2463, Tocris). TLR agonists, FSL-1, HKLM, Loxoribine, and Poly I:C were added to macrophages for 4 hours (Cat#s tlrl-fsl, tlrl-hklm, tlrl-lox, tlrl-pic Invivogen). Cell permeable cAMP analogs, 8-Bromo-cAMP, specific for PKA, and 8-pCPT-2-O-Me-cAMP-AM, specific for Epac, were added to stimulated macrophages for 24 hours (Cat#s 1140 and 4853, respectively, Tocris). 2.5 RNA sequencing sample and library preparation Total RNA was isolated from macrophages using the Trizol reagent. RNA cleanup was done using RNeasy Mini Kit columns (Cat# 74106, Qiagen, Hilden, Germany). RNA 22 quality was determined using an Agilent 2100 bioanalyzer. Poly(A)+-enriched cDNA libraries were generated using the Illumina TruSeq sample preparation kit (Cat#s 16027084, 15027387, 1502062, Illumina, San Diego, CA) and quality of the cDNA was determined again with the bioanalyzer. Paired end reads (100bp) were obtained from an Illumina HiSeq 1500. Reads were aligned to the human genome (Homo_sapiens.GrCh38.79) obtained from the UCSC genome browser (http://genome.ucsc.edu) using Kallisto147. Count tables were restricted to protein-coding genes (34,425) and on-expressed or weakly expressed genes (< 1 read per million in n=5 samples) were removed prior to subsequent analyses, resulting in 12,857 genes analyzed. Quantile normalization and log2-transformation was done on all samples. 2.6 RNA sequencing data assessment, visualization and differential expression analysis Limma, a Bioconductor package, was used to perform differential expression analysis. The voom module was used to transform the data based on observational level weights derived from the mean-variance relationship prior to statistical modeling. Experimental batch effects were adjusted for by including experimental batch as a covariate in our statistical model. Differentially expressed genes were defined as genes with a log2 fold- change > 1 and a Benjamini-Hochberg (BH) multiple-testing adjusted p. value < 0.05. All components of the statistical pipeline, named cbcbSEQ, can be accessed on GitHub (https://github.com/kokrah/cbcbSEQ/). 2.7 Single cell RNA-sequencing sample and library preparation Monocyte derived macrophages generated in M-CSF from a single donor were stimulated for 4 hours with nothing, LPS, LPS + Adenosine and LPS + PGE2 and processed 23 according to the 10x library preparation method. Briefly, Gel Beads-in-emulsion (GEMs) were generated by combining Single Cell 3? v3 Gel Beads, master mix containing cells, and partitioning oil onto Chromium Chip B. Following GEM generation, the Gel Beads were dissolved and mixed with primers containing the 10x Barcode, a TruSeq sequencing primer, a unique molecular identifier and a poly(dT) sequence, in order to produce full-length, barcoded cDNA. This barcoded cDNA was amplified by PCR in order to construct libraries. The above components were included in a library construction kit (Cat# PN-1000075, 10x Genomics, San Francisco, CA). Libraries were sequenced using paired-end Illumina sequencing. 2.8 Single Cell RNA-sequencing analysis The samples were sequenced and processed with cell ranger 3.0.1 at the Johns Hopkins Genetics Resources Core Facility. The resulting outputs were passed to Seurat 3.1.0, merged by sample, and filtered to remove cells with high mitochondrial content (> 15%) and few features (< 200). The remaining data was passed through the default Seurat pipeline. The analysis was done on 17306 cells, with an average of 4327 cells per sample. The analysis entailed normalization, variable feature selection, data scaling, neighbor and cluster searches, the accompanying visualizations, and differential expression of markers across conditions and samples. 2.9 ELISA Cytokine and growth factor levels were measure in the supernatants of 24 hour stimulated macrophages. IL-12p40 and TNF were measured using paired antibody ELISA kits (Cat# BMS2013MST, Invitrogen, Vienna, Austria and Cat# 555212, BD Biosciences, 24 San Diego, CA). GM-CSF, VEGF, and THBS1 were measured using DuoSet ELISA kits (Cat#s DY215, DY293B, DY3074, respectively, R&D, Minneapolis, MN). IL-6 and IL-10 were also measured using OptEIA ELISA sets (Cat#s 555220 and 555157, respectively, BD Biosciences). 2.10 Quantitative real-time PCR RNA was isolated from cells using the Trizol reagent. cDNA was synthesized using Superscript VILO cDNA synthesis kit. Relative quantitation of transcript levels was performed using SYBR-Green. Samples were analyzed in a Roche Light Cycler 480. Expression levels were calculated using the ?Ct method relative to the geometric mean of GAPDH and RAB7 as internal control genes. The primer sequences used to measure transcripts are listed in Table 1. 25 Table 1. Primer Sequences Gene Accession number Sequence GAPDH Forward NM_002046.7 5?-ATAAATTGAGCCCGCAGCC-3? Reverse 5?-CATGTAAACCATGTAGTTGAGGTC-3? RAB7 Forward NM_177403.5 5?-GGTTCCAGTCTCTCGGTGTG-3? Reverse 5?-CGCTTTGTGGCCACTTGTC-3? THBS1 Forward NM_003246.4 5?-GAAGGACTCTGACGGCGATG-3? Reverse 5?-GATGTCCCTTTGGGGTCCAG-3? CD300E Forward NM_181449.3 5?-GTTTCCCCAGCAATTACAACCC-3? Reverse 5?-CAGAAGACAGCACCCAGCAT-3? AREG Forward NM_001657.4 5?-TGTCGCTCTTGATACTCGGC-3? Reverse 5?-GGCATTTCACTCACAGGGGA-3? VEGFA Forward NM_001171623.1 5?-CATGCCAAGTGGTCCCAGG-3? Reverse 5?-GCTGGCTTTGGTGAGGTTTG-3? CD93 Forward NM_012072.4 5?-TGGAGAACCAGTACAGTCCG-3? Reverse 5?-GAGTCACGAAATCCCCACCG-3? CXCL13 Forward NM_006419.2 5?-TCTCTCCAGTCCAAGGTGTTC-3? Reverse 5?-AGCTTGAGGGTCCACACAC-3? MMP10 Forward NM_002425.3 5?-CAGGCATTTGGATTTTTCTACTTCT-3? Reverse 5?-CTGTCTTCCCCCTATCTCGC-3? RGS2 Forward NM_002923.4 5?-AAGAGCGAGGAGAAGCGAG-3? Reverse 5?-GCAAGACCATATTTGCTGGCT-3? TGFA Forward NM_003236.4 5?-CCTGTTCGCTCTGGGTATTGT-3? Reverse 5?-GGTGATGGCCTGCTTCTTCT-3? ADORA2A Forward NM_001278497 5?-CATCCCGCTCCGGTACAATG-3? Reverse 5?-TGGTTCTTGCCCTCCTTTGG-3? ADORA2B Forward NM_000676.2 5?-GACGCCCACCAACTACTTCC-3? Reverse 5?-TTTATACCTGAGCGGGACACA-3? PTGER2 Forward NM_000956.4 5?-GCTCCTTGCCTTTCACGATTT-3? Reverse 5?-AGGATGGCAAAGACCCAAGG-3? PTGER4 Forward NM_000958.3 5?-CCGCTCGTGGTGCGAGTATT-3? 26 Reverse 5?-GGCCTGACATGGCAGAAGAT-3? COX1 Forward NM_000962.4 5?-TGGTTCTTGCTGTTCCTGCT-3? Reverse 5?-CACAGGCCAGGGATGGTG-3? COX2 Forward NM_000963.4 5?-CATCCCCTTCTGCCTGACAC-3? Reverse 5?-TCCTACCACCAGCAACCCTG-3? MPGES1 Forward NM_004878.5 5?-GAAGTGGCTGATGGGAACCA-3? Reverse 5?-GGAGGGAGAGGGAGTGATGT-3? Mouse A2AR Forward NM_009630.3 5?-CCATTCGCCATCACCATCAG-3? Reverse 5?-CCCGTCACCAAGCCATTGTA-3? Mouse A2BR Forward NM_007413.4 5?-GACTCTTCGCCATCCCCTTT -3? Reverse 5?-ACAGCAATGATCCCTCTCGC-3? 27 2.11 Measurement of PGE2 production Prostaglandin E2 levels in the supernatants of 24 hour LPS stimulated M-CSF and GM- CSF macrophages were measured using a monoclonal antibody competitive ELISA kit (Cat # 514010, Cayman Chemical, Ann Arbor, MI). 2.12 Measurement of ATP degradation M-CSF and GM-CSF macrophages were given a spike of 20 ?M ATP (Cat# A6419, Sigma-Aldrich). Supernatants were collected at 2 hours, 1 hour, 30 minutes and 15 minutes following this spike and ATP was measured using the ATPlite reagent (Cat# 6016941, Perkin Elmer, Waltham, MA). Levels of ATP were normalized to the amount of protein in the wells using the Pierce BCA Protein Assay Kit for protein quantification (Cat# 23227, ThermoScientific, Rockford, IL). Luminescence was read in the dark- adapted plate using a luminometer. 2.13 HUVEC tube formation assay Primary human endothelial cells (HUVECs) were obtained and cultured in EGM-2 media (Cat# CC-3162, Lonza) on tissue culture treated plates coated with 1% gelatin from porcine skin (Cat# G1890, Sigma). For the assay, HUVEC cells were distributed at a concentration of 40,000 cells in each well of a 48-well plate. These wells contained growth factor reduced and phenol red-free Matrigel (Cat# 356231, Corning). Supernatants collected from M-CSF and GM-CSF macrophages that were unstimulated, or stimulated with LPS, LPS + Adenosine, and LPS + PGE2 for 24 hours were added to the HUVEC cells on the Matrigel and allowed to incubate for 24 hours. Images of the HUVEC cells were captured in brightfield on an inverted Nikon ECLIPSE Ti2 Microscope at 20x total magnification. Images were converted to high contrast using the 28 ?Find edges? function in ImageJ in order to see the cells. Tube length and number of nodes were assessed manually using the ImageJ software. 2.14 Flow cytometry CD300E and PLAUR surface expression was measured on macrophages stimulated for 24 hours and 8 hours, respectively, using APC conjugated antibodies (Cat# 17-3007-42 and Cat# 17-3879-42, Invitrogen, Carlsbad, CA). Fc block was used to reduce nonspecific binding (Cat# 130-059-901, Miltenyi Biotec). Debris and doublets were removed using gating analysis in FlowJo version X. Surface expression is expressed as median fluorescence intensity (MFI). 29 3 The effects of interferons on purinergic signaling in mouse macrophages Parts of this chapter are adapted from published work: Cohen HB, Ward A, Hamidzadeh K, Ravid K, Mosser DM. IFN-? Prevents Adenosine Receptor (A2bR) Upregulation To Sustain the Macrophage Activation Response. J Immunol. 2015;195(8):3828?3837. doi:10.4049/jimmunol.1501139 3.1 Introduction It has previously been demonstrated in mouse macrophages that purinergic signaling dampens inflammatory responses to LPS29,148,149. Specifically, inflammatory cytokines including TNF and IL-12p40 are significantly reduced in activated macrophages in the presence of ATP or adenosine26,150. Purinergic signaling is thought to be a mechanism to control the level and duration of macrophage inflammation in the host. However, there are situations in which a prolonged inflammatory response is desirable, such as with severe infections. There are a number of signals that are associated with modulating the severity of an immune response, including interferons. Interferons come in different varieties, called Type I, II and III. IFN beta (IFN?) and IFN alpha (IFN?) are Type I, IFN gamma (IFN?) is Type II and IFN lambda (IFN?) is Type III. M1 macrophages, which are generated by stimulation with IFN? + LPS exhibit severe inflammation151. Our lab established a connection between interferons and the purinergic system in the mouse, by demonstrating that IFN? priming of macrophages prevented the upregulation of the A2b receptor (A2br) following LPS stimulation7. We extended this research to further explore the effects of IFN? on adenosine signaling in 30 macrophages, and we also examined the role of Type II interferon, IFN?, in the mouse macrophage response to LPS. 3.2 Results Type II interferon modulation of macrophage activation. The action of IFN? priming of LPS stimulated mouse macrophages has been shown to significantly augment the levels of inflammatory cytokine production. It was demonstrated in our lab that this heightened inflammation was due to IFN? priming preventing the upregulation of the A2b receptor upon LPS stimulation7. As part of this work, we examined the effects of other TLR ligands on A2BR and A2AR mRNA expression (Figure 1). All of the TLR ligands tested (LPS-EK, Pam3Csk4, HKLM, Poly(I:C) LMW, Poly(I:C) HMW, ST-FLA, FSL-1 and ssRNA) which activate TLRs 1-9 upregulated mRNA levels for A2BR. In all stimulations except ssRNA, IFN? priming significantly downregulated expression of the A2BR (Figure 1A). As for A2AR mRNA levels, there was no significant difference between unprimed and IFN? primed macrophages (Figure 1B). In order to further validate the effect of IFN? priming on mouse macrophages, we looked at purinergic receptor expression in STAT1 -/- mouse BMDMs since IFN? is known to signal through STAT1152. The prevention of the upregulation of A2BR mRNA by IFN? priming was not observed in STAT1 -/- macrophages (Figure 2A). Additionally, STAT1 -/- macrophages did not significantly differ in mRNA expression of A2AR (Figure 2B). As expected, the lack of STAT1 abrogated the effect of IFN? priming on TNF production following LPS stimulation and also allowed adenosine to function in decreasing the amount of TNF produced by LPS stimulated macrophages, presumably through the A2b receptor (Figure 3A)7. Preliminary data also suggested that the lack of STAT1 31 A. B. A2BR A2AR 40 4 Unprimed **** Unprimed IFN Primed 30 IFN Primed 3 **** **** 20 2 ** ******** ** 10 1 0 0 ive EK k4- s M A 1 A a KL M W MW L L--F S RN aiv e EK 4N S C L H s S- Cs k KL M W W LA -1 A P 3 H ) M M) T F N 3 H L H -F S L RN L m C C S s LP s Pa (I: (I: am (I:C ) ) C ST F s oly oly P I: P ol y ( P P Po ly Figure 1. Adenosine receptor expression following IFN? priming and stimulation with various TLR ligands. (A) A2BR and (B) A2AR mRNA expression was measured by qPCR following 16 hours of IFN? priming and 4 hours of stimulation with TLR ligands (n=3, ** P-value < 0.01, **** P-value < 0.0001, error bars represent SEM). 32 Expression relative to Naive/GAPDH Expression relative to Naive/GAPDH A. A2BR B. A2AR 40 8 #### 30 6 WT WT STAT1 KO 20 4 STAT1 KO 10 2 **** 0 0 ive Ng PS PS ive Ng PS PS Na IF L g/LN N a IF L g/L IF IFN Figure 2. Adenosine receptor expression in STAT1 knockout BMDMs. (A) A2BR and (B) A2AR mRNA expression was measured in WT (black) and STAT1 KO (grey) macrophages following 16 hours of IFN? priming and 4 hours of LPS stimulation (n=3, **** P-value < 0.0001 relative to WT LPS alone, #### P-value < 0.0001 between WT and KO, error bars represent SEM). Figure generated with Dr. Heather B. Cohen 33 Expression relative to Naive/GAPDH Expression relative to Naive/GAPDH A. TNF B. C.IL12p40 IL10 15 **** 14 WT **** 10 WT 15 13 STAT1 KO STAT1 KO WT 12 8 STAT1 KO1.0 10 0.8 6 0.6 4 0.4 5 2 0.2 0.0 0 0 ive PS do Ng PS do ivea L A IF /L A a LP S o g S o e S o g S o N + g + N +A d IFN /LP A d aivg + N L P +A d IFN g/L P +A d PS SFN S S S S L I g/L P LP IFN g/L P LP IFN /LP IFN g IFN IFN Figure 3. Cytokine levels in WT and STAT1 knockout BMDMs. Macrophages were unprimed or primed for 16 hours with IFN? followed by LPS stimulation for 8 hours at which point supernatants were collected and assayed for (A) TNF by ELISA (n=3, **** P-value < 0.0001, error bars represent SEM). Preliminary data is shown for (B) IL-12p40 and (C) IL-10 levels assayed by ELISA (n=2, error bars represent SD). Figure generated with Dr. Heather B. Cohen 34 TNF (ng/mL) IL-12p40 (ng/mL) IL-10 ( ng/mL) diminished the inhibitory effect of IFN? priming on adenosine signaling in terms of IL- 12p40 (Figure 3B) and IL-10 production (Figure 3C). Next we examined the effect of A2br -/- on inflammatory cytokine production and demonstrated that the downregulation of IL-12p40 by adenosine is mediated largely through the A2b receptor for both unprimed and IFN? primed macrophages (Figure 4A). IFN? primed macrophages stimulated with LPS + Adenosine also significantly differed in terms of TNF production in WT and A2br -/- mice (Figure 4B). A time course experiment indicated that TNF levels were significantly higher in A2br -/- macrophages between 0-2 hours in the presence of LPS, however this difference was not sustained over time in the proceeding absence of LPS (Figure 4C). Type I interferon modulation of macrophage activation. We wondered whether priming with Type I or III interferons had any effects on the purinergic pathway. While IFN? lowers mRNA expression for A2BR, IFN? significantly increased mRNA expression for both A2BR (Figure 5A) and A2AR (Figure 5B) while IFN? had no significant effects. Therefore, we continued to investigate the effect of IFN? by examining adenosine receptor expression in IFNAR -/- mice (Figure 6). mRNA expression of A2BR was higher in LPS stimulated IFNAR -/- BMDMs, but there was no difference in its expression in IFN? primed WT and IFNAR-/- BMDMs (Figure 6A). The upregulation of A2AR mRNA expression by IFN? priming was abolished in IFNAR -/- BMDMs (Figure 6B). IFN? priming did not significantly change the levels of IL- 12p40 or TNF in WT BMDMs but increased the levels of IL-10 (Figure 7). A2AR -/- BMDMs also did not significantly differ in IL-12p40 (Figure 7A) or TNF levels (Figure 7B) relative to WT BMDMs with the exception of IFN? priming which led to lower 35 A. B. IL-12p40 TNF 12 16 10 WT **** 14 WT ** 8 6 A2BR KO 12 10 A2BR KO 2.0 0.4 1.5 * 1.0 0.2 0.5 0.0 0.0 e S o g S o e S o g a?v LP Ad FN LP Ad a? v LP Ad IFN /LP S do N + I / + N + g +Ag LP S IFN S S N LP LP IF /LP S g/ Ng C. I FN IF 8 *** A2BR KO IFNg/LPS 6 A2BR KO LPS WT IFNg/LPS WT LPS 4 2 0 0 2 3 4 6 8 Hours Figure 4. Cytokine levels in WT and A2B receptor knockout BMDMs. Macrophages were unprimed or primed for 16 hours with IFN? followed by LPS stimulation for 8 hours at which point supernatants were collected and assayed for (A) IL-12p40 and (B) TNF (n=3, * P-value < 0.05, ** P-value < 0.01, **** P-value < 0.0001, error bars represent SEM). (C) Unprimed and IFNg primed macrophages were stimulated with LPS for 2 hours followed by a wash. Supernatants were collected at subsequent incubation timepoints following the wash and assayed for TNF levels (n=4, *** P-value < 0.001, error bars represent SEM). 36 IL-12p40 (ng/mL) TNF (ng/mL) TNF (ng/mL) A. A2BR B. A2AR 100 30 **** **** 80 Unprimed Unprimed IFN Primed 20 IFN Primed 60 IFN Primed IFN Primed IFN Primed IFN Primed 40 10 20 *** 0 0 Naive LPS Naive LPS Figure 5. Adenosine receptor expression following priming with Type I, II and III IFNs. mRNA expression of (A) A2BR and (B) A2AR was measured by RT-PCR following 16 hours of priming with IFN? , IFN?, and IFN? and subsequent stimulation for 4 hours with LPS (n=3, *** P-value < 0.001, **** P-value < 0.0001, error bars represent SEM). 37 Expression relative to Naive/GAPDH Expression relative to Naive/GAPDH A. B. A2BR A2AR 0.15 0.020 ** **** 0.015 0.10 WT WT IFNAR KO IFNAR KO 0.010 0.05 0.005 0.00 0.000 Naive IFNb LPS IFNb/LPS Naive IFNb LPS IFNb/LPS Figure 6. Adenosine receptor expession in IFNAR knockout BMDMs. (A) A2BR and (B) A2AR mRNA expression was measured in WT (black) and IFNAR KO (hatched) macrophages following 16 hours of priming with IFN? and 4 hours of LPS stimulation (n=3, ** P-value < 0.01, **** P-value < 0.0001, error bars represent SEM). 38 Expression relative to Naive/GAPDH Expression relative to Naive/GAPDH A. IL-12p40 B. TNF C. IL10 18 **** 18 **** 9 ** WT 15 WT WT **** 16 A2AR KO 12 A2AR KO A2AR KO 14 9 ** 8 6 6 * 6 4 4 3 2 2 0 0 0 ve b g e b g o o o a? FN FN LP S PS SL LP Ad o Ad o o Ad a? ve Nb g S S S do do do ?v N N PS PS PS d d d N I I b/ g/ + + + N I F IFN LP /LP /LP +A +A +A Na IFb g I F L b/L g/L +A +A +A IFN IFN P S S S N N L LP LP IF IF LP S PS PS N N PSF F PS S / / /L /L I I L b/L /L P Nb Ng bFN FN g IFN IFN g IF IF I I Figure 7. Cytokine levels in WT and A2A receptor knockout BMDMs. Macrophages were unprimed or primed for 16 hours with IFN? or IFN? followed by LPS, and LPS + Ado stimulation for 8 hours at which point supernatants were collected and assayed for (A) IL-12p40, (B) TNF and (C) IL-10 secretion (n=3, error bars represent SEM). 39 IL-12p40 (ng/mL) TNF (ng/mL) IL-10 ( ng/mL) levels of these inflammatory cytokines relative to WT. In contrast, A2AR -/- BMDMs produced significantly lower levels of IL-10 following LPS and LPS + Adenosine stimulation in unprimed and IFN? primed macrophages (Figure 7C). Based on our results we concluded that priming with Type I and Type II interferons had different effects on the purinergic pathway in mouse macrophages. 3.4 Discussion It is widely accepted that IFN? treatment of macrophages renders them hyper- inflammatory. We demonstrated that this is partly due to the prevention of the upregulation of the A2B receptor and that this mechanism is universal for a number of pathogenic components that activate different TLR ligands in macrophages7. We also demonstrated that the adenosine A2A receptor is not implicated in the IFN? primed macrophage response. We verified that the effects of IFN? on A2B receptor expression and TNF production were mediated through the STAT1 signaling pathway as its effects were diminished in STAT1 knockout macrophages. This was no surprise since IFN? is known to signal through STAT1, so it should not be able to function if STAT1 is not present. We demonstrated using knockout macrophages that the A2b receptor is critical for the immunosuppressive effects of adenosine on IL-12p40 and TNF production by macrophages, but that its effects are not sustained in the absence of inflammatory stimulus. In the literature, there are conflicting reports of the effects of IFN? treatment on inflammation, including cytokine production76. We observed that IFN? increased transcription of both the A2BR and A2AR in macrophages, which suggests greater susceptibility to a spontaneous reversion to homeostasis. This is in contrast to the IFN? 40 downregulation of the A2B receptor, which we believe indicates that IFN? instructs a milder and more controlled inflammatory response in macrophages. Studies in IFNAR knockout macrophages indicated that the upregulation of A2A receptor expression was mediated through IFN? and not IFN?. However, A2B receptor expression was significantly higher in LPS stimulated IFNAR knockout macrophages compared to WT, at levels roughly equal to the level induced by IFN? priming. This suggested that IFN? may play a role in suppressing A2BR expression in WT cells, as this IFN also signals through the IFNAR receptor, and that perhaps IFN? may oppose this effect. The A2a receptor contributed to the production of increased IL-10 in IFN? primed macrophages and their sensitivity to adenosine, as knockout macrophages produced lower levels of IL- 10 compared to wild type. However, the A2a knockout macrophages did not produce notably different levels of TNF or IL-12p40. Altogether, the data in this chapter implicates a role for adenosine and its receptors in the potentiation of inflammation by IFN? and IFN? in mouse macrophages. 41 4 Characterization of the transcriptome of human M-CSF derived macrophages stimulated with LPS + Adenosine and LPS + PGE2 4.1 Introduction The study of human monocyte derived macrophages is critical, considering the importance of these cells in a number of organs. Monocytes infiltrate tissues and differentiate into macrophages following exposure to a constitutively expressed growth factor, M-CSF. They do so in response to infection, or to replenish the native populations of tissue macrophages that may have become depleted. It has been shown that monocyte derived macrophages are particularly important in the intestine, skin, and peritoneum as well as in disturbances such as atherosclerosis, muscle injury and inflammation153. In this research, we attempt to describe the phenotype of human monocyte derived macrophages stimulated by TLR ligands, such as LPS in the presence of adenosine (LPS + Adenosine) or prostaglandin E2 (LPS + PGE2), using RNA sequencing. We believe that our samples and stimulation conditions mimic physiological environments that macrophages can potentially encounter in the body. For example, the tumor microenvironment contains high levels of purinergic signaling molecules as well as PGE2. These molecules are also produced during inflammatory immune responses by macrophages and other cell types. We use RNA sequencing because it gives us a snapshot of the entire transcriptome at our chosen timepoints. This can allow us to characterize the nature and function of macrophages under different physiological contexts. 4.2 Results RNA-sequencing analysis. RNA-seq was performed on unstimulated, LPS stimulated, LPS + Adenosine and LPS + PGE2 stimulated macrophages from 5 blood donors. 42 Differential expression analysis allowed us to determine the effects of adenosine and PGE2 on LPS stimulation (Figure 8). Our analysis revealed that adenosine and PGE2 have similar effects on the stimulated macrophage transcriptome. 4 of the 10 most highly upregulated genes and 5 of the 10 most downregulated genes in LPS + Adenosine (Figure 8A, starred) and LPS + PGE2 (Figure 8B, starred) versus LPS alone were shared. Venn diagrams of the number of significant differentially expressed genes further highlight the degree of similarity between adenosine and PGE2 stimulation relative to LPS stimulation alone (Figure 9A). 101 of the 259 upregulated genes and 91 of the 294 downregulated genes were shared by LPS + Adenosine and LPS + PGE2 stimulation. For comparison, LPS stimulation alone versus control leads to 1350 significantly differentially expressed genes (data not shown). This highlights the fact that the transcriptomic changes made by the addition of adenosine and PGE2 to TLR stimulated macrophages are quite limited. However, at the same time, the addition of adenosine and PGE2 to TLR stimulated macrophages leads to a similar transcriptomic phenotype as indicated by spearman correlation analysis yielding a correlation coefficient of R= 0.772 (Figure 9B). Single cell RNA sequencing of M-CSF macrophages from 1 donor allowed us to compare the trancriptomic signature of a multitude of individual cells in order to see the variable expression of genes within a population. UMAP analysis was performed as a dimension reduction technique to cluster cells based on the variability of gene expression (Figure 10). LPS, LPS + PGE2 and LPS + Adenosine stimulated macrophages cluster separately from unstimulated cells indicating relatively homogeneous stimulation throughout the macrophage population. There was some overlap between the LPS + PGE2 and LPS + 43 A. B. LPS+Ado/LPS LPS+PGE2/LPS *LYPD3 LYPD3*IGLON5 *ACKR3LIPN AREG K*RT17 ADAMTS17 *KBTBD11 *KBTBD11 * IGLON5 CXCR4 CMSS1 LIPN CREM *GNG4 MYO5C DUSP4 GPR3 ALPK2 CMKLR1 P2RX7 *CCL4L2 CCL15IL6 TNF UNC13A IL36G CSF2 CCL2 *TIFAB *CCL4L2CCL1 TIFAB CCL8 * CCL8 *CCL*3L3/L1 CCL*3L3/L1* IL12B * * IL12B -10 -5 0 5 10 -10 -5 0 5 10 Log2(Fold Change) Log2(Fold Change) Figure 8. Most highly upregulated and downregulated genes by adenosine and PGE2 relative to LPS stimulated macrophages alone. Bulk RNA-seq was done on M-CSF macrphages and differential expression analysis was performed for (A) LPS + PGE2 versus LPS and (B) LPS + Adenosine versus LPS. The top and bottom 10 genes are listed for each comparison. N=5 individuals, P-value < 0.05, log2FC > 2, error bars represent SEM. (*) indicates genes in common between LPS+PGE2/LPS and LPS+Adenosine/LPS. 44 A. B. LPS+PGE2 LPS+Ado r = 0.772 27 101 131 LPS+PGE2 LPS+Ado 37 91 166 Log2FC(LPS+PGE2/LPS) Figure 9. Degree of transcriptional similarity between LPS + Adenosine and LPS + PGE2 stimulated macrophages. (A) The number of unique and shared DEGs of LPS + Adenosine and LPS + PGE2 relative to LPS alone are depicted as venn diagrams. (B) The log2FC of the DEGs of LPS + Adenosine versus LPS + PGE2 relative to LPS alone are depicted on a scatter plot. Spearman correlation was performed to obtain a correlation coefficient (r) printed in the top left quadrant (P-value < 2.2-16). Figure 9B generated with Dr. Ashton Trey Belew 45 Downregulated DEGs Upregulated DEGs Log2FC(LPS+Ado/LPS) 3 LPS Unstimulated 0 L+PGE2 ?3 L+Ado L+Ado L+PGE2 LPS Unstimulated ?6 ?5 0 5 10 UMAP_1 Figure 10. Cluster visualisation of M-CSF macrophage single cell transcriptomes. Uniform manifold approximation and projection (UMAP) was used to arrange points (representing cells) based on their relative transcriptomes. Single cells clustered based on their stimulation conditions: unstimulated, LPS, LPS + Ado and LPS + PGE2. Figure generated with Dr. Ashton Trey Belew 46 UMAP_2 Adenosine clusters, further indicating that a number of cells in these stimulation conditions are transcriptionally similar. Pathway Analysis. In order to explore the functional roles of macrophages stimulated with adenosine and PGE2, we took a closer look at the genes modulated by these conditions. Of the most highly upregulated genes in common between LPS + Adenosine and LPS + PGE2 versus LPS alone, 10 of the top 20 genes have published growth promoting or anti-inflammatory roles (Figure 11A, purple). LYPD3, or C4.4A, deficiency has been implicated in delayed wound healing, and its expression levels correlate with cell invasiveness in numerous cancers154?158. LIPN has been shown to be involved in proper formation of the skin barrier as a 2bp mutation in this gene led to ichthyosis159. AREG is well known for its role in tissue restoration and is an activator of transforming growth factor beta160. ACKR3, also known as CXCR7, is involved in angiogenesis and cell migration, as well as cancer cell invasiveness161,162. KRT17 has recently been found to be overexpressed in a number of cancers and contributes to tumor cell invasiveness163,164. Its primary role is in wound healing, but it also regulates skin inflammation165,166. CXCR4 is a receptor for CXCL12, which has been shown to recruit macrophages to tumor environments and promote M2 phenotypes167. THBS1 is involved in wound healing, the maintenance of homeostasis in the lung, and is an activator of latent TGFbeta168?170. FFAR3 is a short-chain fatty acid receptor and stimulation of this receptor results in decreased inflammatory cytokine production by human monocytes171. CRISPLD2 has been shown to modulate proliferation of fetal lung fibroblasts and is involved in the regulation of extracellular matrix genes during wound healing172. Of the most highly downregulated genes in common between LPS + Adenosine and LPS + 47 A. 7 6 LPS+PGE2 5 LPS+Ado C. 4 3 2 1 0 D3 N5 PN 11 EG R3 G4 17 R4 17 S1 2 MP O I D R K N T C S S K E S 1 P2 F2 D2 R3 BL R3 LY LGL TB A R AC G K CX M T CM AL P R HB HA RL PLC A OT T C IS FF C G P I KB DA CRB. A 0 -1 -2 -3 -4 LPS+PGE2 -5 LPS+Ado -6 -7 2B /L1 L8 AB L2 L1 L2 5 2 F 7 3 1 A H 71 3 C F 4 C C L1 SF TN RX F LR 13 IS IL2 INE 1 AM P NA 3 1A IL 3L C TI CL C C IL L C C C C P2 K NC CM RP T C C C U SE OC S K C Figure 11. Functional characteristics of shared DEGs between LPS + Adenosine and LPS+PGE2 relative to LPS stimulation. Differential expression analysis was performed and the list of shared (A) most highly upregulated genes and (B) most highly downregulated genes are depicted as the Log2FC relative to LPS stimulation alone. The individual Log2FC of each gene for LPS + Adenosine (green bars) and LPS + PGE2 (blue bars) stimulations are shown. The genes colored in purple have roles in growth, proliferation and angiogenesis. The genes colored in red have roles in inflammation. N=5 individuals, P-value < 0.05, log2FC > 1. (C) GO term analysis for molecular function was performed and the top 5 GO terms are plotted. Point size indicates # of DEGs in the GO term category. Point color indicates P-value. Rich factor is the ratio of the # of DEGs per # of genes in the GO term category. 48 Log2(Fold Change/LPS) Log2(Fold Change/LPS) PGE2 versus LPS alone, 13 out of the bottom 20 genes have published roles in inflammation (Figure 11B, red). IL12B is an inflammatory cytokine that promotes the development of Th1 CD4+ T cells173. CCL3L3/L1 are sequence variants of the same gene (CCL3), which is a chemokine that recruits CCR5 expressing cells174. CCL8, CCL4L2, CCL1, CCL2, and CCL15 are also chemokines, which participate in the recruitment of immune cells to sites of inflammation175?177. CSF2 encodes GM-CSF, which can function as a cytokine to promote inflammation178. TNF is a cytokine that mediates inflammation, anti-microbial immunity, and was named after its179 cytotoxicity towards tumors. P2RX7 is a receptor for ATP, which plays a role in the activation of the NLRP3 inflammasome180. CMKLR1 encodes the receptor for chemerin, a potent macrophage chemoattractant181. IL27 is a member of the IL-12 family of cytokines and promotes expansion and IFN? production by CD4+ T cells182. IL1A encodes an inflammatory cytokine that can function as an ?alarmin? and stimulates the production of chemokines183. Our transcriptomic analysis suggests that the M-CSF macrophage response to adenosine and PGE2 during inflammation is highly similar and overlapping. Gene ontology (GO) analysis of the list of shared differentially expressed genes by LPS + Adenosine and LPS+PGE2 relative to LPS alone, revealed a number of processes that were significantly enriched. These included cytokine activity, chemokine activity, and growth factor activity among the top 5 most significantly enriched categories (Figure 11C). Using the pathway analysis software, Cytoscape, the differentially expressed genes shared by LPS + Adenosine and LPS + PGE2 were separated into 5 predicted network clusters based on annotated signaling pathways (Figure 12). The first cluster includes the upregulation of a few growth promoting genes and the downregulation of cytokine genes, 49 A. B. Figure 12. Functional interaction groups in shared DEGs between LPS + PGE2 and LPS + Ado relative to LPS stimulation. Clusters comprised of 47 and 43 genes (A and B, respectively) exhibit functional interactions depicted by edges/arrows. Genes from the shared DEGs in each cluster are depicted by colored circular nodes. The direction of differential expression is depicted by node border color: green represents upregulated and red represents downregulated genes. White diamond nodes represent predicted regulators. (A) Network consisting of genes involved in cytokine signaling and growth factor activity including TNF, IL1A, THBS1, TGFA and AREG. Regulators include AP-1 (Jus/Fos), NFKB1, and EP300. (B) Network consisting of genes involved in tissue repair including TGFB2 and BMP6. Regulators include UBC and GSK3B. 50 C. D. E. Figure 12 continued. Clusters comprised of 32, 26 and 35 genes (C, D, E respectively) exhibit functional interactions depicted by edges/arrows. (C) Network consisting of genes involved in inflammation including IL12B, CSF2, and IL27. Regulators include STAT1, STAT3 and STAT5A. (D) Network consisting of genes involved in chemokine activity including CCL1, CCL15, CCL8, CXCL10, CXCL11, CCR7 and CXCR4. Regulators include GNG2 and ARRB1. (E) Network consisting of growth promoting genes including VEGFA, CD300E, and PDGFB. REgulators include MAPK8, FYN and PIK3CA. 51 and predicts that NF?B, RelA, and AP-1 (fos, jun), among other transcription factors are involved in the regulation of these genes (Figure 12A). The second network consists of genes involved in tissue repair including TGFB2 and BMP6 and implicates UBC and GSK3B as regulators (Figure 12B). The third network includes two growth-promoting genes of interest, VEGFA and CD300E, and implicates the tyrosine kinases MAPK8 and FYN (Figure 12C). The fourth network predicts that STAT1, STAT3 and STAT5A are involved in the regulation of this group of genes including inflammatory CSF2 and IL12B (Figure 12D). The last network includes a number of differentially expressed chemokine genes, which are predicted to be regulated by GNG4, GNG2 and ARRB1 (Figure 12E). Single Cell RNA-Sequencing Analysis. Single cell sequencing was performed in order to enhance our search for marker genes for regulatory macrophages. It allowed us to not only look for expression levels of genes, similar to bulk RNA-sequencing, but also to look at cell numbers expressing select genes. Lists of the most differentially expressed genes by LPS + Adenosine and LPS + PGE2 stimulated cells versus LPS alone as determined by single cell sequencing are available in Table II and Table III, respectively. From these lists we chose a number of candidate marker genes, mainly encoding cell surface or secreted proteins, to examine their expression on a per cell basis for each sample, depicted in violin plots, which allowed us to look at the distribution of gene expression between cells of each stimulation (Figure 13) as well as feature plots which allowed us to see which specific cells had high and low expression of each gene (Figure 14). We hypothesized that some of these genes could serve as potential transcript biomarkers for LPS + Adenosine and LPS + PGE2 macrophages, including THBS1, 52 Table II. Single cell RNA sequencing markers for LPS + PGE2 versus LPS Table III. Single cell RNA sequencing markers for LPS + Ado versus LPS Tables II and III generated with Dr. Ashton Trey Belew 53 THBS1 VEGFA CD300E PLAUR OLR1 G0S2 CREM SAMSN1 INHBA Figure 13. Violin plots of candidate RNA markers for LPS+Adenosine and LPS+PGE2 stimulated M-CSF macrophages. Single cell RNA-seq was performed on M-CSF macrophages and differential expression analysis was done between sample stimulation groups: LPS + Ado (Red), LPS + PGE2 (Green), LPS (Teal), Unstimulated (Purple). From this analysis, 9 genes (A) THBS1, (B) VEGFA, (C) CD300E, (D) PLAUR, (E) OLR1, (F) G0S2, (G) CREM, (H) SAMSN1, and (I) INHBA, were selected based on their similar expression pattern between LPS + PGE2 and LPS + Adenosine. These plots indicate the distribution of individual cells based on their expression level for these genes. 54 LPS Unstimulated LPS+PGE2 LPS+Ado THBS1 VEGFA CD300E 3 3 3 3 3 3 0 2 0 2 0 2 1 1 1 0 0 0 ?3 ?3 ?3 ?6 ?6 ?6 ?5 0 5 10 ?5 0 5 10 ?5 0 5 10 UMAP_1 UMAP_1 UMAP_1 PLAUR OLR1 G0S2 3 3 3 3 3 3 0 2 0 2 0 2 1 1 1 0 0 0 ?3 ?3 ?3 ?6 ?6 ?6 ?5 0 5 10 ?5 0 5 10 ?5 0 5 10 UMAP_1 UMAP_1 UMAP_1 CREM SAMSN1 INHBA 3 3 3 3 3 3 0 2 0 2 0 2 1 1 1 0 0 0 ?3 ?3 ?3 ?6 ?6 ?6 ?5 0 5 10 ?5 0 5 10 ?5 0 5 10 UMAP_1 UMAP_1 UMAP_1 Figure 14. Feature plots of candidate RNA markers for LPS + Adenosine and LPS + PGE2 stimulated M-CSF macrophages. Single cell RNA-seq was performed on M-CSF macrophages and differential expression analysis was done between sample stimulation groups. From this analysis, 9 genes (A) THBS1, (B) VEGFA, (C) CD300E, (D) PLAUR, (E) OLR1, (F) G0S2, (G) CREM, (H) SAMSN1, and (I) INHBA, were selected based on their similar expression pattern between LPS + PGE2 and LPS + Adenosine. These plots indicate which cells are expressing these genes relative to one another on a scale of 0-3 (yellow-blue). The grey plot indicates the identity of the clusters based on stimulation condition. 55 UMAP_2 UMAP_2 UMAP_2 UMAP_2 UMAP_2 UMAP_2 UMAP_2 UMAP_2 UMAP_2 VEGFA, CD300E, PLAUR, OLR1, G0S2, CREM, SAMSN1 and INHBA. A plot of the most variable genes between all 4 samples highlighted a number of genes that we believe comprise a part of the M1 macrophage signature, or the response to LPS, because these should theoretically be most different from both unstimulated and LPS + Adenosine or LPS + PGE2 samples (Figure 15A). The top 30 genes are labeled and include CXCL10, CXCL11, TNF, IL23A, CCL3, CCL4 and a number of other chemokine genes. The genes colored in orange are those that were also found in the top 100 most highly upregulated genes by LPS stimulation versus unstimulated in our conventional RNA- sequencing data. Conventional RNA-sequencing also demonstrated that the majority of the top 25 most highly upregulated genes by LPS relative to unstimulated macrophages were significantly downregulated by either adenosine, PGE2 or both (Figure 15B). This suggests that while the transcriptomic changes by adenosine and PGE2 are limited, they have important consequences in the host. 4.3 Discussion In this chapter we explored the effects of two molecules, adenosine and PGE2, which have previously been shown to modulate the aspects of the inflammatory response induced by LPS. We performed conventional RNA sequencing on macrophages stimulated with LPS in the presence of adenosine and PGE2, and found that their transcriptional program was highly similar using thorough differential expression analysis. This degree of similarity was observed in the number of shared DEGs between the two stimulation conditions. It was also observed in the direction and extent of the changes in gene expression, two parameters that were factored into the Spearman correlation analysis. Single-cell RNA sequencing also supported the likeness between 56 B. ACOD1 CCL20 * IL6 * A. TNIP3 * CSF3 12.5 FABP4 CCL4L2 CXCL10 * * SLAMF1 AKAP12 SERPINB2 10.0 IL19Non?variable * * count: 30738 IL23A PTGS2 ** Variable CCL8 TNFAIP6 7.5 count: 2000 SPP1 CXCL1 CXCL1 ** CCL22 TNF CCL20 IL23A CCL19 CIR1 * CXCL9 FABP5 CCL3 CCL19 5.0 CXCL11 CCL4TFPI2 SPINK1IL19 INHBA FCAMR IL12B CXCL5 CXCL3 SNAI1 TIMP3 TSLP PTX3 *ISG15 *IL12B 2.5 G0S2 * * G0S2 CSF2 ** 0.0 IL27 * * IL1A 1e?03 1e?01 1e+01 * * Average Expression IL1B ** LPS SOCS3 LPS+PGE2 PTX3 * LPS+Ado CCL15 * * CCL3L3/L1 * * 0 3 6 9 12 Log2(Fold Change)/NS Figure 15. M1 genes in M-CSF macrophages. (A) The most significant variable genes among all samples (unstimulated, LPS, LPS + Ado and LPS + PGE2) are indicated with red dots. The top 30 most variable genes are labeled. The genes labeled in orange are also found in the top 100 most differentially expressed genes by LPS versus unstimulated in bulk RNA-seq analysis. (B) The top 25 most highly upregulated genes by LPS (black) versus unstimulated (NS) macrophages and their log2FC are depicted. The corresponding log2FC of LPS + Ado (blue) and LPS + PGE2 (green) versus NS is also depicted. (n=5, * indicates genes with a log2FC > 1 and P-value < 0.05 relative to LPS as determined by differential expression analysis, error bars represent SEM). Figure 15A generated with Dr. Ashton Trey Belew 57 Standardized Variance adenosine and PGE2 stimulated samples. We believe that this high degree of similarity could be attributed to signaling through g-protein coupled receptors (GPCRs) which leads to the intracellular release of cAMP, and downstream activation of transcription factors that control numerous genes involved in immune responses. While similar, PGE2 appeared to have a more pronounced effect on LPS stimulated macrophages than adenosine, due to a higher number of uniquely perturbed transcripts. However, this could be concentration dependent and perhaps it requires more adenosine to achieve even further overlap in transcriptomic phenotype with PGE2. Pathway analysis, including GO, and careful inspection of individual gene changes by LPS + Adenosine and LPS + PGE2 relative to LPS alone led us to conclude that these stimuli lead to what our lab refers to as a ?regulatory? phenotype. This regulatory phenotype was one characterized by the induction of growth promoting genes and the suppression of inflammatory genes. Single-cell RNA sequencing was performed in order to aid in our identification of potential biomarkers for LPS + adenosine and LPS + PGE2 stimulated macrophages. This technique was powerful because it allowed us to look at the gene expression patterns of a large number of cells (roughly 4000 cells per sample). It also indicated to us that in vitro stimulation of macrophages is fairly homogeneous, since individual samples were stimulated in separate wells, but when all the sample data was combined during analysis, the cells clustered together according to their stimulation condition. We identified numerous transcripts that could be used as RNA biomarkers to find regulatory macrophages in tissues including THBS1, VEGFA, CD300E, PLAUR, OLR1, SAMSN1, and G0S2. RNA biomarkers also have the 58 potential to be used to scan transcriptomic data generated from different disease or immune conditions for the likely presence of regulatory macrophages. Single-cell sequencing led us to identify the most variable genes in our data set between all stimulations including unstimulated, LPS, LPS + Adenosine and LPS + PGE2. We propose that these genes represented mainly the M1 phenotype, because M1 genes should be the most changed in the presence of adenosine and PGE2 and relative to no stimulation. This was supported by the fact that 18 out of the 30 most variable genes were also present in the top 100 most highly differentially expressed genes by LPS versus unstimulated macrophages found by conventional RNA sequencing. We then demonstrated with conventional RNA sequencing that the majority of the top 25 most highly upregulated genes by LPS stimulation over resting cells were significantly downregulated by combination with adenosine, PGE2, or both. Many of these genes encode inflammatory cytokines and chemokines. These data imply that while the global transcriptomic changes induced by adenosine and PGE2 are relatively limited in number, they are highly specific and target some of the most important genes that comprise the LPS inflammatory M1 signature. Overall, the work in this chapter describes two highly similar populations of M-CSF macrophages that are characterized by increased expression of tissue repair genes and decreased expression of cytokine and chemokine genes. 59 5 Comparison of human GM-CSF derived macrophages to M-CSF derived macrophages both stimulated with LPS + Adenosine and LPS + PGE2 5.1 Introduction While M-CSF is the growth factor constitutively expressed in a number of tissues that gives rise to macrophages from infiltrating monocytes, there is a second growth factor, GM-CSF, which also leads to differentiated macrophages. GM-CSF is constitutively produced in the lung, but is also induced during inflammatory responses. We wanted to know if macrophages differentiated in GM-CSF are equally as capable of being programmed into ?regulatory? macrophages, as are M-CSF macrophages, in response to LPS + Adenosine and LPS + PGE2 stimulation. This information would be useful as GM-CSF could potentially be used to modulate the macrophage activation response. To explore this question we performed RNA-sequencing on donor matched GM- CSF derived macrophages stimulated with LPS, LPS + Adenosine, and LPS + PGE2 and compared the transcriptomic data to our analysis in the previous chapter for M-CSF derived macrophages. We also did in vitro assays of M-CSF and GM-CSF macrophages side by side to further validate the results from our M-CSF RNA-sequencing analysis, and to compare the in vitro phenotypes of GM-CSF and M-CSF macrophages. Before we began, we examined the expression of macrophage marker CD68 (Figure 16A), dendritic cell marker CD1a (Figure 16B) and macrophage marker CD11b (Figure 16C) to confirm that following 7 days of differentiation in M-CSF and GM-CSF, our blood monocyte samples did fully mature into macrophages and not dendritic cells. 5.2 Results 60 A. CD68 B. CD1a C. CD11b M-CSF M? M-CSF M? M-CSF M? M-CSF Control M-CSF Control M-CSF Control GM-CSF M? GM-CSF M? GM-CSF M? GM-CSF Control GM-CSF Control GM-CSF Control Figure 16. Flow cytometry validation of M-CSF and GM-CSF derived macrophages. Markers for (A) macrophage CD68, (B) dendritic cell CD1a and (C) macrophage CD11b were detected on macrophages differentiated in M-CSF and GM-CSF for 7 days to confirm that our working cells in both differentiation conditions are in fact mature macrophages. Relevant isotype controls are depicted for each antibody. 61 Adenosine and PGE2 sensing in human macrophages. It has recently been published that GM-CSF macrophages degrade less ATP in vitro, and produce less adenosine when stimulated, compared to M-CSF macrophages184. We confirmed these results in our M- CSF and GM-CSF macrophages by examining the kinetics of ATP degradation over a period of 2 hours (Figure 17A). This important finding fits in line with our hypothesis that M-CSF macrophages produce more ATP and convert it to adenosine via CD39 and CD73, which can subsequently act in an autocrine fashion to suppress macrophage inflammation26. Similarly, at 24 hours post-LPS stimulation, supernatants from M-CSF macrophages contained higher levels of PGE2 while the levels of PGE2 in supernatants from GM-CSF macrophages were unchanged with LPS stimulation (Figure 17B). The expression of a number of purinergic and prostaglandin receptor genes was measured by RT-PCR (Figure 18). M-CSF macrophages stimulated with LPS upregulated the expression of the A2a receptor (Figure 18B), EP2 receptor (Figure 18G) and EP4 receptor (Figure 18H) while GM-CSF macrophages did not upregulate any of these receptors to the same extent, and exhibited lower expression levels for these receptors overall. mRNA expression of the A2b receptor (Figure 18C) did not change with LPS stimulation in either M-CSF or GM-CSF, though its expression was higher in M-CSF macrophages overall. The expression of the A1 and A3 receptors decreased following LPS stimulation in both M-CSF and GM-CSF macrophages (Figures 18A and 18D, respectively). These two receptors are coupled to the Gi family of signaling proteins, which typically decrease cAMP release when activated185. The mRNA expression of the two ecto-enzymes involved in the hydrolysis of ATP, CD39 and CD73, did not change with LPS stimulation and were not significantly different between M-CSF and GM-CSF 62 A. B. * M-CSF GM-CSF40 ** * 600 * 600 ns * 500 50030 400 400 20 300 300 200 200 10 M-CSF GM-CSF 100 100 0 0 0 0 15 30 45 60 75 90 105 120 Naive LPS Naive LPS Time (minutes) Figure 17. ATP degradation and PGE2 production by human macrophages. (A) The degradation of 20?M ATP was measured over time and expressed per ?g of protein per sample (n=3 donors, * P-value < 0.05, ** P-value < 0.01, error bars represent SEM). (B) PGE2 was measured in the supernatants of macrophages stimulated with LPS for 24 hours (n=10 donors, * P-value < 0.05, lines connect samples from the same individual). 63 pmoles ATP degraded/ug protein PGE2 pg/mL PGE2 pg/mL A. B. C. D. E. F. G. H. I. Figure 18. mRNA expression of purinergic pathway and PGE2 receptor genes in M-CSF and GM-CSF macrophages. Quantitative RT-PCR was used to measure transcript levels of (A) A1R, (B) A2AR, (C) A2BR, (D) A3R, (E) CD39, (F) CD73, (G) PTGER2, (H) PTGER4, and (I) PTGER3 following stimulation with LPS for 4 hours (n=5 donors, * P-value < 0.05, ** P-value < 0.01, *** P-value < 0.001, **** P-value < 0.0001, error bars represent SEM). 64 macrophages (Figures 18E and 18F, respectively. Lastly, the third receptor for PGE2, PTGER3, did not change at the transcript level following LPS stimulation and was expressed similarly at low levels in M-CSF and GM-CSF macrophages (Figure 18I). The mRNA level of constitutively expressed COX1 did not change with LPS stimulation, nor was it different in M-CSF and GM-CSF macrophages (Figure 19A). However, the increase in PGE2 levels in the supernatants of LPS stimulated M-CSF macrophages correlated with an increase in the expression of COX2 mRNA, which is inducible (Figure 19B). The expression of the prostaglandin synthase gene, MPGES1, seemed to increase slightly in LPS stimulated M-CSF macrophages, but this observation was not found to be significant (Figure 19C). The expression pattern of adenosine and PGE2 pathway genes in M-CSF macrophages was not exclusive to LPS stimulation. We tested various TLR ligands including, FSL-1 (TLR2/6), HKLM (TLR2), Loxoribine (TLR 7), and Poly I:C (TLR 3). We noticed that A2AR expression was significantly different between M-CSF and GM-CSF macrophages (Figure 20A). A2BR expression was not significantly changed (Figure 20B). mRNA expression for PTGER2 (Figure 20C), PTGER4 (Figure 20D), MPGES1 (Figure 20E), COX1 (Figure 20F) and COX2 (Figure 20G) was higher in M-CSF macrophages than in GM-CSF macrophages for most of the TLR ligands with the exception of COX1, which was expected. Transcriptome comparison of M-CSF and GM-CSF macrophages. Principal component analysis (PCA) revealed that M-CSF and GM-CSF macrophages are notably different, even following stimulation, and they separated along principal component 1 (PC1), which explains approximately 54% of the variance among samples (Figure 21). LPS + Adenosine, LPS + PGE2 and LPS stimulated samples differed from each other as 65 COX1 COX2 MPGES1 0.5 50 *** 0.25 0.4 40 0.20 0.3 30 0.15 0.2 20 0.10 0.1 10 0.05 0.0 0 0.00 ive PS ive PS ive PS e S e S e S Na L Na L a L iv N Na L P aiv LP iv PN Na L Figure 19. mRNA expression of PGE2 synthesis pathway genes. M-CSF (blue) and GM-CSF (grey) macrophages were stimulated with LPS for 4 hours. (A) COX1, (B) COX2 and (C) MPGES1 mRNA levels were measured by RT-PCR (n=7, *** P-value < 0.001, error bars represent SEM). 66 2^-dCT 2^-dCT 2^-dCT A. B. A2AR A2BR 2.5 8 M-CSF 2.0 M-CSFGM-CSF 6 GM-CSF 1.5 4 1.0 * 0.5 2* * 0.0 0 ive L-1 LM e C e 1 M e C Na : - : FS HK rib in ly I Na iv n o FS L KL ibi I o H or ol y Lo x P x P Lo C. PTGER2 D. PTGER4 1.5 2.5 M-CSF M-CSF GM-CSF 2.0 GM-CSF 1.0 1.5 1.0 0.5 * * 0.5 0.0 0.0 ive L-1 LM ine I:C iv e L-1a S K L M ine I:C N F H or ib oly N a FS HK ib y x P xo r l o o P o L L E. MPGES1 F. COX1 G. COX2 8 1.5 250 200 M-CSF 150 25 6 GM-CSF * M-CSF 1.0 M-CSF 20 GM-CSF GM-CSF 4 15 0.5 10 2 * 5 * 0 0.0 0 ivea SL -1 LM ine e I:C iv L- 1 M e :C K ib y a S KL bi n y I ai ve L-1 LM ine N I:C F H or ol NP F H or i b x x Po l N FS HK i xo r Po ly Lo Lo Lo Figure 20. Purinergic and PGE2 pathway gene expression following stimulation with various TLR ligands. mRNA expression for (A) A2AR, (B) A2BR, (C) PTGER2, (D) PTGER4, (E) MPGES1, (F) COX1 and (G) COX2 following 4 hours stimulation with FSL-1, HKLM, Loxoribine, and Poly I:C (n=3, * P-value < 0.05, error bars represent SEM). 67 2^-dCT 2^-dCT 2^-dCT 2^-dCT 2^-dCT 2^-dCT 2^-dCT 0.6 Batch Condition GM LPS M LA LP 0.3 0.0 ?0.3 ?0.6 ?0.3 0.0 0.3 0.6 PC1: 54.33% variance Figure 21. PCA plot of stimulated human macrophage samples. M-CSF (squares) and GM-CSF (circles) macrophages were stimulated with LPS (orange), LPS + Ado (purple) and LPS + PGE2 (pink) for 4 hours and bulk RNA-seq was performed. Principal component analysis (PCA) indicating variance among samples is visualized above. Figure generated with Dr. Ashton Trey Belew 68 PC2: 13.68% variance seen on principal component 2 (PC2), which describes approximately 14% of the variance between samples. Also on PC2, we observed a larger spread between LPS, LPS + Adenosine, and LPS + PGE2 samples, indicating greater variance between M-CSF samples than we observed in GM-CSF samples. Volcano plots of all measured transcripts indicated that LPS + Adenosine and LPS + PGE2 stimulation of GM-CSF macrophages resulted in only 7 and 126 differentially expressed genes (DEGs), respectively, compared to LPS alone (Figure 22). This is significantly lower than the 256 and 489 DEGs by LPS + Adenosine and LPS + PGE2, respectively, relative to LPS alone in M-CSF macrophages. We selected the top 20 differentially expressed genes by M-CSF macrophages stimulated with LPS + PGE2 (Figure 23A) and LPS + Adenosine (Figure 23B) versus LPS alone, and compared their fold changes with the corresponding stimulation conditions in GM-CSF macrophages. Many of these transcripts were not as highly upregulated in GM-CSF samples. Similarly, none of the most highly downregulated transcripts by LPS + PGE2 (Figure 23C) and LPS + Adenosine (Figure 23D) versus LPS alone in M-CSF macrophages were as highly downregulated by adenosine and PGE2 in GM-CSF macrophages. The expression of several genes of interest, based on their involvement in cell growth and tissue remodeling, was measured by RT-PCR in M-CSF and GM-CSF macrophages in order to supplement RNA- sequencing data. These genes, including: THBS1, CD93, AREG, VEGFA, CD300E, CXCL13, MMP10, and RGS2, were all significantly upregulated by LPS + Adenosine and LPS + PGE2 stimulation compared to LPS alone in M-CSF macrophages (Figure 24). With the single exception of TGFA, an upregulation of these regulatory transcripts was not observed in GM-CSF macrophages (Figure 24). Altogether, we believe that this 69 LPS+PGE LLPPSS++AAdo2 do 257 232 128 128 ?6 ?4 ?2 0 2 4 6 ?6 ?4 ?2 0 2 4 6 logFC logFC 22 104 2 5 ?6 ?4 ?2 0 2 4 6 ?6 ?4 ?2 0 2 4 6 logFC logFC Figure 22. Volcano plot visualization of DEGs from LPS + Ado and LPS + PGE2 stimulated macrophages relative to LPS alone. Bulk RNA-seq of M-CSF (top) and GM-CSF (bottom) macrophages stimulated with LPS + PGE2 (left) and LPS + Ado (right) was analyzed for differential expression of genes relative to LPS alone. Green points represent significantly changed genes (P-value < 0.05, log2FC > 1). The numbers of significantly upregulated and downregulated genes are indicated in the plot area. Pink points represent genes with a P-value < 0.05 and log2FC < 1. Yellow points represent genes with a P-value > 0.05 and log2FC > 1. Black points represent genes with a P-value > 0.05 and log2FC < 1. 70 GM-CSF M-CSF ?log10(P.Value) ?log10(P.Value) 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 ?log10(P.Value) ?log10(P.Value) 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 A. B. LPS+PGE2 LPS+Ado 40 60 ** 30 * 40 * *** M-CSF M-CSF*** 20 GM-CSF*** *** GM-CSF* 20 ** 10 * *** *** *********** ** *** * ** * *** 0 0 PD 3 R3 G 11 5 4 N 4 4 2 1 17 E 1 2 1K S 1 7 F2 31 3 3 5 N 7 17 1 1 M C 3 4 2 B 9 1 L A G 4 2 Y C AR E D ON R PB L XC L I GN G SP PK BS T 00 S A R D 1U L H R 3 HA R4 MS TS RLD NF 3 FA YP LO NLIP T S D 1 S E 5 R G PR T B MS R YO GP N HA SV 2 SP IPG OB F E R BL A T IG C D A T K C N C M C Z F L A TG R XC GF KB DA I G K M T C C M GA B T C S C A C T A AD K C. LPS+PGE D.2 LPS+Ado 10 10 0 0 -10 *** * ** ***** ** ******** * * * ***** ** ** * * ** ** * ** * ****** * * *** * * *** -20 ** -10 **** * ** *** * -30 M-CSF M-CSF GM-CSF -20 GM-CSF -40 *** *** -50 -30 2B /L1 L8 AB L2 L2 6G NF 15 X7 L3 L1 CN SH 16 L4 F3 27 22 19 2B /L1 L8 L1 AB F2 3A IL6 L2 R1 E1 A3 L2 F3 15 46 S4 MP BA AT IL1 L3 CC IF L4 CC IL3 T C L 2R C I3 T C C C A C GS CC IL RD IL L1 L3 CC CC IF CS C1 L4 KL IN N CC CL PZ I P C A A STA P1 PL CL C C C P R NK 3 T N C M R K C F C S G C A CC L U C C E CS OC Figure 23. Comparison of expression of DEGs by LPS + Adenosine and LPS + PGE2 relative to LPS alone in M-CSF and GM-CSF macrophages. The 20 most upregulated differentially expressed genes in M-CSF (blue) (A) LPS + PGE2 and (B) LPS + Ado stimulated macrophages relative to LPS alone are listed on the x-axis. Their corresponding fold changes in GM-CSF (grey) LPS+ PGE2 and LPS + Adenosine macrophages relative to LPS are plotted (n=5, *P-value < 0.05, ** P-value < 0.01, *** P-value < 0.001). The 20 most downregulated differentially expressed genes in M-CSF (C) LPS + PGE2 and (D) LPS+Adenosine stimulated macrophages relative to LPS alone are listed on the x-axis. Their corresponding fold changes in GM-CSF LPS + PGE2 and LPS + Ado macrophages relative to LPS are plotted (n=5, *P-value < 0.05, ** P-value < 0.01, *** P-value < 0.001). 71 Fold Change/LPS Fold Change/LPS Fold Change/LPS Fold Change/LPS Figure 24. mRNA expression of genes of interest in M-CSF and GM-CSF macrophages. Macrophages were stimulated with LPS, LPS + Adenosine and LPS + PGE2 for 4 hours and the expression for genes THBS1, CD93, AREG, VEGFA, CD300E, CXCL13, MMP10, RGS2, and TGFA was measured by RT-PCR (n=5, * P-value < 0.05, ** P-value < 0.01, *** P-value < 0.001, **** P-value < 0.0001, error bars represent SEM). 72 transcriptomic data indicates a defect in adenosine and PGE2 sensing by GM-CSF macrophages. We hypothesize that this lack of sensing contributes to GM-CSF macrophages being hyper-inflammatory. Functional assays of human macrophages. Human umbilical vein endothelial (HUVEC) cells cultured in conditioned media for 24 hours from stimulated M-CSF and GM-CSF macrophages exhibited tube formation on a Matrigel surface (Figure 25A). HUVEC cells cultured in conditioned media from M-CSF macrophages stimulated with LPS + Adenosine and LPS + PGE2 exhibited the highest levels of tube formation based on tube length (Figure 25B) and the number of nodes between tubes (Figure 25C). HUVEC cells cultured in conditioned media from GM-CSF macrophages stimulated with LPS + Adenosine and LPS + PGE2 showed no increase in tube formation relative to HUVECs cultured in supernatants of LPS stimulated macrophages. In fact, HUVEC cells cultured in media from stimulated GM-CSF macrophages exhibited defects in tube formation relative to those grown in media from unstimulated GM-CSF macrophages and relative to those grown in media from M-CSF macrophages. One of the transcripts that emerged from both our bulk RNA-sequencing and single cell RNA-sequencing analysis as being highly differentially expressed in LPS + Adenosine and LPS + PGE2 stimulated M-CSF macrophages relative to LPS alone was CD300E. CD300E was expressed on the surface of a higher percentage of cells in LPS+ Adenosine and LPS + PGE2 stimulated M-CSF macrophages compared to LPS stimulated M-CSF macrophages (Figure 26A). The level of CD300E expression (median fluorescence intensity) was also increased with LPS + Adenosine and LPS + PGE2 stimulation in M-CSF macrophages, but its expression did not increase beyond baseline 73 A. !"#$%& '!"#$%& !"# $%"# !"# $%"# $&# $%# $&# $%# B. Total Tube Length C. Number of Nodes 20000 60 ## ## M-CSF **** # M-CSF **** GM-CSF **** GM-CSF # 15000 **** 40 ** 10000 20 ** 5000 0 0 e S o 2 e S o 2 Na iv LP +A d GE aiv LP Ad GE PS S+ P N PS + +P L SLP L LP Figure 25. HUVEC cell tube formation in the presence of macrophage conditioned media. Human monocytes were cultured in M-CSF or GM-CSF for 7 days then left unstimulated or stimulated with LPS, LPS + Ado and LPS + PGE2 for 24 hours at which point supernatants were collected for further studies. (A) HUVEC cell tube formation was observed after 24 hour exposure to supernatants harvested from stimulated M-CSF and GM-CSF macrophages from one representative donor (n=3 donors total). Representative images were captured by brightfield microscopy with ?find edges? contrast applied in ImageJ in order to be able to see the tubes. (B) Total tube length was measured in pixels manually using ImageJ software on various images of HUVEC cells exposed to supernatants of macrophages from multiple donors (n=3 donors, ** P-value ?0.01, **** P-value <0.0001 between M-CSF and corresponding GM-CSF samples; # P-value <0.05, ## P-value <0.01 for M-CSF samples relative to NS supernatants; error bars represent SEM). (C) The number of nodes, defined as 3 or more tubes originating from one point, was counted manually using ImageJ software on various images of HUVEC cells exposed to supernatants of macrophages from multiple donors (n=3 donors, ** P-value ?0.01, **** P-value <0.0001 between M-CSF and corresponding GM-CSF samples; # P-value <0.05, ## P-value <0.01 for M-CSF samples relative to NS supernatants; error bars represent SEM). 74 Tube Length (Pixels) Nodes A. Nonstim LPS LPS+Ado LPS+PGE2 B. CD300E 11000 10000 9000 8000 6000 # ** 4000 M-CSF # GM-CSF 2000 0 aiv e PS ML 0? 0n M aiv e PS ?M M N 5 5 N L 50 50 n do E 2 do E 2A G A G PS + +PS PS + +P L LP L LP S Figure 26. Flow cytometry of CD300E surface expression. Macrophages were untreated or stimulated with LPS, LPS + Ado and LPS + PGE2 for 24 hours. (A) Dot plots of CD300E expression from 1 representative donor are shown and gates indicate the percentage of cells that are CD300E+ and CD300E-. (B) Median fluorescence intensity of CD300E levels on stimulated macrophages was calculated (n=5, ** P-value < 0.01 versus LPS stimulated samples alone; # P-value < 0.05, ## P-value < 0.01 between M-CSF and GM-CSF samples, error bars represent SEM). 75 GM-CSF M-CSF Median Fluorescence Intensity (MFI) in stimulated GM-CSF macrophages (Figure 26B). This lead us to conclude that CD300E is a suitable biomarker unique to M-CSF macrophages stimulated with LPS + Adenosine and LPS + PGE2. PLAUR was another one of the transcripts that was highly differentially expressed in our single cell RNA-sequencing analysis. We measured the surface expression of PLAUR by flow cytometry and found that its expression was indeed increased by LPS + Adenosine and LPS + PGE2 stimulation in M-CSF macrophages (Figure 27). We believe that PLAUR could be used in combination with CD300E as a secondary marker for these regulatory cells; however, it is also expressed in GM-CSF macrophages so it would not be a suitable marker to use on its own. We also looked at the levels of secreted, soluble proteins from M-CSF and GM-CSF macrophages. Thrombospondin-1 (Figure 28A) and VEGF? (Figure 28B), both growth- promoting proteins, were secreted at higher levels by M-CSF macrophages than by GM- CSF macrophages. The cytokine IL-6 was significantly decreased with adenosine and PGE2 stimulation in M-CSF macrophages but not in GM-CSF macrophages (Figure 28C). To our surprise, anti-inflammatory IL-10 levels were not increased by adenosine and PGE2 in M-CSF cells, and were actually decreased by PGE2, which is contrary to the behavior of mouse macrophages (Figure 28D). However, the levels of IL-10 were significantly higher in M-CSF macrophages than in GM-CSF macrophages. Preliminary data indicated that stimulation of M-CSF macrophages with LPS in combination with a cell permeable cAMP analog specific for protein kinase A (PKA) activation led to increased levels of both THBS1 and VEGF? (Figure 28E). However, stimulation with a cell permeable cAMP analog specific for exchange protein activated by cAMP (Epac) did not affect the levels of these two cytokines. 76 PLAUR 10000 ** M-CSF 8000 * GM-CSF 6000 4000 2000 0 ive PSa L 0? M M 0n aiv e PS ML 0? 0n M N do 5 5 N 5 5 A GE 2 Ad o E 2 S+ +P S+ +P G LP LP S LP SLP Figure 27. Flow cytometry of PLAUR surface expression. Macrophages were untreated or stimulated with LPS, LPS + Ado and LPS + PGE2 for 8 hours. The median fluorescence intensity of PLAUR levels on stimulated macrophages was calculated (n=3, * P-value < 0.05, ** P-value < 0.01 versus LPS stimulated samples alone, error bars represent SEM). 77 Median Fluorescence Intensity (MFI) A. B. THBS1 VEGF # 100 ## 2.0 * ** # ** 80 * # # M-CSF 1.5 GM-CSF 60 M-CSF 1.0 GM-CSF 40 # # 0.5 20 0 0.0 ive PS Ma L 0? 0n M e S aiv LP 0? M M e S 0n aiv LP 0? M nM0 aiv e PS M M N o 5 5 N ? n 2 o 5 2 5 N o 5 5 N L d E d o 50 50 +A PG +A PG E 2 2 S + S + +A d GE Ad E S +P + +P G LP PLP L LP S S S LP PS LP SL LP C. hIL6 D. hIL10 50 10 ### ## # 40 M-CSF 8 * M-CSF GM-CSF GM-CSF 30 ** 6 20 * 4 10 2 0 0 ive PS ?M nM ive PS ?M nM ive PS M M e S M M Na L 50 50 Na L 50 50 Na L 50 ? 0n iv P ? n o o o 5 N a L o 5 0 50 2 2 2 2 +A d GE Ad E dP + PG +A PG E Ad E S + S + S + S+ +P G LP SLP L P S P LP L LP S LP PSL E. THBS1 VEGFa 40 3 M-CSF M-CSF GM-CSF GM-CSF 30 2 20 1 10 0 0 ive S c ea LP pa PK A aiv LP S pa c KA N +E + N +E +P LP S LP S LP S SLP Figure 28. Growth promoting, anti-inflammatory cytokine secretion by M-CSF and GM-CSF macrophages. Macrophages were differentiated for 7 days in M-CSF or GM-CSF and then stimulated with LPS, LPS + Adenosine, and LPS + PGE2 for 24 hours. (A) THBS1, (B) VEGFa, (C) IL-6 and (D) IL-10 levels were measured in the supernatants by ELISA (n=4-7; * P-value < 0.05, ** P-value < 0.01 relative to LPS stimulation alone; # P-value < 0.05, ## P-value < 0.01, ### P-value < 0.001 between M-CSF and GM-CSF samples; error bars represent SEM; points are color coded by donor). (E) Preliminary data depicting THBS1 and VEGFa levels in macrophages stimulated with LPS coupled to cell permeable Epac selective (8-pCPT-2-O-Me-cAMP-AM) cAMP analog or PKA selective (8-Br-cAMP) cAMP analog (n=2, error bars represent SD). 78 THBS1 (ng/mL) hIL-6 ng/mL hTHBS1 ng/mL VEGFa (ng/mL) hIL-10 (ng/mL) hVEGF ng/mL The levels of inflammatory cytokines TNF (Figure 29A), IL-12p40 (Figure 29B) and GM-CSF (Figure 29C) were higher in supernatants collected from stimulated GM- CSF derived macrophages than in supernatants from M-CSF macrophages. IL-12p40 and GM-CSF levels were unchanged by LPS + Adenosine and LPS + PGE2 relative to LPS stimulation alone in GM-CSF derived macrophages, indicating a resistance of these macrophages to regulatory stimuli. To further explore the extent of GM-CSF resistance to adenosine and PGE2, we use IL-12p40 production as a readout in response to increasing concentrations of adenosine and PGE2 coupled to LPS stimulation (Figure 30). M-CSF macrophages responded with a dose dependent decrease in IL-12p40 production with a significant decrease with just 25 ?M of adenosine and 1 nM of PGE2, while GM- CSF macrophages did not have a significant decrease in IL-12p40 production in the presence of concentrations as high as 50 ?M adenosine and 50 nM PGE2. Kinetics and modulation of cytokine production. We sought to gain insight regarding the kinetics of responses for both M-CSF and GM-CSF macrophages in response to LPS stimulation and also to examine the effects of different priming conditions on cytokine production. First we investigated endotoxin tolerance, a mechanism by which innate immune cells limit their inflammation186?188. We demonstrated that LPS priming of M- CSF and GM-CSF macrophages both led to the tolerance of a second LPS exposure in terms of TNF production and that there was no difference in tolerance between the two populations of macrophages (Figure 31A). Next, we examined whether GM-CSF priming had any effect on the cytokine production of M-CSF macrophages. Priming M- CSF derived macrophages with GM-CSF led to an increase in TNF (Figure 31B) and IL- 12p40 (Figure 31C) production that correlated with the length of priming time. However, 79 hTNF hIL12p40 hGMCSF # 18 ## 18 7 # # # 16 16M-CSF 6 14 GM-CSF 14 M-CSF M-CSF GM-CSF 5 GM-CSF12 12 10 ## # 10 4 8 8 3 6 6 2 4 4 * * 2 2 1 0 0 0 ive Sa LP d o E 2 ive PS do E 2 ive PS do E 2 ive PS do E 2 ive PS do E 2 ive PS do E 2 N S+ A PG Na L +A PG Na L +A PG+ S + S + N a L +A PG Na LS + S+ A PG Na L +A PG LP PS LP PS P + S + L L L LP S LP S PLP L LP S LP SLP Figure 29. Inflammatory cytokine secretion by M-CSF and GM-CSF macrophages. Macrophages were differentiated for 7 days in M-CSF or GM-CSF and then stimulated with LPS, LPS + Adenosine, and LPS + PGE2 for 24 hours. (A) TNF, (B) IL-12p40 and (C) GM-CSF levels were measured in the supernatants by ELISA (n=5-9, * P-value < 0.05 relative to LPS stimulation alone; # P-value < 0.05, ## P-value < 0.01 between M-CSF and GM-CSF samples, error bars represent SEM, points are color coded by donor). 80 hTNF ng/mL hIL-12p40 ng/mL hGM-CSF (ng/mL) human IL-12p40 24h 2.0 12 M-CSF 1.5 GM-CSF 9 * 1.0 6 0.5 *** ******* **** **** 3**** 0.0 0 ive PS ?M ?M ?M ?M ?M nM nM nM nM nM ive PS ?M ?M ?M ?M ?M nM M M M M Na L o 1 o 5 10 25 50 2 1 2 5 10 n n n n o o o E E 2 5 50 Na L 1 5 10 25 50 1 5 10 25 50 Ad Ad d d d G G E 2 E 2 E 2 Ad o Ad o do do o E 2 E 2 E 2 P P G G G E 2 E 2 S+ S+ +A +A +A + + +P +P +P S+ S+ +A +A +A d PG PG G G G LP LP PS PS PS PSL LP S PS PS PS LP LP S S + + P P P L L L LP LP LP S PS S +L LP PS PS + PS + L L L L L L Figure 30. Modulation of macrophage IL-12p40 secretion by increasing concentrations of adenosine and PGE2. M-CSF (blue) and GM-CSF (black) macrophages were stimulated with LPS coupled with various concentrations of adenosine and PGE2, indicated on the x-axis, for 24 hours and IL-12p40 levels were measured in the supernatants by ELISA (n=5 M-CSF, n=4 GM-CSF, * P-value < 0.05, *** P-value < 0.001, **** P-value < 0.0001, error bars represent SEM). 81 hIL-12p40 (ng/mL) hIL-12p40 (ng/mL) A. TNF 25 M-CSF * GM-CSF 20 15 10 5 ns 0 ive PS S Na L e/L P im S p r LP B. TNF C. IL-12p40 D. IL-10 6 15 25 **** ** 20 4 10 15 * 10 2 5 **** * 5 * 0 0 0 e S h h m S e S h h m S e S h h S a?v LP 2 4 4 mF 30 LP a? v 4 LP 2 F 4 v 4 4 30 LP a? LP 2 F 30 LP SF n SF SF CS F SF F n SF SF CS F F F n F SF S -C -C M- -C S -C S -C C - S S S C SF SF -C M M G M M -C M M - GM M- C -C S-C -C - C - -C -C M - G - M F - G G M - G - M G G M M M - G - G M M F S F F SF F - F F G S S S F - G -C -C CS C -C CS CS CM M M- M - SM M- - M- M M- C Figure 31. Priming in M-CSF and GM-CSF macrophages. (A) M-CSF (blue) and GM-CSF (grey) macrophages were unprimed or primed for 24 hours with LPS followed by a wash and subsequent LPS stimulation for an additional 24 hours. Supernatants were collected and assayed for TNF levels by ELISA (n=3, * P-value < 0.05, error bars represent SEM). Macrophages were grown for 7 days in M-CSF and then unprimed (black) or primed with GM-CSF for different lengths of time as indicated on the x-axis (purple) or grown in GM-CSF alone for 7 days (grey). Following a wash, macrophages were stimulated with LPS for 24 hours and supernatants were collected to assay for (B) TNF, (C) IL-12p40 and (D) IL-10 by ELISA (n=3, * P-value < 0.05, ** P-value < 0.01, **** P-value < 0.0001 relative to M-CSF LPS stimulation alone, error bars represent SEM). 82 TNF ( ng/mL) IL-12p40 (ng/mL) TNF (ng/mL) IL-10 ( ng/mL) the levels of TNF and IL-12p40 in GM-CSF primed macrophages were not as high as in macrophages that were derived in GM-CSF alone for 7 days. IL-10 production by M- CSF macrophages was not affected by various lengths of GM-CSF priming time (Figure 31D). Altogether, this data indicates that although GM-CSF is able to alter the M-CSF macrophage response, it is most effective at macrophage polarization when present as a growth factor during the monocyte to macrophage differentiation process. We next observed that TNF production was sustained longer in LPS stimulated GM-CSF macrophages compared to M-CSF macrophages following removal of the LPS stimulus (Figure 32A). Additionally, the accumulated levels of TNF (Figure 32B) and IL-12p40 (Figure 32C) were higher over the collected timepoints in LPS stimulated GM-CSF macrophages compared with M-CSF macrophages. In order to examine the role of endogenously produced adenosine and PGE2 during inflammatory contexts, we made use of pharmacological inhibitors of adenosine and PGE2 receptors. Preliminary data suggested that simultaneous pharmacological blockade of the A2A receptor, EP2 receptor and EP4 receptor appeared to prevent the upregulation of transcript levels of three growth promoting and candidate marker genes for regulatory macrophages, CD300E (Figure 33A), VEGFA (Figure 33B) and THBS1 (Figure 33C) at later (12 hour) but not earlier (4 hour) time points. Pharmacological blockade of the even the EP4 receptor alone led to a significant increase in inflammatory TNF levels in LPS stimulated M-CSF macrophage supernatants after 24 hours (Figure 33D). Conversely, the EP4 antagonist had virtually no effect on LPS stimulated GM-CSF macrophages. This suggests that M-CSF macrophages are sensitive to endogenously produced PGE2 in their environment, but GM-CSF macrophages are not. 83 A. human TNF LPS timecourse 4 **** GM-CSF 3 M-CSF 2 ** 1 0 0h -2h -4h -8h 0h 4h0 2 4 8-2 0-22 B. C. hTNF hIL-12p40 4 M-CSF 25 4 M-CSF 50 GM-CSF **** GM-CSF 3 **** 20 **** 3 40**** **** 15 30 2 2 ** 10 * 20 1 5 1 10 0 0 0 0 0h 2h 4h 8h 2h 4h 0h 2h 4h 8h 2h 4h 0h 2h 4h 8h 2h 4h 0h1 2 1 2 1 2 2 h 4h 8h 12 h 24 h Stimulation time (hours) Stimulation time (hours) Figure 32. Kinetics of inflammatory TNF and IL-12p40 cytokine secretion by M-CSF and GM-CSF macrophages. (A) Human macrophages were stimulated for 2 hours with LPS and supernatants were collected and assayed for TNF levels by ELISA. Macrophages were washed and media was replaced at each timepoint following supernatant collection (n=7, ** P-value < 0.01, **** P-value < 0.0001, error bars represent SEM). The accumulation of (B) TNF and (C) IL-12p40 was measured by ELISA over a period of 24 hours (n=7, * P-value < 0.05, ** P-value < 0.01, **** P-value < 0.0001, error bars represent SEM). 84 TNF (ng/mL) hTNF ng/mL IL-12p40 (ng/mL) IL-12p40 (ng/mL) TNF (ng/mL) CD300E VEGFA THBS1 3 2.0 4h 84h 4h 12h 12h 12h 1.5 6 2 1.0 4 1 0.5 2 0 0.0 0 aiv e LP S k loc aiv e LP S k loc aiv e PS ck ive PS ck ive PS ck ive PS ck N L lo a L lo a L lo a L lo tor b N r b No r b N r b N b N b p pt to r r e e ep ep to pto pto rec rec c c c e ce + + + re re re re PS PS + + PS PS + S S L L L L LP LP TNF 10 #### M-CSF #### GM-CSF 8 6 4 * 2 0 ive PS M Na L O 5n ON PS + L Figure 33. Pharmacological inhibition of adenosine and PGE2 receptors. M-CSF macrophages were stimulated with LPS, and LPS + a cocktail of small molecule antagonists (against A2a, A2b, EP2, and EP4 receptors) for 4 and 12 hours. (A) CD300E, (B) VEGFA and (C) THBS1 mRNA was measured (n=4, error bars represent SEM). (D) M-CSF and GM-CSF macrophages were stimulated with LPS and LPS + ONO AE3 208 5nm (an EP4 receptor antagonist) for 24 hours and TNF levels in the supernatants were measured by ELISA (n=7, * P-value < 0.05 relative to LPS stimulation alone; #### P-value < 0.0001 between M-CSF and GM-CSF samples, error bars represent SEM). 85 2^-dCT hTNF ng/mL 2^-dCT 2^-dCT 5.3 Discussion In this chapter, we elaborated on the findings pertaining to the human M-CSF macrophages described in chapter 4. We also made side-by-side comparisons of M-CSF and GM-CSF macrophages in order to explore whether these two growth factors lead to macrophages that adopt similar phenotypes upon stimulation. It is common to use either M-CSF or GM-CSF to generate monocyte-derived macrophages for further study. However, the combination of GM-CSF with IL-4 is used to obtain monocyte derived dendritic cells189, which may lead to questioning of whether our differentiation protocol resulted in mature macrophage populations. The expression of CD68 and CD11b on both M-CSF and GM-CSF derived cells and the lack of expression of CD1a led us to believe that our working cells were in fact macrophages. This is in line with results from other labs190,191. We demonstrated that M-CSF and GM-CSF macrophages differed with respect to the purinergic and prostaglandin E2 pathways. We hypothesized that M-CSF macrophages may produce more ATP upon TLR stimulation than GM-CSF macrophages, but ATP secretion is difficult to measure due to its rapid conversion to adenosine by enzymes on the macrophage surface. Therefore, we measured the degradation of high concentrations of exogenously added ATP by M-CSF and GM-CSF macrophages to find that M-CSF macrophages degraded higher levels of ATP over the chosen timepoints. This supported similar results found by another group who demonstrated that M-CSF macrophages degraded more ATP than GM-CSF macrophages in 30 minutes regardless of stimulation condition184. Increased ATP degradation suggests that there is more endogenous, immunosuppressive adenosine available adjacent to the 86 macrophage surface to signal through the adenosine receptors to promote a transition to a regulatory phenotype. We also demonstrated that LPS stimulated M-CSF macrophages secrete more PGE2, which we believe can act in an autocrine fashion in order to promote the transition to a regulatory phenotype in M-CSF but to a lesser extent in GM-CSF macrophages. This was further supported by mRNA expression of COX2, which was significantly induced in LPS stimulated M-CSF macrophages but not in GM-CSF macrophages. Not only were these molecules produced at lower levels in GM-CSF macrophages, but the receptors for sensing them, A2a receptor, A2b receptor, EP2 receptor and EP4 receptor, were also transcribed at significantly lower levels following TLR stimulation by LPS and various other TLR ligands. Notably, these 4 receptors mediate the immunosuppressive effects of adenosine and PGE 56,192,1932 . This also suggests that the differential regulation of the purinergic and PGE2 pathway genes by M- CSF and GM-CSF macrophages is consistent in different contexts of pathogenic infection. Transcriptomic data allowed us to compare global genetic changes between stimulated M-CSF and GM-CSF macrophages. The PCA analysis alone indicated to us that M-CSF and GM-CSF macrophages are transcriptionally different even under similar stimulation conditions. Stimulation with LPS, LPS + Adenosine, and LPS + PGE2 exhibited more variance among samples in M-CSF than in GM-CSF macrophages suggesting that GM-CSF macrophages are less susceptible to phenotypic modulation by adenosine and PGE2. This was further supported by the number of DEGs by LPS + Adenosine and LPS + PGE2 relative to LPS alone, which was significantly higher in M- CSF macrophages than GM-CSF macrophages. Many of the upregulated and 87 downregulated genes by LPS + Adenosine and LPS + PGE2 relative to LPS alone in M- CSF macrophages were not up- or downregulated to the same extent in GM-CSF macrophages subject to the same conditions. Many of these upregulated genes had growth promoting functions, demonstrating that GM-CSF macrophages are not easily programmed to promote tissue repair. This was highlighted by the fact that M-CSF macrophages stimulated with adenosine and PGE2 secreted factors that promoted tube formation by HUVEC cells, while stimulated GM-CSF macrophages secreted factors the inhibited tube formation. Tube formation has been proposed to be a reliable in vitro assay for angiogenesis as it involves adhesion, migration and tubule formation all in one experiment194. Surface proteins CD300E and PLAUR were found to have increased expression on adenosine and PGE2 stimulated macrophages. CD300E in particular was not expressed above baseline on GM-CSF macrophages, making it a more suitable biomarker for M-CSF regulatory macrophages. Secreted proteins THBS1 and VEGF? were also higher in adenosine and PGE2 stimulated M-CSF macrophages, suggesting that they could also be used as biomarkers for M-CSF regulatory macrophages. Both of these proteins are well known contributors to the wound healing process195,168. On the other hand, levels of inflammatory cytokines, TNF, IL-12p40 and GM-CSF were secreted at higher levels in LPS stimulated GM-CSF macrophages, and the addition of adenosine and PGE2 did not dampen their secretion. This implies that GM-CSF macrophages are programmed to resist phenotypic change in response to resolving molecules in the inflammatory milieu in order to maintain higher levels of activation. Because macrophages are highly plastic in nature, we wanted to know if adding GM-CSF to M- 88 CSF differentiated macrophages could skew their phenotype. This had previously been shown to be true by another group who demonstrated that the M-CSF and GM-CSF differentiation of macrophages was not end stage, and that a subsequent period of 6 days in the opposite growth factor could reverse original phenotypes196. We demonstrated that this was the case for the secretion of inflammatory cytokines TNF and IL-12p40, and the extent of skewing correlated with the length of GM-CSF priming time. However, GM- CSF priming did not negatively affect the levels of IL-10 secretion. Additionally, GM- CSF priming did not restore TNF and IL-12p40 levels to the levels seen in GM-CSF differentiated macrophages. Therefore, it appears that exposure longer than 24 hours to GM-CSF is most effective in programming macrophages to reach a more inflammatory potential. While total amounts of inflammatory cytokine secretion contribute to immunopathology, the duration of cytokine secretion also has an important role in immune responses. GM-CSF macrophages secreted TNF at higher levels than M-CSF macrophages for up to 18 hours following the removal of stimulus. GM-CSF macrophages also had significantly higher steady state levels of TNF and IL-12p40 in their supernatants at different time points up to 24 hours. Together these data suggest that M-CSF macrophages terminate their activation more effectively and faster than their GM-CSF counterparts and transition to a resolving phenotype. The expression of marker gene candidates CD300E, VEGFA and THBS1 were measured at early (4 hour) and later (12 hour) time points following LPS stimulation in the presence of pharmacological inhibitors of the A2ar, EP2 and EP4 receptors. This preliminary data suggested to us that pharmacological blockade of these receptors may prevent the upregulation of these 89 transcripts at later time points, but may not have an effect on the upregulation of these transcripts at early time points. We hypothesize that this may be due to the fact that the ligands for these receptors need some time to be produced endogenously in order for them to act in an autocrine manner. The pharmacological blockade of the EP4 receptor alone led to more than double an increase in TNF production by M-CSF macrophages in response to LPS, suggesting that under normal conditions, endogenous PGE2 helps to limit inflammatory TNF. The pharmacological blockade of the EP4 receptor had no effect on the high levels of TNF produced by GM-CSF macrophages, which is not surprising, due to the presumed lack of EP4 receptor expression by GM-CSF macrophages based on mRNA data. Overall, the data in this chapter combined with the data in chapter 4 led us to propose a model for both the regulation of M-CSF macrophage activation and the lack of regulation in GM-CSF macrophages (Figure 34). We hypothesize that M-CSF macrophages are better equipped to turn off inflammation and initiate a program of tissue repair due to the production and increased sensitivity to the resolving molecules, adenosine and PGE2. We demonstrate that a number of components proposed in our model were lacking or present at lower levels in GM-CSF macrophages, which we believe helps to explain why GM-CSF macrophages are known to be hyper- inflammatory. In M-CSF but not GM-CSF macrophages, the expression of A2ar, A2br, EP2 and EP4 receptors is upregulated following TLR stimulation, making them more ready to sense adenosine and PGE2 in their environment. The degradation of ATP and production of PGE2 by GM-CSF macrophages is also lower than in M-CSF macrophages. We propose that together these differences in the purinergic and PGE2 pathways, between 90 "&/"$ X &1"$ 12($ "0!#$ &/"$ X *+-.$ &78$ "'3456$ *+,-$ ":89;:8<=;=7$ &#)($ /("%$ !"#$ &#'($ X !"%$ &1"$ "0!X #$&>'?:=783=?$ 0 X9$ 09$ $'?=7$ 09$ /"0!A6$08IG:$J'?G8>9$ Figure 34. Proposed pathway for endogenous regulation of M-CSF macrophage activation and lack of regulation of GM-CSF macrophages. TLR activation by PAMPs leads to the transcription and production of inflammatory mediators including the cytokines and chemokines. However, TLR activation also leads to the production of ATP which is degraded to adenosine. Adenosine signals through A2aR and A2bR to inhibit inflammation and initiate production of growth promoting proteins. Similarly, TLR activation leads to the production of PGE2,via COX and MPGES proteins, which then signals through EP2 and EP4 receptors to inhibit inflammation and initiate production of growth promoting proteins. We propose that these two molecules contribute to the resolution and control of M-CSF macrophage activation in response to pathogens. Components marked with a red (X) are those that are inhibited in macrophages differentiated in GM-CSF. Therefore, we propose that GM-CSF macrophages are unable to respond to adenosine and PGE2 in order to limit their activation or contribute to tissue homeostasis. 91 M-CSF and GM-CSF macrophages, are contributing factors to the propensity of these cells to either perpetuate inflammation or promote tissue repair. 92 6 Conclusions and Future Directions Macrophages are highly responsive to their tissue environments. In this research, we explore the effects of a number of different stimuli that macrophages may encounter in the host under different contexts. We demonstrate that Type I and Type II IFNs modulate the expression of adenosine receptors in mouse macrophages. We have also characterized a population of human M-CSF macrophages with growth promoting and pro-angiogenic activity that we believe arises following the termination of every immune response as a mechanism to restore tissue homeostasis. We demonstrate that GM-CSF human macrophages exhibit prolonged inflammatory responses because they are defective in this transition. These observations have several potential implications for influencing immunity and inflammatory responses. First, they predict that IFN inflammatory responses are partially regulated in macrophages by the purinergic system. Second, they suggest that M-CSF macrophages are poised to promote tissue repair and that a lack of this growth factor has the potential to lead to chronic inflammatory conditions. Third, they suggest that GM-CSF may prolong immunity and delay immune resolution not only by increasing inflammation but also by delaying its resolution and preventing the upregulation of genes critical for tissue repair. The failure of GM-CSF macrophages to transition to a growth-promoting phenotype could explain the mechanism of action of this protein in disease. Tissue GM-CSF levels are elevated in numerous autoimmune/inflammatory conditions including multiple sclerosis197, rheumatoid arthritis198, systemic inflammation112, and allergic responses113. A multitude of clinical trials for monoclonal antibodies to GM-CSF have been undertaken in the context of these diseases102,199. 93 Exploring macrophage activation on a spectrum by examining responses to molecules aside from the usual M1 and M2 stimuli, furthers our understanding of macrophage function in numerous physiological and disease environments. For example, our results are relevant in the context of certain cancers, as adenosine and PGE2 are present at high levels in the tumor microenvironment, known to harbor numerous tumor associated macrophages47,57,200?202. Because macrophages are highly sensitive to small changes in their surroundings, it is important to continue to investigate macrophage responses to a wide range and combination of stimuli. The findings presented in this work illuminate the similarity of the macrophage response to adenosine and PGE2 during inflammation. We believe that this could be attributed to the signaling of these molecules through g-protein coupled receptors (GPCRs) and downstream cAMP production. It is possible that this macrophage phenotype extends to stimulation with numerous other GPCR ligands, which should be further explored. GPCRs make up the largest class of receptors for approved membrane drug targets203. Therefore, it is possible that there are existing drugs that can be used in contexts that we have not yet discovered. Much of the research on human monocyte-derived macrophages is highly variable as different labs have multitudes of protocols for generating macrophages from monocytes. This work shows that using GM-CSF alone as a differentiation factor can skew or bias the resulting macrophages to be inflammatory and resistant to the transition to a growth promoting phenotype. We propose that M-CSF is the growth factor most ?neutral? to generate human macrophages. 94 Our observation that stimulated GM-CSF macrophages are resistant to the anti- inflammatory effects of adenosine and PGE2 illuminates the potential use of GM-CSF in contexts in which it would be beneficial for macrophages to be hyper-inflammatory. One example would be the use of GM-CSF as a vaccine adjuvant. There are recent clinical trials testing GM-CSF as an adjuvant in cancer vaccines due to its anti-tumor properties204,205. GM-CSF could also potentially be used in parasitic diseases such as leishmaniasis in which cell mediated immune responses are needed for pathogen killing130. In fact, topical application of GM-CSF to lesions has been demonstrated to reduce the healing time in cutaneous leishmaniasis patients, due to increased parasite killing206. We identified promising protein biomarkers for regulatory macrophages including, THBS1, VEGFA, CD300E and PLAUR which could potentially be used in combination with cell specific markers such as CD68 and CD11b to identify growth promoting macrophages in histological samples or in vivo. Exploring where these macrophages are located in the host can help us better understand their functional roles and allow us to target them in different diseases. Along with this, another potential use for these biomarkers is during therapeutic testing in order to determine if certain drugs are effective in producing the intended phenotype in macrophages. This work raises new questions that would benefit from further research. Pathway analysis led to the prediction of many transcriptional regulators of the genes modulated by regulatory macrophages. It would be beneficial to identify specific transcriptional regulators using pharmacological tools or protein interaction studies. This way, regulatory macrophage phenotypes could be mimicked with the modulation of a few key 95 proteins. Additionally, further exploration of the mechanism of M-CSF and GM-CSF control of the purinergic and PGE2 pathways is needed, including how the expression of the receptors for these molecules is regulated. We highlighted the pro-angiogenic nature of LPS + Adenosine and LPS + PGE2 macrophages, which has implications for macrophage actions in the tumor microenvironment as well as in wound healing or tissue repair. It would be interesting to compare the transcriptomic data generated in this work with transcriptomic data generated by other researchers on macrophages in known disease environments. 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