THE ROLE OF MACROPHAGE POLARIZATION IN LIVER REPAIR FOLLOWING AN ACUTE INJURY By Akie Jessica Mochizuki A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Biochemistry and Molecular Biology- Master of Scien ce 2014 ABSTRACT THE ROLE OF MACROPHAGE POLARIZATION IN LIVER REPAIR FOLLOWING AN ACUTE INJURY By Akie Jessica Mochizuki Acute liver failure is a condition in which the liv er loses of function and the ability to repair. ALF is hallmarked by the inability of mo nocytes and macrophages to become classically activated. The mechanisms by which macr ophages may contribute to the liver injury and their role in the subsequent repai r are not well characterized. The purpose of these studies was to determine the r ole of macrophages in acute liver injury and repair in response to carbon tetra chloride treatment. We found that compared to control mice, the ALT activity level wa s not significantly different in macrophage-depleted mice, while an increased area o f necrosis was observed in macrophage-depleted mice 72hrs following injury. Ho wever, a decreased amount of Type I collagen staining was seen in macrophage-dep leted mice while no change was seen in the mRNA levels of Type I collagen and fact ors related to collagen production. Collectively, these studies suggest that macrophage s may play a role in the clearance of necrotic cells and collagen remodeling to aid in liver repair. Macrophages are known to polarize into a pro-inflam matory phenotype termed M1 macrophages and an anti-inflammatory phenotype, M2 macrophages. Currently, the role of M1 and M2 macrophages in liver injury and r epair is not clear. I have conducted preliminary studies that determined the validity an d feasibility of an in vitro macrophage polarization and the localization of adoptively tra nsferred macrophages to the liver into macrophage depleted mice following the induction of an acute liver injury. ACKNOWLEDGEMENTS Firstly, I would like to thank my mother, Bette Jo Nakahara, my sister, Naomi Mochizuki, my grandmother, Betty Nishimoto and my s tep-father, Keith Nakahara, for their unwavering love and support despite windy roa ds and late revelations. My sincere appreciation is extended to my co-mentor s, Professors Bryan Copple and Cheryl Rockwell for their guidance, flexibility and continued support. Thank you for the experience and expertise that will be the found ation for my future career. I would also like to thank my colleagues in both the Copple and Rockwell labs for their friendship and shared supplies. Thank you to everyo ne who helped me on a daily basis, Heather Dover, Kara Kelly, Aaron Pace, Ryan Albee, Keara Towery and Holly Cline. To my lab siblings, Kate O™Brien, Alexandra Turley and Joseph Zagorski, thank you for the all the academic and emotional support throughout t his experience. Lastly, I would like to thank all the people in at Michigan State University who have inspired, challenged and shaped me into the pe rson I am today. In the past two and a half years, I was driven to think more deeply , work harder and grow more quickly than I ever have before. My graduate experience at Michigan State University has truly been an experience that has had a lasting impact on my personal and professional development. TABLE OF CONTENTS LIST OF TABLES ––––––––.––––––––––...–––––––––...–vi LIST OF FIGURES –––––––––.––––––––––––...––––––....vii KEY TO ABBREVIATIONS –––..–––––––––––––––..–––..–......viii CHAPTER 1 ––– ––.–––––––– ––.––––– ––––––––––––1 INTRODUCTION –––––––– ––––––––––––––––––––––..1 INTRODUCTION––––––––––––––––––––––––––– ..2 CHAPTER 2 –– ––––––––––––..– ...––––––––––––––––7 THE IMPACT OF MACROPHAGES ON MECHANISMS OF LIVER RE PAIR FOLLOWING CARBON TETRACHLORIDE INDUCED LIVER –––––.––––– .7 ABSTRACT–––––––––––––––..––––– –..–––––– .–..8 INTRODUCTION–––––––––– –––––––––..–– ––...– ––.9 MATERIALS AND METHODS––––...–––––––––– ––......–– –12 Animals–––––––––––––––––––––– .––––––12 Carbon Tetrachloride Treatment–––––––––––– .–––––.12 RNA Isolation and qRT-PCR–––––––––––––––––––12 Immunohistochemistry and Immunofluorescence––––– .–––––13 Macrophage Depletion––––––––––––––––––––..–13 Quantification of Necrosis––––––––––––––––––––.13 Alanine Aminotransferase Activity––––––––––––– .––......14 Statistics–––––––––––––––––––––––– .–––..14 RESULTS–––––––––––––––...–––––––––– ––..–...16 DISCUSSION–– ––––––––––––––––––––––– ..––– 30 CHAPTER 3 ––––––––––––––– –––––––––––––––––33 MACROPHAGE POLARIZATION IN ACUTE LIVER INJURY: A PR OSPECTIVE ADOPTIVE TRANSFER STUDY ––––.–––––––––––––––––––33 ABSTRACT––––– –.––––––.–––––––– –– –..––...– .–.34 INTRODUCTION––––.–––––––––––..–––––– –..–––..35 MATERIALS AND METHODS––––––––––––...–– ––– –..––39 Animals––––––––––––––––––––––––– –––.39 Macrophage Depletion–––––––––––––––––––––..39 Macrophage Adoptive Transfer––––––––––––––––––39 In vitro Bone Marrow-Derived Macrophage Polarization––––––– .39 RNA Isolation and qRT-PCR––––––––––––––––––....40 Immunohistochemistry and Immunofluorescence–––––– ––––.40 Statistics––––––––––––––––––––––––––...–41 RESULTS–––––...––––––––––––––..–––––– –...–...42 DISCUSSION–––––––––– ––..–––––..––––––––––.47 REFERENCES –––––––––––––––––..–––––––––...–– –..49 LIST OF TABLES Table 1: Summary of Primer Sequences used for qRT-P CR–––––––––– ..–15 LIST OF FIGURES Figure 2-1: Macrophages accumulate in the liver fol lowing carbon tetrachloride treatment–––––––––––––––––––––––––––––––– ..–19 Figure 2-2: Macrophage depletion 72 hours after car bon tetrachloride treatment.–.....20 Figure 2-3: Serum ALT activity of mice following tr eatment with carbon tetrachloride– 21 Figure 2-4: Quantification of necrotic areas follow ing macrophage depletion and carbon tetrachloride treatment–––––––––––––––––––––––––––. ..22 Figure 2-5: The effect of macrophage depletion on i nflammatory mediators in the liver following carbon tetrachloride treatment––––––––––– ––––––––– .23 Figure 2-6: The effect of macrophage depletion on h epatocyte proliferation in the liver following carbon tetrachloride treatment––––––––––– ––––––––– .24 Figure 2-7: Quantification of PCNA staining of hepa tocyte nuclei following macrophage depletion and carbon tetrachloride treatment––––––– ––––––––––....25 Figure 2-8: Quantification of Type I Collagen accum ulation following macrophage depletion and carbon tetrachloride treatment– ––––––––––––––––– 26 Figure 2-9: The effect of macrophage depletion on p ro-fibrotic gene expression in the liver following carbon tetrachloride treatment––––– ––––––––––––– 28 Figure 2-10: The effect of macrophage depletion on matrix metalloproteinase expression in the liver following carbon tetrachlor ide treatment..––– –––– –......29 Figure 3-0: Adoptive transfer of M1 and M2 polarize d macrophages into macrophage- depleted mice following acute liver injury––––––––– –––––––––...–38 Figure 3-1: Hepatic macrophage depletion of the liv er following liposomal clodronate treatment–––––––––––––––––––––––––––––– –..––44 Figure 3-2: GFP-expressing macrophage adoptive tran sfer into macrophage depleted livers–––––––––––––––––––––––––––––––.............. ..45 Figure 3-3: Bone marrow derived macrophage in vitro polarization––––––...–...46 KEY TO ABBREVIATIONS ALF Acute Liver Failure ALT Alanine Aminotransferase APAP Acetaminophen CCl 4 Carbon Tetrachloride CCND1 Cyclin D1 ECM Extracellular matrix FGF2 Fibroblast growth factor 2 H&E Hematoxylin and Eosin HGF Hepatocyte Growth Factor HSCs Hepatic Stellate Cells IFN Interferon gamma IL Interleukin iNOS Inducible Nitric Oxide Synthase I.P. Intraperitoneal LAP Latency associated peptide LPS Lipopolysaccharide LTBP Latent TGF-beta binding protein Ly6C Lymphocyte antigen 6C MMP Matrix Metallopeptidase MT-MMP Membrane type -MMP NF- B Nuclear Factor kappa-light-chain-enhancer of Ac tivated B-cells NKT Natural Killer T-cells NO Nitric Oxide PAI-1 Plasminogen Activator Inhibitor-1 PAMP Pathogen Associated Molecular Pattern PCNA Proliferating Cell Nuclear Antigen PDGF Platelet derived growth factor qRT-PCR Quantitative Real-Time Polymerase Chain Re action TGF- Transforming Growth Factor alpha TGF- Transforming Growth Factor beta TH1 Type-1 T-Helper Cell TH2 Type-2 T-Helper Cell TIMP Tissue Inhibitor of Metalloproteinase TNF Tumor Necrosis Factor alpha TNFR-1 Tumor Necrosis Factor Receptor-1 TSP-1 Thrombospondin-1 uPA Urokinase-type Plasminogen Activator CHAPTER 1 INTRODUCTION INTRODUCTION Liver regeneration is an amazing process that has been known to occur since ancient times. Following partial hepatectomy, the l iver will regenerate proportionally to the amount of liver removed (Kawasaki et al., 1992) . The liver has also been shown to regenerate proportionally to the size of the animal following transplantation of a donor liver of a different sized animal (Vine & Kier, 199 3). These observations have led to the desire to fully understand the cellular mechanisms behind liver regeneration in order to treat people suffering from various liver diseases that impair normal liver repair and regeneration. Most of the previous work done on liv er repair has been focused on the process of hepatocyte proliferation and the molecul ar signals that mediate this process. Hepatocytes are the main cell type that comprise t he liver and are of critical importance for liver function and maintenance of ho meostasis. Hepatocytes are responsible for glucose regulation, synthesis of a variety of proteins found in blood, synthesis and secretion of bile as well as drug and toxin metabolism. During liver repair, hepatocytes are the first cells in the liver to res pond by synthesizing DNA and mitogenic stimuli for other cell types in the liver (Adachi e t al., 1995). However, the trigger for hepatocyte synthesis itself remained elusive until 1984, when it was found in the serum of rats that had undergone partial hepatectomy. Hep atocyte Growth Fator (HGF) concentrations reach 20-fold above normal levels in the blood 1 hour following partial hepatectomy and reduce slowly until 72 hours (Naldi ni et al., 1991; Lindroos et al., 1991). HGF is known to play an essential role in li ver development. Complete deletion of this gene in mice results in embryonic lethality (Schmidt et al., 1995). The source of HGF in the liver following partial hepatectomy is t he result of the disruption in the biomatrix of the liver that contains a large amount of HGF (Matsumoto & Yamamoto, 1991). Once HGF is released into the serum, it is d irectly involved in the induction of hepatocyte mitosis (Kim et al., 1997). Although much is known about hepatocyte proliferat ion during liver repair, much remains unclear about the roles that other non-pare nchymal cells play in liver repair. In addition, much of what we know about liver repair h as been determined from studies of partial hepatectomy. However, most instances of liv er repair naturally occur as a result of mild and acute liver injury. Drug Induced Liver Injury (DILI) is a common cause of mild and acute liver injury but also accounts for h alf of the acute liver failure cases in the United States (Abboud & Kaplowitz, 2007). This sugg ests that there is a threshold for injury that can be repaired by normal liver regener ation and beyond this threshold, the liver fails to repair itself and results in failure (Chaudhuri et al., 2011). By illuminating the mechanisms normally involved in DILI repair, furthe r research can then be conducted to identify deviations from this repair and potential therapeutic targets. My goal is to determine the role of macrophages in normal liver r epair as a result of DILI. Macrophages are innate immune cells of the myeloid lineage which play an important role in liver injury (Geissmann et al., 2 010). They are phagocytic cells and are the first line of defense against bacteria and othe r infectious microbes (Liaskou et al., 2012). Macrophages are known to undergo classical a nd alternative activation (Laskin, 2009). During infection, macrophages and monocytes are induced by pathogen associated molecular patterns (PAMPs) such as lipop olysaccharide (LPS) to polarize and express pro-inflammatory cytokines during class ical activation (Bieghs & Trautwein, 2013). Classically activated or M1 macrophages expr ess pro-inflammatory cytokines such as tumor necrosis factor (TNF ), interleukin-1 (IL-1) and interleukin-6 (IL-6) (Mantovani et al., 2004). TNF and IL-6 have been shown to be important signals involved in early signaling pathways leading to liv er regeneration (Fujiyoshi & Ozaki, 2011). The concentration of both cytokines increase in the liver following injury. And the ablation of both cytokines results in severe impair ment of liver repair (Bourdi et al., 2007; Kovalovich et al., 2000). However, the mecha nism for classical activation in DILI remains unclear. Macrophages can also be alternatively activated by exposure to anti- inflammatory cytokines such as IL-4 and IL-13 (Loke et al., 2002). These alternatively activated macrophages or M2 macrophages express ant i-inflammatory cytokines such as IL-10 and transforming growth factor- (TGF- ) (Gong et al., 2012). Kupffer cells, resident liver macrophages, express more of an anti -inflammatory or M2-like phenotype (Bieghs & Trautwein, 2013). The liver serves as a f ilter for the products of the digestive system before nutrients and other macromolecules en ter the systemic circulation (Vollmar & Menger, 2009). Kupffer cells phagocytose bacteria and other microbes that arrive from the gut and serve as the liver™s first line of defense (Parker & Picut, 2005). Following liver injury, monocytes infiltrate into t he liver and differentiate into macrophages (Holt et al., 2008). The phenotype of t hese monocytes can be characterized by the level of Lymphocyte antigen 6C (Ly6C) expressed on the cell surface (Geissmann et al., 2003). Ly6C hi monocytes circulate in the blood, are recruited to sites of inflammation and express pro-inflammato ry mediators (Gordon & Taylor, 2005). Ly6C lo monocytes also circulate throughout the body, but differentiate into resident tissue macrophages such as Kupffer cells. Ly6C lo monocytes are phenotypically anti-inflammatory and M2-like (Geiss mann et al., 2003). Ly6C hi monocytes are recruited to the liver following tox icant exposure and contribute to the inflammatory response by expressi ng Thrombospondin-1 (TSP-1), TNF and Nitric Oxide (NO) in the first 12 hours after toxicant exposure (Helk et al., 2013; Ramachandran et al., 2012). As necrotic hepat ocyte debris accumulates in the liver, the M1-like cells phagocytose the debris whi ch induces the macrophages to polarize into an M2-like phenotype (Ramachandran et al., 2012). The polarization of M1-like macrophages into M2-like macrophages could be due to the exposure of the M1-like macrophages to IL-4 that is expressed by Ty pe-2 T-Helper cells (T H2) and Natural Killer T-cells (NKT) and to TGF- that is expressed by Kupffer cells (Gong et al., 2012; Pollard, 2009). By a combination of these two mechanisms, an increase in the population of Ly6C lo or M2-like macrophages in the liver arises from th e polarization of infiltrated Ly6C hi monocytes (Ramachandran et al., 2012). In addition to anti- inflammatory factors, Ly6C lo macrophages are known to express factors involved in scar resolution and tissue repair such as Matrix Metallo proteinase (MMP) 9 and 12 (Sobrevals et al., 2010). The role of these different macrophage populations in liver repair is not well understood. Ly6C lo macrophages may contribute to liver repair by thei r increased phagocytic activity and production of growth factor activators and ECM components. However factors produced by Ly6C hi macrophages also induce hepatocyte proliferation although some cytokines may exacerbate the injury. In the following studies, I investigated the role of macrophages in liver injur y, inflammation, hepatocyte regeneration and matrix deposition involved in live r repair. I also generated preliminary data to support future studies to identify the role of M1 and M2 macrophages in liver repair. CHAPTER 2 THE IMPACT OF MACROPHAGES ON MECHANISMS OF LIVER REPAIR FOLLOWING CARBON TETRACHLORIDE INDUCED LIVER INJURY ABSTRACT Acute Liver Failure is a condition in which the li ver loses function as well as the ability to repair itself. There are a number of stu dies that implicate the dysfunction of macrophages in ALF. However, not much is known abou t the role of macrophages in normal liver repair. In order to determine if macro phages are involved in liver repair following an acute injury, macrophages were deplete d from mice treated with carbon tetrachloride to induce an acute liver injury. The livers of the macrophage depleted mice had decreased mRNA levels of pro-inflammatory genes typically produced by macrophages. However, the ALT activity level of the control mice and macrophage- depleted mice were consistent between both groups w hile the areas of necrosis observed in the macrophage-depleted mice were incre ased at 72 hours following injury. This suggests that macrophages are involved in the clearance of dead hepatocytes following injury. We also found that macrophages ma y not be required for hepatocyte proliferation possibly due to compensatory redundan t mechanisms. This was determined by observing similar levels of PCNA staining of macrophage depleted and control mice. The mRNA levels of genes involved in hepatocyte proliferation were also unchanged in the macrophage depleted mice as compared to the control mice. A notable decrease in Type I collagen deposition was observed in the macrophage depleted mice. However, the mRNA levels of Type I collagen were consistent between groups. In addition, the mRNA le vels of genes involved in ECM production were all unchanged by macrophage depleti on. However, the decreased collagen deposition could be accounted for by a cha nge in the activation states of TGF- or other regulators of collagen deposition such as MMPs, MT-MMPs or TIMPS. INTRODUCTION Acute liver failure is a condition in which the liv er undergoes a massive and rapid loss of function following injury. A variety of tox ic agents can damage the liver sufficiently to cause ALF such as drugs, alcohol, v iral hepatitis, autoimmune liver disease and septic shock. These agents induce hepat ic damage that can lead to wide- spread hepatic necrosis. Normally, the liver has an incredible ability to repair itself. However, the livers of patients with ALF have lost the ability to carry out this essential function. Instead, patients have elevated levels of anti-inflammatory cytokines and decreased levels of pro-inflammatory cytokines in t heir serum which correlates with poor outcomes (Antoniades et al., 2006). In additio n, ALF patients with adverse outcomes exhibit compensatory anti-inflammatory res ponse syndrome in which their monocytes and macrophages lose the ability to becom e activated (Wasmuth et al., 2005). Together these data imply that the immune sy stem and especially macrophages may play an important role in the pathogenesis of A LF (Antoniades et al., 2008). However, in order to fully understand the mechanism s that underlie ALF, it is important to also understand the role of macrophages in norma l liver repair. The purpose of the following studies was to investigate the role of ma crophages in liver repair. Kupffer cells, the resident liver macrophages, serv e as the first line of defense against pathogens arriving from the gut. In respons e to PAMPs and other molecular signals such as toxin-induced injury, macrophages c an become classically activated to produce pro-inflammatory cytokines and anti-microbi al superoxides. One mechanism by which macrophages may contribute to liver repair is through the production of the pro- inflammatory cytokines, TNF and IL-6. Both cytokines are known to stimulate hepatocyte proliferation and are necessary for live r repair (Cressman et al., 1996; Yamada et al., 1997). Studies have demonstrated tha t these cytokines prime hepatocytes to be more responsive to growth factors such as HGF and transforming growth factor-alpha (TGF- ) by inducing transcription factors involved in cel l cycle progression (Columbia & Clinic, 1998; Libermann & B altimore, 1990). For example, IL-6 activates the STAT3 pathway which increases Cyclin D1 (CCND1) expression, a key regulatory protein for cells to pass from G1 to S p hase during mitosis (Li et al., 2002) . Once primed, hepatocytes achieve a maximal level o f mitotic activity in response to the mitogens HGF and TGF- (Adachi et al., 1995; Tomiya et al., 2000). Macrophages can also regulate availability of these growth factors to hepatocytes by the production of MMPs and other factors that regul ate extracellular matrix (ECM) remodeling (Valentin et al., 2009). The ECM is the structural scaffold of the liver and is mainly comprised of collagens Type I, III, IV and V as well as various glycoproteins and proteoglycans (Ashkenas et al., 1996). MMPs degrade collagens and thereby release HGF that was previously sequestered in the ECM (Skr tic et al., 1999). Macrophages also regulate ECM synthesis by producin g growth factors that stimulate collagen synthesis and stimulate prolifer ation of activated HSCs and fibroblasts. These growth factors include platelet derived growth factor (PDGF), fibroblast growth factor (FGF), and TGF- (Khalil et al., 1989). In addition to producing growth factors, macrophages can produce proteins th at affect the activity of these growth factors, such as TSP-1 and MMPs which can ac tivate latent-TGF- . Macrophages also aid in tissue recovery by clearing pathogens as well as cellular debris by phagocytosis. During this proces s, macrophages have the ability to distinguish pathogens from necrotic and apoptotic c ellular debris through binding of different cell surface receptors (Allen & Aderem, 1 996). Following recognition, the target material is then engulfed by the cell and transport ed to the phagosome where it awaits fusion with a lysosome in order for the engulfed ma terial to be fully degraded (Poon, Hulett, & Parish, 2010). However, it is not known if the phagocytic function of macrophages contributes to normal liver repair. Although not much is known about the distinct func tions of macrophages during ALF, there is evidence that the production of pro-i nflammatory cytokines and macrophage activation may be essential to proper li ver repair. In the following experiments, liver injury was produced by carbon te trachloride in normal mice and macrophage depleted mice and mechanisms of liver re pair were assessed. My hypothesis was that following liver injury caused b y carbon tetrachloride treatment in mice, livers depleted of macrophages would not unde rgo proper liver repair. MATERIALS AND METHOD Animals Mice used in these studies are all male C57BL/6 (Ja ckson Laboratories, Bar Harbor, ME) mice from 8 to 10 weeks of age. All mic e were maintained on a 12-h light/dark cycle under controlled temperature (18-2 1ºC) and humidity. Food (Rodent Chow; Harlan-Teklad, Madison, WI) and tap water wer e allowed ad libitum . All procedures on animals were carried out in accordanc e with the Guide for the Care and Use of Laboratory Animals promulgated by the Nation al Institutes of Health. Carbon Tetrachloride Treatment Mice were treated by intraperitoneal injection with 1 ml/kg of carbon tetrachloride (CCl 4, Sigma Chemical Company). The carbon tetrachlorid e was diluted 1:10 in corn oil (Sigma Chemical Company) prior to injection. RNA Isolation and qRT-PCR Liver tissue was homogenized in 1mL of TRI reagent (Sigma Chemical Company, St. Louis, MO) and total RNA was isolated per manufacturer™s instructions. Total RNA was quantified spectrophotometrically usi ng the Infinite 200PRO (Tecan Group Ltd., Männedorf, Switzerland). 125ng of total RNA was reverse transcribed into cDNA in a volume of 12 L containing PCR buffer (166 mM (NH 4)2SO 4, 50 mM -mercaptoethanol, 67 M EDTA, 0.67 M Tris buffer, pH 8.8, 0.8 mg/ml BSA, 5 mM MgCl 2), 1 mM each dNTP (Promega, Madison, WI), 10 units rRNasin (Promega), 125 ng oligo(dT) 15 (Promega) and 50 units of Moloney Murine Leukemia Virus reverse transcriptase (MMLV-RT; Promega). The cDNA reaction was heated to 37°C for 55 min and then 70°C for 5 min. Next, 10 L of iTaq Universal SYBR Green Supermix (Bio- Rad, Hercules, CA) and 5 M of each primer pair was added to each cDNA sample for a final volume of 20 L. Real-time PCR was performed on an ABI 7900 real- time PCR instrument (Applied Biosystems, Foster City, CA). Primers used for PCR are shown in Table 1. Immunohistochemistry and Immunofluorescence Immunofluorescence was used to detect and quantify type I collagen and macrophages in 8 m frozen sections of liver. The sections were fixed in 4% formalin and then incubated with rat anti-CD68 antibody dilu ted 1:100 (Novus, Littleton, CO) or rat anti-F4/80 antibody diluted 1:25 (AbD Serotec, Raleigh, NC) and rabbit anti-type I collagen antibody diluted 1:400 (Millipore, Billeri ca, MA). The sections were then incubated with secondary antibodies conjugated to e ither Alexa Fluor 488 or Alexa Fluor 594 (Life Technologies, Grand Island, NY). Proliferating cell nuclear antigen (PCNA) was detec ted in formalin-fixed, paraffin- embedded sections of liver using the Vectastain Eli te ABC Kit and Vector DAB (Vector Laboratories, Burlingame, CA). Anti-PCNA antibody (1:8000, Abcam, Cambridge, MA) was added to the tissues and incubated overnight at 4 degrees C. Macrophage Depletion Mice were treated with 200 l liposome encapsulated clodronate (ClodronateLiposomes.com, The Netherlands) or PBS-c ontaining liposomes by i.p. injection. After 48 hours, the mice were treated w ith carbon tetrachloride. Quantification of Necrosis The area of necrosis was quantified in 15, 200X fi elds per hematoxylin and eosin-stained liver section using Image J Software (National Institutes of Health). The analysis was performed in a blinded fashion. Alanine Aminotransferase Activity Hepatocyte injury was evaluated by measuring the se rum activity of alanine aminotransferase (ALT) (Pointe Scientific Inc., Can ton, MI). Statistics Results are presented as the mean + SEM. 5-7 mice were used for all in vivo studies. 3 mice were used for all in vitro studies. Data were analyzed by Analysis of Variance (ANOVA). Data expressed as a fraction wer e transformed by arc sine square root prior to analysis. Comparisons among group mea ns were made using the Student- Newman-Keuls test. The criterion for significance w as p < 0.05 for all studies. Table 1: Summary of Primer Sequences used for qRT-P CR Gene Forward Primer Reverse Primer Rpl13a 5'-TCCTTGTTCCACTGTGCCTTG-3' 5'-TGCTTCCACATGT CCTCACAA-3' Col1a1 5'-TTGACGGAAGGGCACCACCAG-3' 5'-GCACCACCACCCA CGGAATCG-3' MIP-2 5'-CTCAGACAGCGAGGCACATC-3' 5'-CCTCAACGGAAGAAC CAAAGAG-3' PDGF-B 5™-CCCACAGTGGCTTTTCATTT-3™ 5™-GTGGAGGAGCAGACTGAAGG- 3™ FGF2 5™-AGCGGCTCTACTGCAAGAAC-3™ 5™-GCCGTCCATCTTCCTT CATA-3™ PAI-1 5™-AGTCTTTCCGACCAAGAGCA-3™ 5™-ATCACTTGCCCCATG AAGAG-3™ -SMA 5™-CCACCGCAAATGCTTCTAAGT-3™ 5™-GGCAGGAATGATTTGGAAAGG-3™ iNOS 5™-TTCTGTGCTGTCCCAGTGAG-3' 5™-TGAAGAAAACCCCTTG TGCT-3' IL-6 5™-ACCAGAGGAAATTTTCAATAGGC-3™ 5™-TGATGCACTTGCA GAAAACA-3™ TNF- 5'- TGGCTGTGACTCCCCTTCTTT-3' 5'-AGAGCTCAACACAAGCGT GGA-3' TGF- 1 5™-CAACCCAGGTCCTTCCTAAA-3™ 5™-GGAGAGCCCTGGATACCAA C-3™ MMP2 5™-GGGGTCCATTTTCTTCTTCA-3' 5™-CCAGCAAGTAGATGCT GCCT-3' MMP3 5™CCCACCAAGTCTAACTCTCTGGAA-3™ 5™-GGGTGCTGACTGCATCAAAGA-3™ MMP9 5™-CTGTCGGCTGTGGTTCAGT-3' 5™-AGACGACATAGACGGCA TCC-3' MMP13 5™-GGTCCTTGGAGTGATCCAGA-3' 5™-TGATGAAACCTGGAC AAGCA-3' RESULTS The extent of macrophage accumulation in the liver after carbon tetrachloride was quantified by immunohistochemical staining of t he macrophage cell surface markers, CD68 and F4/80. CD68 is a 110-kD transmemb rane glycoprotein that is expressed in blood monocytes and resident tissue ma crophages in mice and humans. (Holness & Simmons, 1993). F4/80 is a highly specif ic cell surface marker of resident tissue macrophages found in mice (Austyn & Gordon, 1981). Following carbon tetrachloride treatment, CD68 and F4/80 positive ma crophages accumulated within centrilobular regions of liver (Figure 2-1). In order to probe the role of macrophages in liver repair after injury, macrophages were depleted from the livers of mice p rior to carbon tetrachloride treatment with liposomal clodronate. Treatment of m ice with liposomal clodronate decreased hepatic macrophages 72 hours following ca rbon tetrachloride treatment (Figure 2-2). ALT is a hepatocyte protein released into the blood after hepatocyte cell death. Its activity in serum is measured clinically to est imate the extent of liver injury (Karmen et al., 1954; Snell & Jenkins, 1959). ALT activity was not different between mice treated with liposomal clodronate and carbon tetrachloride and mice treated with liposomal PBS and carbon tetrachloride at 48 hours (Figure 2-3). At 72 hours after carbon tetrachloride, there was a modest decrease in ALT activity in mice treated with liposomal clodronate (Figure 2-3). Next, the area of necrosis was quantified in sectio ns of hematoxylin and eosin (H&E) stained liver. Liposomal clodronate treated m ice had a significantly larger area of necrosis in the liver 72 hours after carbon tetrach loride when compared to mice treated with liposomes containing PBS (Figure 2-4). This su ggested that clearance of necrotic tissue may be defective in the mice depleted of mac rophages. Expression of pro-inflammatory genes was analyzed t o determine if macrophage depletion affects their upregulation (Figure 2-5). TNF- , inducible nitric oxide synthase (iNOS) and IL-6 were all ablated in liposomal clodr onate treated mice as compared to mice treated with liposomal PBS. Hepatocyte proliferation is another key process in liver repair. Genes involved in hepatocyte proliferation were analyzed to determine whether macrophages contribute to their upregulation. As shown in Figure 2-6 there w as no difference in the levels of these genes between control mice and macrophage depleted mice. Proliferating cell nuclear antigen (PCNA) protein was quantified in liver sect ions following macrophage depletion and carbon tetrachloride treatment as a measure of hepatocyte proliferation. There was no significant difference in PCNA staining between the macrophage depleted and non- depleted mice (Figure 2-7). A critical component of the tissue regeneration is synthesis of extracellular matrix, such as Type I collagen. Therefore, we stai ned livers of macrophage-depleted and control mice for Type I collagen using immunofl uorescence, in order to determine if macrophages affect this critical process (Figure 2- 8). Interestingly, hepatic Type I collagen was decreased in macrophage depleted mice as compared to control mice. We next verified this finding by analyzing the mRNA levels of Type I collagen (Figure 2- 9A). Interestingly, the mRNA levels of this gene we re not affected by macrophage depletion. Since Type I collagen is mainly produced by hepatic stellate cells in the liver, the mRNA levels of other genes involved in hepatic stel late cell activation and collagen production were analyzed to identify any difference s that may explain the difference seen in Type I collagen accumulation (Figure 2-9). All of the genes that were analyzed were unchanged in the macrophage depleted mice as c ompared to the control mice. Macrophages also produce members of the MMP family that are involved in the degradation of different components of the liver EC M. mRNA levels of MMP-13, a collagenase, and MMP-2, a gelatinase, were both sig nificantly decreased as a result of macrophage depletion (Figure 2-10). Figure 2- 1: Macrophages accumulate in the liver treatment. Male C57BL/6 mice were treated with either (A and C ) vehicle or (B and D) 1 ml/kg carbon tetrachloride for 72 hours. Representative C D68 immunofluorescence in frozen liver sections from (A) vehicle or (B) carbon t F4/80 immunofluorescence in livers sections from (C ) vehicle or (D) carbon tetrachloride treated mice. Positive staining is shown in red for both CD68 and F4/80. The area of immunofluorescence of (E) CD68 and (F) F Image J analysis. aSignificantly different from control mice. E 1: Macrophages accumulate in the liver following carbon tetrachloride Male C57BL/6 mice were treated with either (A and C ) vehicle or (B and D) 1 ml/kg carbon tetrachloride for 72 hours. Representative C D68 immunofluorescence in frozen liver sections from (A) vehicle or (B) carbon t etrachloride treated mice. Representative F4/80 immunofluorescence in livers sections from (C ) vehicle or (D) carbon tetrachloride treated mice. Positive staining is shown in red for both CD68 and F4/80. The area of immunofluorescence of (E) CD68 and (F) F 4/80 was quantified in liver sections by Significantly different from control mice. F following carbon tetrachloride Male C57BL/6 mice were treated with either (A and C ) vehicle or (B and D) 1 ml/kg carbon tetrachloride for 72 hours. Representative C D68 immunofluorescence in frozen etrachloride treated mice. Representative F4/80 immunofluorescence in livers sections from (C ) vehicle or (D) carbon tetrachloride treated mice. Positive staining is shown in red for both CD68 and F4/80. The area of 4/80 was quantified in liver sections by Figure 2- 2: Macrophage depletion 72 hours after carbon tetra chloride treatment. Male C57BL/6 mice were treated with either (A and C ) PBS and D) clodronate- containing liposomes for 72 hours. Mice were then t reated with carbon tetrachloride for 72 hours. Representative C D68 immunofluorescence in frozen liver s ections, positive staining shown in black, from (A) PBS treated mice or (B) clodronate immunofluorescence in livers sections, positive sta ining shown in black, from (C) PBS containi ng liposome treated mice or (D) clodronate Area of immunofluorescence of (E) CD68 and (F) F4/8 0 was quantified in liver sections. aSignificantly different from PBS E 2: Macrophage depletion 72 hours after carbon tetra chloride treatment. Male C57BL/6 mice were treated with either (A and C ) PBS -containing liposomes or (B containing liposomes for 72 hours. Mice were then t reated with carbon tetrachloride for 72 hours. Representative C D68 immunofluorescence in frozen ections, positive staining shown in black, from (A) PBS -containing liposome treated mice or (B) clodronate -containing liposome treated mice. Representative F4 /80 immunofluorescence in livers sections, positive sta ining shown in black, from (C) PBS ng liposome treated mice or (D) clodronate -containing liposome treated mice. Area of immunofluorescence of (E) CD68 and (F) F4/8 0 was quantified in liver sections. Significantly different from PBS -containing liposome treated mice. 2: Macrophage depletion 72 hours after carbon tetra chloride treatment. containing liposomes or (B containing liposomes for 72 hours. Mice were then t reated with carbon tetrachloride for 72 hours. Representative C D68 immunofluorescence in frozen containing liposome containing liposome treated mice. Representative F4 /80 immunofluorescence in livers sections, positive sta ining shown in black, from (C) PBS -containing liposome treated mice. Area of immunofluorescence of (E) CD68 and (F) F4/8 0 was quantified in liver sections. Figure 2-3: Serum ALT activity in mice following treatment with carbo n tetrachloride. Serum from mice treated with liposomal PBS or lipos omal clodronate was collected and analyzed for ALT activity. aSignificantly different from PBS mice. ALT activity in mice following treatment with carbo n Serum from mice treated with liposomal PBS or lipos omal clodronate was collected and Significantly different from PBS -containing liposome treated ALT activity in mice following treatment with carbo n Serum from mice treated with liposomal PBS or lipos omal clodronate was collected and containing liposome treated Figure 2- 4: Quantification of necrotic areas following macro phage depletion and carbon tetrachloride treatment. Male C57BL/6 mice were treated with either (A) PBS clodronate- containing liposomes for 72 hours. Mice were then t rea tetrachloride for 72 hours. Liver sections were sta ined with H&E and (C) area of necrosis was quantified using Image J analysis. containing liposome treated mice. 4: Quantification of necrotic areas following macro phage depletion and carbon tetrachloride treatment. Male C57BL/6 mice were treated with either (A) PBS -containing liposomes or (B) containing liposomes for 72 hours. Mice were then t rea ted with carbon tetrachloride for 72 hours. Liver sections were sta ined with H&E and (C) area of necrosis was quantified using Image J analysis. aSignificantly different from PBS containing liposome treated mice. 4: Quantification of necrotic areas following macro phage depletion and containing liposomes or (B) ted with carbon tetrachloride for 72 hours. Liver sections were sta ined with H&E and (C) area of Significantly different from PBS - Figure 2-5: The effect of macrophage depletion on i nflammatory mediators in the liver following carbon tetrachloride treatment. mRNA levels of pro-inflammatory genes were measured in liver samples from mice following liposomal PBS or liposomal clodronate tre atment prior to carbon tetrachloride treatment. mRNA expression was quantified 72 h afte r carbon tetrachloride treatment. aSignificantly different from PBS-containing liposom e treated mice. Figure 2-6: The effect of macrophage depletion on e xpression of genes associated with hepatocyte proliferation in the liv er following carbon tetrachloride treatment. mRNA levels of genes involved in hepatocyte prolife ration from whole liver samples of mice following liposomal PBS or liposomal clodronat e treatment prior carbon tetrachloride treatment. mRNA expression was quanti fied 72 h after carbon tetrachloride treatment. Figure 2-7: Quantification of PCNA staining of hepa tocyte nuclei following macrophage depletion and carbon tetrachloride treat ment. Male C57BL/6 mice were treated with either (A) PBS- containing liposomes or (B) clodronate-containing liposomes for 72 hours. Mice were then treated with carbon tetrachloride for 72 hours. Paraffin embedded liver sections were stained for PCNA. (C) PCNA positive hepatocyte nuclei were quantified. Figure 2- 8: Quantification of Type I Collagen accumulation f ollowing macrophage depletion and carbon tetrachloride treatment. Male C57BL/6 mice were treated with either (A) PBS clodronate- containing liposomes for 72 hours. Mice were then t reated with carbon tetrachloride for 72 hours. Representative CD68 imm unofluorescence in frozen liver E 8: Quantification of Type I Collagen accumulation f ollowing macrophage depletion and carbon tetrachloride treatment. Male C57BL/6 mice were treated with either (A) PBS -containing liposomes or (B) containing liposomes for 72 hours. Mice were then t reated with carbon tetrachloride for 72 hours. Representative CD68 imm unofluorescence in frozen liver 8: Quantification of Type I Collagen accumulation f ollowing macrophage containing liposomes or (B) containing liposomes for 72 hours. Mice were then t reated with carbon tetrachloride for 72 hours. Representative CD68 imm unofluorescence in frozen liver Figure 2-8 (cont™d) sections, positive staining shown in red, from (A) PBS-containing liposome treated mice or (B) clodronate-containing liposome treated mice. Representative F4/80 immunofluorescence in livers sections, positive sta ining shown in red, from (C) PBS- containing liposome treated mice or (D) clodronate- containing liposome treated mice. All frozen sections were also stained for Type I co llagen with positive staining shown in green. (E) Area of Type I collagen positive stainin g was quantified using Image J software. Figure 2-9: The effect of macrophage depletion on p ro-fibrotic gene expression in the liver following carbon tetrachloride treatment. mRNA levels of (A) genes involved in fibrosis and ECM repair and (B) TGF- and TSP- 1 in macrophage depleted livers as compared to cont rol livers following 72 hours of carbon tetrachloride treatment. A B Figure 2-10: The effect of macrophage depletion on matrix metalloproteinase expression in the liver following carbon tetrachlor ide treatment. mRNA levels matrix metalloproteinase proteins from whole liver samples of mice following liposomal PBS or liposomal clodronate tre atment prior carbon tetrachloride treatment. mRNA expression was quantified 72 h afte r carbon tetrachloride treatment. aSignificantly different from PBS-containing liposom e treated samples. DISCUSSION M2 macrophages are known to produce anti-inflammato ry cytokines that counteract the effects of the inflammatory response carried out by M1 macrophages (Duffield et al., 2005). Although the pro-inflammat ory response is known to have a number of beneficial effects, it also has the poten tial to cause additional injury to the rest of the liver (Zimmermann et al., 2010). It was interesting to find that by depleting mice of macrophages, the ALT activity level and thu s the level of hepatocyte injury was only modestly reduced when compared to non-depleted mice. Surprisingly, although the extent of liver injury as measured by ALT was modes tly reduced, the area of necrosis was larger in the livers of mice depleted of macrop hages when compared to non- depleted mice. This suggested that although the sam e level of hepatocyte death had occurred in the two groups, dead hepatocytes were n ot being cleared from the livers of macrophage depleted mice, and suggested that the ph agocytic clearance of necrotic debris from the liver is primarily conducted by mac rophages. Another possibility is that the rate of hepatocyte proliferation in the livers of macrophage depleted mice is reduced as compared to the non-depleted mice. Macrophages produce the pro-inflammatory cytokines TNF and IL-6 that have been shown to induce hepatocyte proliferation (Bradham e t al., 1998; Zimmers, McKillop et al., 2003). Both cytokines were shown to be signifi cantly decreased in the livers of macrophage depleted mice. However, the mRNA levels of genes involved in hepatocyte proliferation were unchanged in the macrophage depl eted mice as compared to control mice. Also, the level of PCNA staining seen in the livers of macrophage depleted mice was not significantly different from control livers . Together, this showed that reducing the levels of TNF and IL-6, by depleting macrophages, did not alter the rate of hepatocyte proliferation. Since a number of redunda nt mechanisms control hepatocyte proliferation, it is possible that the decrease in TNF and IL-6 were not significant enough to inhibit hepatocyte proliferation during n ormal liver repair. Macrophages may also contribute to liver repair by the production of cytokines that induce production of various ECM components (C ordeiro-da-Silva et al., 2004). M2 macrophages are known to produce TGF- , a potent inducer of collagen type I deposition, and other pro-fibrotic genes (Nunes et al., 1995). TGF- is produced in a latent form, noncovalently associated with the late ncy associated peptide (LAP) (Koli et al., 2001). This complex is secreted from cells wit h LAP bound by disulfide linkages to latent TGF- binding protein (LTBP) (Miyazono et al., 1991). LT BP localizes the complex to the ECM. In order to become biologically active, TGF- is cleaved from LAP by proteins, such as plasmin, MMP-2, MMP-9 and TSP- 1. Since macrophages are an important source of MMPs, these cells may affect ma trix deposition by regulating activation of TGF- . In addition to activating TGF- , MMPs produced by macrophages can breakdown Type I collagen directly. It was int eresting to find that in the livers of macrophage depleted mice, collagen type I was reduc ed at 72 hours following carbon tetrachloride treatment. This demonstrates that mac rophages are involved in regulating the production of collagen Type I in the liver afte r an acute injury. In order to determine the mechanism by which macrop hages induce collagen production, the mRNA levels of genes that are class ically known to be induced during fibrosis such as PDGF, FGF-2 and plasminogen activa tor inhibitor-1 (PAI-1) were investigated. However, all pro-fibrotic genes remai ned unchanged in macrophage depleted mice along with TGF- . In contrast, MMP-2 and MMP-13 were significantly decreased in the livers of macrophage depleted mice as compared to control mice. Since MMPs are known activators of TGF- , it is possible that although the mRNA level of TGF- was unchanged, the concentration of activated TGF- in the liver following macrophage depletion was decreased as compared to c ontrol livers resulting in the decreased collagen production. While a lack of TGF- activation could be contributing to the decreased expression of Type I collagen in macrophage deplete d livers, this effect should have been reflected by a decreased level of Col1a1 mRNA in the mice treated with clodronate liposomes. Instead, the mRNA levels of C ol1a1 were unaffected by the depletion of macrophages. This suggests either a po st-transcriptional inhibition of Col1a1 production or an increased degradation of Ty pe I collagen protein. However, MMP-2 and MMP-13 mRNA expression were significantly decreased in macrophage depleted mice, while MMP-3 and MMP-9 mRNA levels we re unchanged in the same mice. MMP-2 requires cleavage by membrane-type MMP- 1 (MT-MMP-1) in order to gain activity. In addition, the family of tissue in hibitors of metalloproteinases (TIMPs) is known to inhibit MMP activity. Although the mRNA le vels of MMP-2 and MMP-13 were lowered, the level of activity of MMPs could be inc reased by a decreased level of TIMPs or an increased level of MT-MMPS. In summary, macrophages are involved in normal live r repair by phagocytic clearance of necrotic hepatocyte debris and regulat ion of ECM deposition. Although macrophage depletion did not exacerbate injury or d ecrease hepatocyte proliferation, the mechanisms by which macrophages regulate collag en deposition in normal liver repair will be an important topic for further inves tigation. CHAPTER 3 MACROPHAGE POLARIZATION IN ACUTE LIVER INJURY: A PROSPECTIVE ADOPTIVE TRANSFER STUDY ABSTRACT The role of macrophage polarization in acute liver injury is not well understood. Macrophages have a high level of phenotypic plastic ity and undergo polarization in order to carry out discrete functions as a part of the innate immune system. M1 macrophages are known to produce pro-inflammatory m ediators such as IL-1, IL-6 and TNF . On the other hand, M2 macrophages exhibit a high phagocytic activity and produce anti-inflammatory cytokines such as TGF- and IL-10. In previous studies, M1 macrophages have been indirectly implicated in the exacerbation of liver injury following APAP treatment while cytokines produced by M2 macro phages have been shown to play a protective role against injury in this model . We are proposing to conduct a study that would directly demonstrate the involvement of M1 and M2 macrophages in liver injury and repair following acute exposure to a tox in. Mice will be depleted of macrophages and will receive either M1 or M2 macrop hages by adoptive transfer following induction of acute liver injury. In order to determine the feasibility of this experiment, I have conducted the macrophage depleti on, macrophage adoptive transfer, macrophage isolation and subsequent in vitro polarization. However, the ideal timeline in which the adoptive transfer should take place relative to the induction of acute injury has yet to be determined. INTRODUCTION Macrophage polarization is important for the inflam matory response and for the subsequent resolution of inflammation following hep atic injury (Edwards et al., 1992). M1 and M2 macrophages contribute to these processes by producing distinct classes of cytokines. Although there is indirect evidence that M1 macrophages exacerbate liver injury following exposure to a hepatotoxicant and t hat M2 macrophages protect the liver from injury, these distinct roles have not been sho wn directly (Bourdi et al., 2007; Ishida et al., 2002). The following studies are preliminar y work in preparation for an experiment that would employ adoptive transfer to directly inv estigate the role of M1 and M2 macrophages on liver injury and repair following an acute exposure to a liver toxin. Macrophages derive from the myeloid lineage and d evelop directly from the differentiation of monocytes. Monocytes are induced to polarize into an M1 phenotype by exposure to cytokines that typically activate T H1 cells such as, interferon-gamma (IFN ) and TNF (Gordon & Taylor, 2005). M1 macrophages produce th e pro- inflammatory factors IL-6, TNF and nitric oxide (NO) (J. P. Edwards, Zhang, Frauw irth, & Mosser, 2006). Monocytes can also polarize into M 2 macrophages after exposure to IL-4 and IL-13; cytokines that are involved in the initiation of differentiation and maintenance of T H2 activation (Sica & Mantovani, 2012). M2 macrophag es produce anti-inflammatory cytokines such as IL-10 and TGF- (Sutterwala, Noel, Salgame, & Mosser, 1998). By the production of these cytokines , M1 and M2 macrophages can potentiate either a pro- or anti-inflammatory respo nse by autocrine and paracrine signaling (Biswas & Mantovani, 2010). However, the distinct roles of M1 and M2 macrophage populations in the liver following acute injury remain unclear. There has been some indirect evidence arguing that M1 macrophages contribute to liver injury while M2 macrophages aid in the res olution of liver inflammation and repair. In rodents depleted of TNF- and IFN , pro-inflammatory cytokines that polarize monocytes into M1 macrophages, the level of injury in APAP treated mice was decreased as compared to control mice (Ishida et a l., 2002; Morio et al., 2001). Also in IL-10 and IL-4 knockout mice as well as in wild typ e mice depleted of IL-13, the level of injury was increased as compared to controls follow ing an acute liver injury (Bourdi et al., 2007; Yee, Bourdi, Masson, & Pohl, 2007). This suggests that cytokines produced by M1 macrophages exacerbate acute liver injury whi le cytokines produced by M2 macrophages protect the liver from injury. In our studies, we will test this hypothesis by iso lating macrophages from mice and polarizing them into either an M1 or M2 phenoty pe (Figure 3-0A). Next, separate mice will be depleted of macrophages and treated wi th a hepatotoxicant to induce acute liver injury (Figure 3-0B). These mice will then re ceive either M1 or M2 macrophages by adoptive transfer (Figure 3-0C). By polarizing M1 a nd M2 populations in vitro and adoptively transferring them into macrophage deplet ed mice that have been treated with a hepatotoxicant, this will directly demonstrate th e effects of M1 and M2 macrophages in the liver following an acute injury. The following preliminary work was necessary to ens ure that each step of the experiment was yielding the expected results in ord er to maintain the integrity of the experiment as a whole. First, we determined that ma crophages could be isolated and polarized in vitro properly and in adequate numbers (Figure 3-0A). Nex t, we determined that mice were in fact depleted of macrophages foll owing an i.p. injection of liposomal clodronate (Figure 3-0B). And finally we demonstrat ed the adoptive transfer of GFP- expressing macrophages into macrophage-depleted mic e and their migration to the liver. The adoptive transfer of M1 and M2 polarized macrophages into the livers of mice depleted of macrophages following acute liver injur y would provide a direct method to determine the effect of macrophage polarization fol lowing acute liver injury. Figure 3- 0: Adoptive transfer of M1 and M2 polarized macroph ages into macrophage- depleted mice following acute liver injury. macrophages will be isolated from male C57BL/6 mice and polarized into M1 and M2 macrophages in vitro (A). Mice macrophages or liposomal PBS as a control. The mic e will also be treated with a hepatotoxic agent such as APAP or CCl previously depleted of macrophages wil adoptively transferred by retro 0: Adoptive transfer of M1 and M2 polarized macroph ages into depleted mice following acute liver injury. Bone marrow macrophages will be isolated from male C57BL/6 mice and polarized into M1 and M2 (A). Mice will then be treated with liposomal clodronate to d eplete macrophages or liposomal PBS as a control. The mic e will also be treated with a hepatotoxic agent such as APAP or CCl 4 in order deplete macrophages (B). Mice previously depleted of macrophages wil l then have either M1 or M2 macrophages adoptively transferred by retro -orbital injection (C). 0: Adoptive transfer of M1 and M2 polarized macroph ages into Bone marrow -derived macrophages will be isolated from male C57BL/6 mice and polarized into M1 and M2 will then be treated with liposomal clodronate to d eplete macrophages or liposomal PBS as a control. The mic e will also be treated with a in order deplete macrophages (B). Mice l then have either M1 or M2 macrophages MATERIALS AND METHODS Animals Mice used in these studies were male C57BL/6 (Jacks on Laboratories, Bar Harbor, ME) and GFP-expressing mice (C57BL/6-Tg(ACT B-EGFP)131Osb/LeySopJ, Jackson Laboratories) from 8 to 10 weeks of age wer e used in this study. All mice were maintained on a 12-h light/dark cycle under control led temperature (18-21ºC) and humidity. Food (Rodent Chow; Harlan-Teklad, Madison , WI) and tap water were allowed ad libitum . All procedures on animals were carried out in acc ordance with the Guide for the Care and Use of Laboratory Animals promulgated by the National Institutes of Health. Macrophage Depletion Mice were treated with 200 l of liposome encapsulated clodronate (ClodronateLiposomes.com, The Netherlands) or PBS-c ontaining liposomes by i.p. injection. Macrophage Adoptive Transfer Peritoneal macrophages were isolated from GFP mice 72 hours after i.p. injection with 2 mL of 3% Brewer™s thioglycollate. Macrophages were isolated as previously described (Zhang, Gonclaves, & Mosser, 2 008). 5x10 6 macrophages per mouse were injected retro-orbitally into 5 mice. In vitro Bone Marrow-derived Macrophage Polarizatio n Bone marrow-derived macrophages were cultured as p reviously described (Zhang et al., 2008). Briefly, bone marrow cells we re isolated from the femurs of C57BL/6 mice by flushing with 5mL of sterile PBS wi th 2% FBS. Following centrifugation at 50g for 10 minutes at RT, bone ma rrow cells were resuspended and plated in complete growth media (DMEM +10% FBS +10 ng/ml M-CSF) at a concentration of 4x10 4 cells/ mL. Cells were incubated in 37°C and 5% CO2 . On day 3, the old complete media was aspirated and new comple te growth media was added to each plate. On day 7, media was changed to either M 1 differentiation media (DMEM +10% FBS +50ng/mL IFN ) or M2 differentiation media (DMEM +10%FBS +15ng/m L IL-4). M1 macrophages were incubated for 24 hours a nd M2 macrophages were 48 hours prior to characterization. RNA Isolation and qRT-PCR Macrophages were lysed in 500 L of TRI reagent (Sigma Chemical Company, St. Louis, MO) and total RNA was isolated per manuf acturer™s instructions. Methods for qRT-PCR were conducted as previously described in C hapter 2, Materials and Methods Section, pages 9-10. PCR primers were used as follo ws: mouse iNOS forward: 5™- TTCTGTGCTGTCCCAGTGAG-3' and reverse: 5™-TGAAGAAAACC CCTTGTGCT-3'; mouse Arg1 forward: 5™-TTTTTCCAGCAGACCAGCTT-3' and reverse: 5™- AGAGATTATCGGAGCGCCTT-3™; mouse TNF forward: 5™- AGGGTCTGGGCCATAGAACT-3™ and reverse: 5™-CCACCACGCTC TTCTGTCTAC-3™; mouse TGF- forward: 5™-CAACCCAGGTCCTTCCTAAA-3™ and reverse: 5 ™- GGAGAGCCCTGGATACCAAC-3™. Immunohistochemistry and Immunofluorescence Immunofluorescence was used to detect and quantify macrophages in 8 m frozen sections of liver. The sections were fixed in 4% formalin and then inc ubated with rat anti-CD68 antibody diluted 1:100 (Novus, Little ton, CO) or rat anti-F4/80 antibody diluted 1:25 (AbD Serotec, Raleigh, NC) and chicken anti-GFP antibody diluted 1:500 (Aves Labs, Tigard, OR). The sections were then in cubated with secondary antibodies conjugated to either Alexa Fluor 488 or Alexa Fluor 594 (Life Technologies, Grand Island, NY). Statistics Results are presented as the mean + SEM. 5-7 mice were used for all in vivo studies. 3 mice were used for all in vitro studies. Data were analyzed by unpaired Student™s t-test. The criterion for significance wa s p < 0.05 for all studies. RESULTS In order to carry out the adoptive transfer experim ent, macrophages were first depleted from mice by i.p. injection of liposomal c lodronate (Figure 3-1). To confirm depletion of macrophages in the liver, macrophages were detected by immunofluorescent staining of the macrophage marker s CD68 and F4/80 as described in Chapter 2. CD68 and F4/80-positive macrophages w ere detected in liver sections from mice treated with PBS-containing liposomes (Fi gure 3-1A and 3-1C). In contrast, staining of both markers was decreased in livers fo llowing liposomal clodronate treatment (Figure 3-1B and 3-1D). Next, we determined whether macrophages injected re tro-orbitally migrate to the liver of macrophage-depleted mice. For this study, macrophages were isolated from mice that ubiquitously express green fluorescent pr otein (GFP) in all cell types, and injected retro-orbitally into macrophage depleted m ice. Adoptively transferred macrophages were then detected in the liver by co-i mmunofluorescence staining for GFP and the macrophage markers CD68 and F4/80. As s hown in Figure 3-2, GFP expressing cells in the liver also expressed F4/80 (Figure 3-2A) or CD68 (Figure3-2B) indicating that the macrophages administered to mic e by retro-orbital injection repopulated the livers of macrophage-depleted mice. Finally, M1 and M2 macrophages were polarized in vitro from bone-marrow derived macrophages. Bone marrow cells were isolate d from the femur of C57BL/6 mice. Hematopoietic stem cells were then differenti ated into macrophages by incubation with monocyte colony stimulating factor (M-CSF). An d the mature macrophages were then polarized into either M1 or M2 cells by IFN and IL-4 treatment, respectively. Both the M1 and M2 cultures were analyzed for specific m arkers indicating both classical and alternative activation. As expected, mRNA levels of iNOS, TNF and HIF-1 were highest in M1 macrophages (Figures 3-3A, B & C), wh ereas mRNA levels of HIF-2 and TGF- were highest in M2 macrophages (Figures 3-3D & E). Figure 3-1: Hepatic macrophage depletion in the liver following liposomal clodronate treatment. Male C57BL/6 mice were treated with either (A and C ) PBS containing liposomes or (B and D) clodronate Representative CD68 immunofluoresce (A) PBS- containing liposomes or (B) clodronate F4/80 immunofluorescence (red staining) in livers s ections from (C) PBS liposomes or (D) clodronate- containing Hepatic macrophage depletion in the liver following liposomal Male C57BL/6 mice were treated with either (A and C ) PBS containing liposomes or (B and D) clodronate -containing liposomes for 72 hours. Representative CD68 immunofluoresce nce (green staining) in frozen liver sections from containing liposomes or (B) clodronate -containing liposomes. Representative F4/80 immunofluorescence (red staining) in livers s ections from (C) PBS -containing liposomes. Hepatic macrophage depletion in the liver following liposomal Male C57BL/6 mice were treated with either (A and C ) PBS -containing liposomes for 72 hours. nce (green staining) in frozen liver sections from containing liposomes. Representative -containing Figure 3- 2: Adoptive transfer of GFP depleted mice. Male C57BL/6 mice were treated with 200 72 hours to deplete macrophages. Thioglycollate isolated from GFP- expressing mice and adoptively transferred into mac rophage depleted mice by retro- orbital injection. Macrophages and GFP were then de tected by immunofluorescence. (A) Representative F4/80 (red staining) and GFP (green staining) imm unofluorescence in liver sections. (B) Representati ve CD68 (red staining) and GFP (green staining) immunofluorescence in liver sectio ns. Yellow indicates co of GFP with macrophage markers. 2: Adoptive transfer of GFP -expressing macrophages into macrophage Male C57BL/6 mice were treated with 200 L of liposomal clodronate for 72 hours to deplete macrophages. Thioglycollate -elicited peritoneal macrophages expressing mice and adoptively transferred into mac rophage orbital injection. Macrophages and GFP were then de tected by immunofluorescence. (A) Representative F4/80 (red staining) and GFP (green staining) unofluorescence in liver sections. (B) Representati ve CD68 (red staining) and GFP (green staining) immunofluorescence in liver sectio ns. Yellow indicates co of GFP with macrophage markers. expressing macrophages into macrophage L of liposomal clodronate for elicited peritoneal macrophages were expressing mice and adoptively transferred into mac rophage -orbital injection. Macrophages and GFP were then de tected by immunofluorescence. (A) Representative F4/80 (red staining) and GFP (green staining) unofluorescence in liver sections. (B) Representati ve CD68 (red staining) and GFP (green staining) immunofluorescence in liver sectio ns. Yellow indicates co -localization Figure 3-3: Polarization of bone marrow derived mac rophages in vitro . Macrophages were isolated, differentiated and polar ized, as previously described. Quantitative real-time PCR was used to measure mRNA levels of the indicated genes. Data represent means ± SEM, n=3. aSignificantly different from M1 macrophages. DISCUSSION In the future, the adoptive transfer of M1 and M2 m acrophages is highly feasible. I have demonstrated that macrophages can be success fully depleted from the liver and that macrophages injected retro-orbitally into a ho st mouse, will migrate to the liver. Also M1 and M2 macrophages can be successfully different iated and polarized in vitro. However, the ideal time for the adoptive transfer to take place relative to the induction of acute liver injury is another factor t hat will require optimization prior to conducting the study. Other cell types in the liver produce chemotactic factors in order to induce monocyte migration into the liver followi ng injury. Circulating macrophages infiltrate into the liver by the binding of chemoki ne (C-C motif) ligand 2 (CCL2) also called, monocyte chemoattractant protein-1 (MCP-1) to the C-C chemokine receptor type 2 (CCR2) (Karlmark, Wasmuth, & Trautwein, 2008 ). Activated stellate cells, hepatocytes, macrophages and endothelial cells prod uce CCL2 following acute liver injury (Baeck et al., 2012). For this reason, macro phages may migrate to the liver in larger numbers if the adoptive transfer follows the induction of the liver injury. However, depleting the liver of macrophages prior to causing acute injury may cause a higher degree of liver injury than would be present by hav ing the adoptive transfer precede the induction of acute liver injury (Ju et al., 2002). More studies will need to be conducted in order to determine the ideal time course of this ex periment. Another confounding factor could include the high degree of phenotypic plasticity of macrophages. Macrophage phenotype can be altered by a change in the cytokine milieu in the extracellular environment (Davis, Tsa ng, & Qiu, 2013). For example, M1 macrophages can switch to an M2 phenotype following exposure to IL-4, IL-13 or after phagocytosis of necrotic tissue (Ramachandran et al ., 2012). While this property of macrophages aids in the in vitro polarization, this property may be a problem once in vivo. Since the cytokine milieu of the liver and circulat ory system changes throughout the progression of acute liver injury and subsequen t repair, it is possible that macrophages may undergo another phenotypic switch a t different stages of the acute liver injury and repair (Porcheray et al., 2005). I t will be important to determine if this occurs. This in itself may provide insight into the mechanisms that drive macrophage polarization during liver injury and repair. In summary, this experiment provides a method to t est the effects of macrophage polarization in the liver following acute injury. T his experiment also provides a method to test the impact of macrophage polarization on liver repair. REFERENCES REFERENCES Abboud, G., & Kaplowitz, N. (2007). Drug-induced li ver injury. Drug safety : an international journal of medical toxicology and dru g experience , 30 (4), 277Œ94. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/2 3453390 Adachi, T., Nakashima, S., Saji, S., Nakamura, T., & Nozawa, Y. (1995). Roles of prostaglandin production and mitogen-activated prot ein kinase activation in hepatocyte growth factor-mediated rat hepatocyte pr oliferation. Hepatology (Baltimore, Md.) , 21 (6), 1668Œ74. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/7539396 Allen, L.-A., & Aderem, A. (1996). Molecular defini tion of distinct cytoskeletal structures involved in complement- and Fc receptor-mediated ph agocytosis in macrophages. The Journal of experimental medicine , 184 (2), 627Œ37. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi ?artid=2192718&tool=pmcentre z&rendertype=abstract Antoniades, C. G., Berry, P. a, Davies, E. T., Huss ain, M., Bernal, W., Vergani, D., & Wendon, J. (2006). Reduced monocyte HLA-DR expressi on: a novel biomarker of disease severity and outcome in acetaminophen-induc ed acute liver failure. Hepatology (Baltimore, Md.) , 44 (1), 34Œ43. doi:10.1002/hep.21240 Antoniades, C. G., Berry, P. a, Wendon, J. a, & Ver gani, D. (2008). The importance of immune dysfunction in determining outcome in acute liver failure. Journal of hepatology , 49 (5), 845Œ61. doi:10.1016/j.jhep.2008.08.009 Ashkenas, J., Muschler, J., & Bissell, M. J. (1996) . The Extracellular Matrix in Epithelial Biology: Shared Molecules and Common Themes in Dist ant Phyla. Developmental Biology , 180 (2), 1Œ19. doi:10.1006/dbio.1996.0317.The Austyn, J. M., & Gordon, S. (1981). F4/80, a monocl onal antibody directed specifically against the mouse macrophage. European journal of immunology , 11 (10), 805Œ15. doi:10.1002/eji.1830111013 Baeck, C., Wehr, A., Karlmark, K. R., Heymann, F., Vucur, M., Gassler, N., – Tacke, F. (2012). Pharmacological inhibition of the chemokine CCL2 (MCP-1) diminishes liver macrophage infiltration and steatohepatitis in chro nic hepatic injury. Gut , 61 (3), 416Œ26. doi:10.1136/gutjnl-2011-300304 Bieghs, V., & Trautwein, C. (2013). The innate immu ne response during liver inflammation and metabolic disease. Trends in immunology , 34 (9), 446Œ52. doi:10.1016/j.it.2013.04.005 Biswas, S. K., & Mantovani, A. (2010). Macrophage p lasticity and interaction with lymphocyte subsets: cancer as a paradigm. Nature Immunology , 11 (10), 889Œ896. Bourdi, M., Eiras, D. P., Holt, M. P., Webster, M. R., Reilly, T. P., Welch, K. D., & Pohl, L. R. (2007). Role of IL-6 in an IL-10 and IL-4 dou ble knockout mouse model uniquely susceptible to acetaminophen-induced liver injury. Chemical research in toxicology , 20 (2), 208Œ16. doi:10.1021/tx060228l Bradham, C. a, Plümpe, J., Manns, M. P., Brenner, D . a, & Trautwein, C. (1998). Mechanisms of hepatic toxicity. I. TNF-induced live r injury. The American journal of physiology , 275 (3 Pt 1), G387Œ92. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/9724248 Chaudhuri, S., McCullough, S. S., Hennings, L., Let zig, L., Simpson, P. M., Hinson, J. a, & James, L. P. (2011). Acetaminophen hepatotoxicity and HIF-1 induction in acetaminophen toxicity in mice occurs without hypox ia. Toxicology and applied pharmacology , 252 (3), 211Œ20. doi:10.1016/j.taap.2011.02.005 Columbia, B., & Clinic, H. (1998). Tumor Necrosis F actor Primes Hepatocytes for DNA Replication in the Rat, (206), 1226Œ1234. Cordeiro-da-Silva, A., Tavares, J., Araújo, N., Cer queira, F., Tomás, A., Kong Thoo Lin, P., & Ouaissi, A. (2004). Immunological alterations induced by polyamine derivatives on murine splenocytes and human mononuc lear cells. International immunopharmacology , 4(4), 547Œ56. doi:10.1016/j.intimp.2004.02.009 Davis, M. J., Tsang, T. M., & Qiu, Y. (2013). Macro phage M1/M2 Polarization Dynamically Adapts to Changes in Cytokine Microenvi ronments in Cryptococcus neoformans Infection. mBio , 4(3), e00264Œ13. doi:10.1128/mBio.00264-13.Editor Duffield, J. S., Forbes, S. J., Constandinou, C. M. , Clay, S., Partolina, M., Vuthoori, S., – Iredale, J. P. (2005). Selective depletion of mac rophages reveals distinct , opposing roles during liver injury and repair, 115 (1). doi:10.1172/JCI200522675.56 Edwards, J. P., Zhang, X., Frauwirth, K. A., & Moss er, D. M. (2006). Biochemical and functional characterization of three activated macr ophage populations. Journal of leukocyte biology , 80 (6), 1298Œ1307. doi:10.1189/jlb.0406249.Biochemical Edwards, M. J., Keller, B. J., Kauffman, F. C., & T hurman, R. G. (1992). The Involvement of Kupffer Cells in Carbon Tetrachlorid e Toxicity. Toxicology and Applied Pharmacology , 119 (December), 275Œ279. Fujiyoshi, M., & Ozaki, M. (2011). Molecular mechan isms of liver regeneration and protection for treatment of liver dysfunction and d iseases. Journal of hepato-biliary- pancreatic sciences , 18 (1), 13Œ22. doi:10.1007/s00534-010-0304-2 Geissmann, F., Jung, S., & Littman, D. R. (2003). B lood monocytes consist of two principal subsets with distinct migratory propertie s. Immunity , 19 (1), 71Œ82. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/1 2871640 Geissmann, F., Manz, M. G., Jung, S., Sieweke, M. H ., Merad, M., & Ley, K. (2010). Development of monocytes, macrophages, and dendriti c cells. Science (New York, N.Y.) , 327 (5966), 656Œ61. doi:10.1126/science.1178331 Gong, D., Shi, W., Yi, S., Chen, H., Groffen, J., & Heisterkamp, N. (2012). TGF signaling plays a critical role in promoting altern ative macrophage activation. BMC immunology , 13 (1), 31. doi:10.1186/1471-2172-13-31 Gordon, S., & Taylor, P. R. (2005). Monocyte and Ma crophage Heterogeneity. Nature Reviews Immunology , 5(December), 953Œ964. doi:10.1038/nri1733 Helk, E., Bernin, H., Ernst, T., Ittrich, H., Jacob s, T., Heeren, J., – Lotter, H. (2013). TNF -mediated liver destruction by Kupffer cells and Ly 6Chi monocytes during Entamoeba histolytica infection. PLoS pathogens , 9(1), e1003096. doi:10.1371/journal.ppat.1003096 Holness, C. L., & Simmons, D. L. (1993). Molecular cloning of CD68, a human macrophage marker related to lysosomal glycoprotein s. Blood , 81 (6), 1607Œ13. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/7 680921 Holt, M. P., Cheng, L., & Ju, C. (2008). Identifica tion and characterization of infiltrating macrophages in acetaminophen-induced liver injury. Journal of leukocyte biology , 84 (6), 1410Œ21. doi:10.1189/jlb.0308173 Ishida, Y., Kondo, T., Ohshima, T., Fujiwara, H., I wakura, Y., & Mukaida, N. (2002). A pivotal involvement of IFN- in the pathogenesis of acetaminophen-induced acute liver injury. The FASEB Journal , 16 (August), 1227Œ1236. Ju, C., Reilly, T. P., Bourdi, M., Radonovich, M. F ., Brady, J. N., George, J. W., & Pohl, L. R. (2002). Protective role of Kupffer cells in a cetaminophen-induced hepatic injury in mice. Chemical research in toxicology , 15 (12), 1504Œ13. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/12482232 Karlmark, K., Wasmuth, H., & Trautwein, C. (2008). Chemokine-directed immune cell infiltration in acute and chronic liver disease. Expert Reviews Gastroenterology and Hepatology , April (2), 233Œ242. Karmen, A., Wroblewski, F., & Ladue, J. S. (1954). Transaminase Activity in Human Blood. Journal of Clinical Investigation , 34 (1), 126Œ133. Kawasaki, S., Makuuchi, M., Ishizone, S., Matsunami , H., Terada, M., & Kawarazaki, H. (1992). Liver regeneration in recipients and donors after transplantation. Lancet , 339 (8793), 580Œ1. Retrieved from http://www.ncbi.nlm.n ih.gov/pubmed/19025926 Khalil, N., Bereznay, O., Sporn, M., & Greenberg, A . H. (1989). Macrophage Production of Transforming Growth Factor-beta and Fibroblast C ollagen Synthesis in Chronic Pulmonary Inflammation. The Journal of Experimental Medicine , 170 (September), 727Œ737. Kim, T. H., Mars, W. M., Stolz, D. B., Petersen, B. E., & Michalopoulos, G. K. (1997). Extracellular matrix remodeling at the early stages of liver regeneration in the rat. Hepatology (Baltimore, Md.) , 26 (4), 896Œ904. doi:10.1002/hep.510260415 Koli, K., Saharinen, J., Hyytiäinen, M., Penttinen, C., & Keski-Oja, J. (2001). Latency, activation, and binding proteins of TGF-beta. Microscopy research and technique , 52 (4), 354Œ62. doi:10.1002/1097-0029(20010215)52:4<35 4::AID- JEMT1020>3.0.CO;2-G Kovalovich, K., DeAngelis, R. A., Li, W., Furth, E. E., Ciliberto, G., & Taub, R. (2000). Increased toxin-induced liver injury and fibrosis i n interleukin-6-deficient mice. Hepatology , 31 (1), 149Œ 159. Laskin, D. L. (2009). Macrophages and Inflammatory Mediators in Chemical Toxicity: A Battle of Forces. Chemical research in toxicology , 22 (8), 1376Œ1385. doi:10.1021/tx900086v.Macrophages Li, W., Liang, X., Kellendonk, C., Poli, V., & Taub , R. (2002). STAT3 contributes to the mitogenic response of hepatocytes during liver rege neration. Journal of Biological Chemistry , 32 (277), 28411Œ7. Liaskou, E., Wilson, D. V, & Oo, Y. H. (2012). Inna te Immune Cells in Liver Inflammation, 2012 . doi:10.1155/2012/949157 Libermann, T. A., & Baltimore, D. (1990). Activatio n of Interleukin-6 Gene Expression through the NF-KB Transcription Factor. Molecular and Cellular Biology , 10 (5), 2327Œ2334. Loke, P., Nair, M. G., Parkinson, J., Guiliano, D., Blaxter, M., & Allen, J. E. (2002). IL-4 dependent alternatively-activated macrophages have a distinctive in vivo gene expression phenotype. BMC immunology , 3(7), 1Œ11. Mantovani, A., Sica, A., Sozzani, S., Allavena, P., Vecchi, A., & Locati, M. (2004). The chemokine system in diverse forms of macrophage act ivation and polarization. Trends in immunology , 25 (12), 677Œ86. doi:10.1016/j.it.2004.09.015 Matsumoto, A., & Yamamoto, N. (1991). Sequestration of a hepatocyte growth factor in extracellular matrix in normal adult rat liver. Biochemical and biophysical research communications , 174 (1), 90Œ95. Retrieved from http://www.sciencedirect.com/science/article/pii/00 06291X9190489T# Miyazono, K., Olofsson, a, Colosetti, P., & Heldin, C. H. (1991). A role of the latent TGF- beta 1-binding protein in the assembly and secretio n of TGF-beta 1. The EMBO journal , 10 (5), 1091Œ101. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi ?artid=452762&tool=pmcentrez &rendertype=abstract Morio, L. a, Chiu, H., Sprowles, K. a, Zhou, P., He ck, D. E., Gordon, M. K., & Laskin, D. L. (2001). Distinct roles of tumor necrosis factor- alpha and nitric oxide in acute liver injury induced by carbon tetrachloride in mice. Toxicology and applied pharmacology , 172 (1), 44Œ51. doi:10.1006/taap.2000.9133 Nunes, I., Shapiro, R., & Rifkin, D. B. (1995). Cha racterization of Latent TGF-beta Activation by Murine Peritoneal Macrophages. The Journal of Immunology , (155), 1450Œ1459. Parker, G. a, & Picut, C. a. (2005). Liver immunobi ology. Toxicologic pathology , 33 (1), 52Œ62. doi:10.1080/01926230590522365 Pollard, J. W. (2009). Trophic macrophages in devel opment and disease, 9(APRIl), 259Œ270. doi:10.1038/nri2528 Poon, I. K. H., Hulett, M. D., & Parish, C. R. (201 0). Molecular mechanisms of late apoptotic/necrotic cell clearance. Cell death and differentiation , 17 (3), 381Œ97. doi:10.1038/cdd.2009.195 Porcheray, F., Viaud, S., Rimaniol, a-C., Léone, C. , Samah, B., Dereuddre-Bosquet, N., – Gras, G. (2005). Macrophage activation switching: an asset for the resolution of inflammation. Clinical and experimental immunology , 142 (3), 481Œ9. doi:10.1111/j.1365-2249.2005.02934.x Ramachandran, P., Pellicoro, A., Vernon, M. A., Bou lter, L., Aucott, R. L., & Ali, A. (2012). Differential Ly-6C expression identifies th e recruited macrophage phenotype, which orchestrates the regression of mur ine liver fibrosis. Proceedings of the National Academy of Sciences of the United S tates of America , 109 (46), E3186ŒE3195. doi:10.1073/pnas.1119964109 Sica, A., & Mantovani, A. (2012). Macrophage plasti city and polarization : in vivo veritas. Journal of Clinical Investigation , 122 (3), 787Œ795. doi:10.1172/JCI59643DS1 Skrtic, S., Wallenius, V., Ekberg, S., Brenzel, a, Gressner, a M., & Jansson, J. O. (1999). Hepatocyte-stimulated expression of hepatoc yte growth factor (HGF) in cultured rat hepatic stellate cells. Journal of hepatology , 30 (1), 115Œ24. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/9927158 Snell, E. E., & Jenkins, W. T. (1959). The Mechanis m of the Transamination Reaction. Journal of Cellular and Comparative Physiology , 54 (Supplement S1), 161Œ177. Sobrevals, L., Rodriguez, C., Romero-Trevejo, J. L. , Gondi, G., Monreal, I., Pañeda, A., – Fortes, P. (2010). Insulin-like growth factor I g ene transfer to cirrhotic liver induces fibrolysis and reduces fibrogenesis leading to cirrhosis reversion in rats. Hepatology (Baltimore, Md.) , 51 (3), 912Œ21. doi:10.1002/hep.23412 Sutterwala, B. F. S., Noel, G. J., Salgame, P., & M osser, D. M. (1998). Reversal of Proinflammatory Responses by Ligating the Macrophag e Fc-g Receptor Type I. The Journal of Experimental Medicine , 188 (1), 217Œ222. Tomiya, T., Ogata, I., Yamaoka, M., Yanase, M., Ino ue, Y., & Fujiwara, K. (2000). The mitogenic activity of hepatocyte growth factor on r at hepatocytes is dependent upon endogenous transforming growth factor-alpha. The American journal of pathology , 157 (5), 1693Œ701. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi ?artid=1885723&tool=pmcentre z&rendertype=abstract Valentin, J. E., Stewart-Akers, A. M., Gilbert, T. W., & Badylak, S. F. (2009). Macrophage participation in the degradation and rem odeling of extracellular matrix scaffolds. Tissue engineering. Part A , 15 (7), 1687Œ94. doi:10.1089/ten.tea.2008.0419 Vine, W., & Kier, a. (1993). Baboon-to-human liver transplantation. Lancet , 341 (8853), 1158. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi ?artid=3154767&tool=pmcentre z&rendertype=abstract Vollmar, B., & Menger, M. D. (2009). The Hepatic Mi crocirculation : Mechanistic Contributions and Therapeutic Targets in Liver Inju ry and Repair, 1269Œ1339. doi:10.1152/physrev.00027.2008. Wasmuth, H. E., Kunz, D., Yagmur, E., Timmer-Strang höner, A., Vidacek, D., Siewert, E., – Lammert, F. (2005). Patients with acute on ch ronic liver failure display fisepsis-likefl immune paralysis. Journal of hepatology , 42 (2), 195Œ201. doi:10.1016/j.jhep.2004.10.019 Yee, S. B., Bourdi, M., Masson, M. J., & Pohl, L. R . (2007). Hepatoprotective role of endogenous interleukin-13 in a murine model of acet aminophen-induced liver disease. Chemical research in toxicology , 20 (5), 734Œ44. doi:10.1021/tx600349f Zhang, X., Gonclaves, R., & Mosser, D. M. (2008). T he Isolation an Characterization of Murine Macrophages. Current Protocols in Immunology , 1Œ18. doi:10.1002/0471142735.im1401s83.The Zimmermann, H. W., Seidler, S., Nattermann, J., Gas sler, N., Hellerbrand, C., Zernecke, A., – Tacke, F. (2010). Functional contri bution of elevated circulating and hepatic non-classical CD14+ CD16+ monocytes to inflammation and human liver fibrosis. PloS one , 5(6), e11049. doi:10.1371/journal.pone.0011049 Zimmers, T. a, McKillop, I. H., Pierce, R. H., Yoo, J.-Y., & Koniaris, L. G. (2003). Massive liver growth in mice induced by systemic in terleukin 6 administration. Hepatology (Baltimore, Md.) , 38 (2), 326Œ34. doi:10.1053/jhep.2003.50318