Fibrosis development requires mitochondrial Cu,Zn-superoxide dismutase-mediated macrophage polarization

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1 University of Iowa Iowa Research Online Theses and Dissertations Spring 2014 Fibrosis development requires mitochondrial Cu,Zn-superoxide dismutase-mediated macrophage polarization Chao He University of Iowa Copyright 2014 Chao He This dissertation is available at Iowa Research Online: Recommended Citation He, Chao. "Fibrosis development requires mitochondrial Cu,Zn-superoxide dismutase-mediated macrophage polarization." PhD (Doctor of Philosophy) thesis, University of Iowa, Follow this and additional works at: Part of the Other Biochemistry, Biophysics, and Structural Biology Commons

2 FIBROSIS DEVELOPMENT REQUIRES MITOCHONDRIAL CU,ZN- SUPEROXIDE DISMUTASE-MEDIATED MACROPHAGE POLARIZATION by Chao He A thesis submitted in partial fulfillment of the requirements for the Doctor of Philosophy degree in Free Radical and Radiation Biology in the Graduate College of The University of Iowa May 2014 Thesis Supervisor: Professor A. Brent Carter

3 Copyright by CHAO HE 2014 All Rights Reserved

4 Graduate College The University of Iowa Iowa City, Iowa CERTIFICATE OF APPROVAL PH.D. THESIS This is to certify that the Ph.D. thesis of Chao He has been approved by the Examining Committee for the thesis requirement for the Doctor of Philosophy degree in Free Radical and Radiation Biology at the May 2014 graduation. Thesis Committee: A. Brent Carter, Thesis Supervisor Garry R. Buettner Douglas R. Spitz Fayyaz Sutterwala Joseph Zabner

5 Tsze-hsia said, "He, who from day to day recognizes what he has not yet, and from month to month does not forget what he has attained to, may be said indeed to love to learn." The Analects ii

6 ACKNOWLEDGEMENTS I would like to thank the members of my examining committee: Drs. Garry Buettner, Douglas Spitz, Fayyaz Sutterwala, and Joseph Zabner. Their guidance, discussion, and helpful suggestions have helped my research and academic development. I would like to thank all of the past and present members of the Carter laboratory for helping me in all aspects of my research. In particular I would like to thank Drs. Shubha Murthy and Alan Ryan, for bearing with me through all the ups and downs of research, and for their guidance on my endeavor as a scientist. I would also like to thank Ana-Monica Racila and Kaleen Balun for helping me with the genotyping. In addition I would like to thank Drs. Jenna Casey, Heather Osborn, Omar Jaffer, Brad Rosen, and Taajwar Khan for their support on my professional development. I am also grateful to all faculty and colleagues in the Free Radical and Radiation Biology program who helped me along the way. I would like to thank Mr. Brett Wagner and Dr. Michael McCormick for their technical support, and Dr. Frederick Domann for scientific discussion. Special thanks to Laura Hefley for administrative support. Thank my parents for their endless love and continuous support. Also, I would like to thank all of my friends for helping make my time at Iowa so enjoyable. Finally, the most sincere thanks to my thesis advisor, Dr. A. Brent Carter, for his trust in my becoming of a good scientist. His guidance and mentorship has aided me to mature intellectually and emotionally. His passion for education and dedication to my training has tremendously helped me to approach my career goal. I could not have asked for a better or more supportive mentor and I will cherish the tools he has taught me for the rest of my career. The work was supported, in whole or in part, by National Institute of Health Grants 2R01ES and R01ES The work was also supported by a Merit Review from the Department of Veterans Affairs, Veterans Health Administration, iii

7 Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development Grant 1BX iv

8 ABSTRACT Hydrogen peroxide (H 2 O 2 ) generated by alveolar macrophages has been linked to the development of pulmonary fibrosis, but little is known about the source, mechanism of production and exact role of H 2 O 2 upon alveolar macrophage activation. In this study, we found that alveolar macrophages from asbestosis patients spontaneously produce high levels of H 2 O 2 and have high expression of Cu,Zn-SOD. Cu,Zn-SOD localized to the mitochondrial intermembrane space (IMS) in asbestosis patients and asbestos-induced the translocation of Cu,Zn-SOD to the IMS. This process was unique to macrophages and dependent on functional mitochondrial respiration. The presence of at least one of the two conserved cysteines (Cys57 and Cys146) was required for disulfide bond formation and mitochondrial translocation. These conserved cysteine residues were also necessary for Cu,Zn-SOD enzymatic activation and H 2 O 2 generation. Cu,Zn-SOD-mediated H 2 O 2 generation was inhibited by knockdown of the iron-sulfur protein, Rieske, in complex III. The role of Cu,Zn-SOD was biologically relevant as Cu,Zn-SOD -/- mice generated significantly less H 2 O 2, had less oxidative stress, and were protected from developing pulmonary fibrosis. This protective mechanism is closely related to the alveolar macrophage activation and polarization in Cu,Zn-SOD -/- mice, as they had a dominant pro-inflammatory phenotype. Macrophages not only initiate and accentuate inflammation after tissue injury, but they are also involved in resolution and repair. The proinflammatory M1 macrophages have microbicidal and tumoricidal activity, whereas the M2 macrophages are involved in tumor progression and tissue remodeling, and can be pro-fibrotic in certain settings. We demonstrate that overexpression of Cu,Zn-SOD promoted macrophage polarization into an M2 phenotype. Furthermore, overexpression of Cu,Zn-SOD in mice resulted in a pro-fibrotic environment and accelerated the development of pulmonary fibrosis. The mechanism which Cu,Zn-SOD-mediated H 2 O 2 utilizes to modulate macrophage M2 polarization is through redox regulation of a critical cysteine (Cys528) in STAT6. The polarization process, at least partially, was regulated v

9 by epigenetic modulation. We show that STAT6 was indispensable for Cu,Zn-SODmediated M2 polarization. STAT6 bound to Jmjd3, a histone H3 lysine 27 demethylase, promoter region, upregulated Jmjd3, and initiated M2 gene transcriptional activation. Targeting STAT6 with leflunomide, which can reduce cellular ROS production and inhibit STAT6 phosphorylation, abolished M2 polarization and ameliorated the fibrotic development. Taken together, these observations provide a novel mechanism for the pathogenesis of pulmonary fibrosis whereby the antioxidant enzyme Cu,Zn-SOD plays a paradoxical role. The study highlights the importance of mitochondrial Cu,Zn-SOD and redox signals in macrophage polarization and fibrosis development. These observations demonstrate that the Cu,Zn-SOD-STAT6-Jmjd3 pathway is a novel regulatory mechanism for M2 polarization and that leflunomide is a potential therapeutic agent in the treatment of pulmonary fibrosis. vi

10 TABLE OF CONTENTS LIST OF TABLES..ix LIST OF FIGURES...x LIST OF ABBREVIATIONS...xii CHAPTER I: INTRODUCTION AND BACKGROUND...1 Copper, Zinc-Superoxide Dismutase...1 Asbestos...6 Macrophages...10 CHAPTER II: MITOCHONDRIAL CU,ZN-SOD PROMOTES PULMONARY FIBROSIS BY INCREADING H 2 O Abstract...21 Introduction...22 Methods and Materials...23 Results...30 Discussion...38 CHAPTER III: CU,ZN-SOD-MEDIATED H 2 O 2 PROMOTES MACROPHAGE ALTERNATIVE ACTIVATION...64 Abstract...64 Introduction...64 Methods and Materials...66 Results...68 Discussion...73 CHAPTER IV: CU,ZN-SOD MEDIATES MACROPHAGE M2 POLARIZATION VIA REDOX MODULATION OF STAT Abstract...93 Introduction...93 Methods and Materials...95 Results...98 Discussion APPENDIX: OLIGONUCLEOTIDE TABLE REFERENCES vii

11 LIST OF TABLES Table 1-1. Classification and chemical structure of asbestos fiber Table A-1. Comprehensive oligonucleotide table for PCR reactions viii

12 LIST OF FIGURES Figure 1-1. The disulfide relay in the mitochondrial IMS faciliates protein folding and localization...15 Figure 1-2. Proposed mechanism for the generation of superoxide at the surface of asbestos Figure 2-1. Alveolar macrophages from asbestos patients produce high levels of H 2 O Figure 2-2. Asbestos patients have increased expression and activity of Cu,Zn- SOD in alveolar macrophages Figure 2-3. Cu,Zn-SOD in the mitochondrial intermembrane spaces contributes to the increased level of H 2 O 2 production Figure 2-4. Alveolar macrophages from asbestosis patients have increased mitochondrial Cu,Zn-SOD Figure 2-5. Asbestos induced translocation of Cu,Zn-SOD into the mitochondrial IMS in macrophages Figure 2-6. Mitochondrial Cu,Zn-SOD activation and translocation requires conserved cysteines Figure 2-7. Translocation and activation of Cu,Zn-SOD is redox sensitive Figure 2-8. Cu,Zn-SOD modulates asbestos-induced oxidative stress in vivo Figure 2-9. Cu,Zn-SOD -/- mice were protected from developing pulmonary fibrosis after asbestos exposure Figure Cu,Zn-SOD alters pro-fibrotic cytokine TGF- production Figure Knocking down Cu,Zn-SOD in macrophages decreases collagen production by fibroblasts Figure 3-1. Cu,Zn-SOD -/- macrophages have increased M1 markers Figure 3-2. Cu,Zn-SOD induces macrophage M2 polarization Figure 3-3. Cu,Zn-SOD induces macrophage M2 polarization in vivo Figure 3-4. Cu,Zn-SOD induces macrophage M2-pattern metabolic changes Figure 3-5. Cu,Zn-SOD modulates M1/M2 polarization via H 2 O 2 levels Figure 3-6. Cu,Zn-SOD-mediated M2 polarization accelerates pathogenesis of asbestos-induced pulmonary fibrosis Figure 3-7. Cu,Zn-SOD-mediated M2 polarization promotes pulmonary fibrosis ix

13 Figure 3-8. Alveolar macrophages from asbestosis patients have an M2- phenotype Figure 4-1. Cu,Zn-SOD-mediated H 2 O 2 activates STAT6 nuclear translocation Figure 4-2. STAT6 nuclear translocation requires functional active Cu,Zn-SOD Figure 4-3. STAT6 is indispensable for Cu,Zn-SOD-mediated M2 polarization Figure 4-4. Cys 528 in STAT6 is critical for redox activation of STAT Figure 4-5. STAT6 and M2 gene transcription can be activated by general redox signals Figure 4-6. STAT6 depletion leads to an M1-dominant phenotype Figure 4-7. STAT6 -/- mice are protected from developing pulmonary fibrosis Figure 4-8. Collagen production is decreased in WT fibroblasts treated with BAL fluid from STAT6 -/- mice Figure 4-9. STAT6 regulates jmjd3 gene expression Figure Overexpression Cu,Zn-SOD up-regulates Jmjd Figure Jmjd3 activity is increased in macrophages from asbestosis patients Figure Leflunomide abolishes H 2 O 2 generation and inhibits STAT6 activation Figure Leflunomide treatment inhibits M2 polarization Figure Leflunomide treatment attenuates pulmonary fibrosis Figure Leflunomide treatment decreases jmjd3 gene expression x

14 LIST OF ABBREVIATIONS AAM: alternatively activated macrophage ALL: acute lymphoblastic leukemia ALS: amyotrophic lateral sclerosis AML: acute myeloid leukemia ATP: adenosine triphosphate BAL: bronchoalveolar CAM: classically activated macrophage CCL: CC chemokine ligand COPD: chronic obstructive pulmonary disease Cu,Zn-SOD: copper- and zinc-containing SOD ec-sod: extracellular SOD EPA: Environment Protection Agency ESR: electron spin resonance ETC: electron transport chains FAD: flavin adenine dinucleotide FIZZ: found in inflammatory zone GSH: glutathione HDAC: histone deacetylase HO : hydroxyl radical H 2 O 2 : hydrogen peroxide HRP: horseradish peroxidase IARC: International Agency for Research on Cancer IL: interleukin IL-1ra: interleukin-1 receptor antagonist IMS: intermembrane space INF: interferon xi

15 inos: inducible nitric oxide synthase IRF: interferon regulatory factor Jmjd3: jumanji domain containing 3 LPS: lipopolysaccharide Mn-SOD: manganese superoxide dismutase mtdna: mitocondrial DNA (mtdna) NAC: N-acetyl-cysteine OCR: oxygen consumption rates O - 2 : superoxide phpa: p-hydroxylphenyl acetic acid PPAR: peroxisome proliferator-activated receptor RNS: reactive nitrogen species ROS: reactive oxygen species SOD: superoxide dismutase STAT: signal transducer and activator of transcription TAM: tumor-associated macrophage TGF: transforming growth factor Th: T-helper TIM: the inner membrane TNF: tumor necrosis factor TOM: the outer membrane VEGF: vascular endothelial growth factor WT: wild type xii

16 1 CHAPTER I INTRODUCTION AND BACKGROUND Copper, Zinc-Superoxide Dismutase (Cu,Zn-SOD) The basics Reactive oxygen species (ROS) are a group of short-lived and highly active molecules including superoxide, hydroxyl radical, and hydrogen peroxide. Low levels of ROS are pivotal for intra- and extra-cellular signaling, however, excessive amounts of ROS will cause toxicity 1. Oxidative stress is defined as the imbalance between reactive oxygen species (ROS) or reactive nitrogen species (RNS) and biological anti-oxidant system, the defending mechanism our bodies utilize to mitigate the damaging effects of oxidants. Superoxide (O - 2 ) is continuously generated in vivo, primarily produced within mitochondria as by-product of oxygen metabolism through the electron transport chain 2. The superoxide dismutase family specialize in eliminating superoxide anion radicals and catalyzing the dismutation of superoxide to form hydrogen peroxide (H 2 O 2 ) 3. Three distinct isoforms of SOD have been identified in humans. sod1 gene encodes the intracellular copper-zinc superoxide dismutase; sod2 gene encodes manganese superoxide dismutase which locates in the mitochondrial matrix; sod3 gene encodes extracellular superoxide dismutase (ecsod) which also has copper and zinc. Cu,Zn-SOD is initially isolated from bovine blood as a blue-green colored protein containing copper, thus being named as hemocuprein in Thirty years later, Drs. McCord and Fridovich found that the protein maintained the catalytic function of removing superoxide and named it superoxide dismutase 4,5. sod1 gene is located on chromosome 21 (21q22) and is consisted of five exons interrupted by four introns. The Cu,Zn-SOD protein is a 32 kda homodimer, with each monomer is built upon a -barrel motif and possesses two large functionally important loops--the electrostatic and zinc

17 2 loops 6,7. Each subunit has 153 amino acid residues and harbors an intramolecular disulfide bond between Cys 57 and Cys 146, which are crucial for the enzymatic activity. The active site of Cu,Zn-SOD includes the His 63 and imidazolate structure that actively linked copper and zinc. The inner surface of each active site is coated with a positive charge that sucks superoxide into the bottom of the funnel. The chemical reactions that Cu,Zn-SOD reacts with superoxide are as follows. - O 2 + Cu 2+,Zn-SOD O 2 + Cu +,Zn-SOD (1) O H + + Cu +,Zn-SOD H 2 O 2 + Cu 2+,Zn-SOD (2) 2 O H + O 2 + H 2 O 2 (3) The copper ion is catalytically active. His 53 anchors zinc during transformation. Zinc itself does not take part in the catalysis but stabilizes the enzyme. Cu,Zn-SOD is very efficient catalyst for this reaction, reactions (1) and (2) have near diffusion-limited rate constant at physiological ph, and the activity is nearly independent of ph over the range of 5.0 to 9.5 for the holoenzyme. In the presence of H 2 O 2 at mm concentration, Cu,Zn-SOD has also shown to harbor pro-oxidant effects, leading to the generation of HO Although it is still controversial whether the source would be CO 2 or bicarbonate, it is accepted that ph and the concentration of CO 2 /HCO - 3 will have an impact on the peroxidase activity of Cu,Zn- SOD. Cu,Zn-SOD and human diseases Cu,Zn-SOD is implicated in multiple disease conditions. Both Cu,Zn-SOD overexpressing and Cu,Zn-SOD knockout animal models have been generated, and they are viable and generally phenotypically normal 11. This distinguishes them from Mn-SOD knockout mice, which usually die within10 days after birth with significant mitochondrial damage in hearts and various organs 12.

18 3 Patients with Down s syndrome, who have an extra copy of the chromosome 21, usually have fifty percent increase of Cu,Zn-SOD activity 13,14. Brain and neural samples collected from Down syndrome patients and animal models showed exacerbated lipid oxidation, mitochondrial damage, and DNA abnormalities. Worthy noticing is that blood malignancies are prevalent in Down s syndrome patients. Acute lymphoblastic leukemia (ALL) is 20 times higher and acute myeloblastic leukemia (AML), acute megakaryocytic leukemia in particular, is 500 times higher, suggesting that the level of Cu,Zn-SOD might closely related to hematopoietic stem cell function Moreover, when Cu,Zn-SOD activity level is measured in ALL and AML patients, AML cells have 1.7-fold increase in Cu,Zn-SOD activity while ALL cells have same Cu,Zn-SOD activity compared with control cells 19, underscoring the role of Cu,Zn-SOD in granulocyte lineage differentiation. Another disease related with Cu,Zn-SOD function is amyotrophic lateral sclerosis (ALS), known as Lou Gehrig s disease in the United States. ALS can be classified into two categories: the familiar type (fals), which accounts for 5%~10% of total cases, and the sporadic type (sals), which accounts for most instances (90%~95%) 20. Cu,Zn-SOD mutations are implicated in about 20% of the fals cases, most of them are single amino acid mutation (missense). In ALS, the mutated Cu,Zn-SOD can have various levels of enzymatic activity. In the three most commonly used mice models, the G93A mice have elevated SOD1 activity, the G73R mice retain full specific activity, and G85R mice have low SOD1 activity 21,22. Recent advances in this field also proposed that misfolded Cu,Zn-SOD protein may act like a prion and cause protein aggregation and microfilament sediment in neurons 23,24. The lungs are directly exposed to higher oxygen concentrations than most other tissues. Increased oxidative stress plays a significant part in the pathogenesis of almost every respiratory disease. Cu,Zn-SOD acts an important role in lung biology. Although Cu,Zn-SOD has relatively low concentrations in the lungs compared with other tissue

19 4 (such as liver and kidney), Cu,Zn-SOD is still the dominant lung SOD, constituting around 70 percent of the total SOD activity. Meanwhile Mn-SOD and ECSOD each represents ~15 to 20% of the SOD activity 3. Mitochondrial Cu,Zn-SOD and mitochondrial protein trafficking At the time of its discovery, Cu,Zn-SOD had been found to be not only present in the cytosol, but also located in other cellular compartments including the mitochondrial intermembrane space, a compartment bordered by the mitochondrial inner and outer membranes. While the subjects has been controversial over decades, recent progresses in the field have highlighted an important role of mitochondrial Cu,Zn-SOD Mammalian mitochondria consist of approximately 1500 proteins and most of them are synthesized in the cytosol and transported into mitochondria 29,30. Precursors of mitochondrial matrix proteins have N-terminal signal sequence that will be recognized by receptors on the membranes, and pass through the translocase of the outer membrane (TOM) complex and the translocase of the inner membrane (TIM) complex to residue in the matrix. The targeting signals then will be cleaved in the matrix. On the contrary, most of proteins in the mitochondrial IMS lack any cleavable N-terminal targeting sequence and, unlike the counterparts in the matrix, are independent on adenosine triphosphate (ATP) or the inner membrane electric potential 31. Intramolecular disulfide bonds are relatively common in secreted proteins, where their primary purpose is protein stabilization. However, they are rare in intracellular proteins because of the highly reducing environment and low concentration of dioxygen in the cytosol. For this reason, when intramolecular disulfide bonds do occur in intracellular proteins, such as SOD1, they are usually predicted to play more than just a structural role and to have functional significance.

20 5 Proteins in the mitochondrial IMS that lack N-terminal signals are small in size, usually between 7 and 16 kda and share the feature of harboring a conserved pattern of cysteine residues 32. The cysteine residues are crucial for the folding and further activation of proteins in the mitochondrial IMS. Recently, a disulfide relay system has been discovered and elucidates the mechanism of how mitochondrial IMS proteins be imported and activated (Figure 1-1). Two proteins are central for this disulfide relay system, Mia40 and Erv1. Mia40 is a conserved mitochondrial IMS protein and contains an essential redox-active disulfide bond in a cysteine-proline-cysteine (CPC) pattern, which can oxidize the cysteine residues of substrate proteins 33. Oxidation introduced by Mia40 locks the substrate proteins in a stably folded state which they cannot travel back to the cytosol. Mia40 is then reoxidized by Erv1, a sulfhydryl oxidase containing flavin adenine dinucleotide (FAD). The importance of Erv1 lies in its catalytic ability to overcome the kinetic barrier caused by the incompatibility of one- and two-electron transfer process. In mitochondrial IMS, the unique feature of coupling Erv1 directly to the respiratory chain, a process to pass electrons to molecular oxygen to form water instead of H 2 O 2, avoids the damage to mitochondrial components. This detoxifying mechanism not only scavenges reactive oxygen species at proximal sites of their production, but also increases the efficiency of Erv1 reoxidation. Although not much information have obtained on protein oxidation in mitochondrial IMS, some initial studies have shown it contributes to neurodegradative diseases, apoptosis, and aging. Cu,Zn-SOD, as previously mentioned, is also imported into mitochondrial IMS and mutation in Cu,Zn-SOD leads to protein misfolding, which contributes to pathogenesis of amyotrophic lateral sclerosis 34. One of fals mutant, C146R, which lacks the intramolecular disulfide bond, was ~9% as active of the wildtype protein 20.

21 6 Asbestos The basics Asbestos is a generic term for two classes of naturally occurring hydrated silicate fibers whose tensile strength, and resilient structural and chemical properties are ideally suited for various construction and insulating purpose. This group of six types of crystalline hydrated silicate fibers that are classified into two major classes: serpentine family, which includes chrysotile, and the amphibole family, which includes crocidolite, amosite, anthophyllite, tremolite and actinolite. The U. S. Environmental Protection Agency (EPA) defines asbestos fibers as fibers that have the ratio of length to diameter greater than 3:1 35. The chemical structure of each asbestos fiber is given in Table 1. Among the six types of asbestos fibers, chrysotile is the most common and commercially important one in the United States, contributing to 95% of total asbestos consumption 36. The serpentine fibers have a curly stranded structure, where amphiboles are straight, rod-like fibers. The difference in physical properties leads to the accumulation of amphibole fibers in the distal lung parenchyma, difficulties in clearance, and thus a longer half-life. However, a general consensus is that structural characteristics of fibers alone are insufficient to account for the pulmonary toxicity of asbestos. Asbestos and free radicals The mineral surfaces of asbestos fibers are dynamic and complex, and they can be modified in the body. Iron from amosite or crocidolite, or magnesium from chrysotile may be leached or mobilized after absorption or uptake by cells, which leads to the generation of ROS. By using electron spin resonance (ESR) spin trap method, chrysotile, amosite, and crocidolite asbestos were found to be able to catalyze the production of hydroxyl radicals from hydrogen peroxide. Applying an iron chelator, desferrioxamine, significantly inhibited the production of the hydroxyl radical spin adduct 37. These observations

22 7 highlighted the role of iron in asbestos-induced ROS generation. In a chemical setting without additional reactants, iron catalyzes the generation of hydroxyl radical (HO ) from hydrogen peroxide (H 2 O 2 ) via the Fenton reaction (Reaction (1) ), in which ferrous ion is oxidized by hydrogen peroxide to ferric ion. The hydroxyl radical can then react with H 2 O 2 to produce O - 2, which can conversely reduce ferric iron back to ferrous iron to initiate a new cycle of reactions. The net process is converting H 2 O 2 to HO, known as iron-catalyzed Haber-Weiss reaction. However, this set of reactions play a minimal role in biology. Rather, ferrous iron in the labile iron pool can participate in the Fenton reaction or react with dioxygen, whose concentration is at least fold greater than H 2 O 2, to generate strong one-electron oxidants 38. The resulting Fe 3+ can readily be reduced to Fe 2+ by any of the many abundant cellular reducing agents. The very low steady-state level of superoxide suggests that it would play an insignificant role in reducing ferric iron. Instead, superoxide may serve as an oxidant to oxidize specific ironcontaining enzymes, such as aconitase and prolyl hydroxylase domain-containing proteins (PHDs) to modulate enzyme activity and the labile iron pool in cells and tissues 39. Fe 2+ + H 2 O 2 Fe 3+ + OH - + HO (1) Fe 3+ + O - 2 Fe 2+ + O 2 (2) Net reaction: O H 2 O 2 OH - + HO + O 2 (3) Amphibole fibers such as crocidolite and amosite typically have high iron content (~27%), whereas chrysotile has a lower but significant iron content (~1-6%), primarily as a surface contaminant 40. However, it is interesting that chrysotile and crocidolite, despite the difference in iron concentration, were all highly effective in producing free radicals. The iron chemistry of asbestos relies on the amount of catalytic or free iron, which consists of two components: redox active and diffusible. Fe 3+ is more stable but relatively less water-soluble at neutral ph, compared to ferrous ions. Thus, the rate of

23 8 mobilization of iron from asbestos and its reactivity is higher at ph 5 than ph 7, and varies in the presence of different chelators 41,42. However, though it is well known that iron catalyzes the degradation of H 2 O 2 to HO, it is still not clear how asbestos generates O - 2 in cell free systems. Zalma et al. proposed an electron transfer reaction from a ferrous ion in the asbestos surface to oxygen to produce ferric ions and superoxide, which involves a water molecule localized at asbestos surface (Figure 1-2) 43. The second mechanism implicates the release of ROS upon activation of inflammatory cells such as pulmonary alveolar macrophages and neutrophils. During phagocytosis of asbestos fibers, macrophages and neutrophils generate O - 2 and H 2 O 2 that lead to the production of HO. Oxidant release from phagocytic cells occurs rapidly (peaks between less than 1 min to 4 h) after in vitro exposure to asbestos fibers and is dose-dependent ( µg/ml) 44. Moreover, multiple studies showed that alveolar macrophages isolated from asbestosis patients can spontaneously produce high levels of H 2 O 45,46 2. However, the mechanism of how asbestos stimulates ROS formation from inflammatory cells is not completely clarified. One hypothesis is that oxidant release occurs nonspecifically during frustrated phagocytosis by macrophages and neutrophils that are unable to ingest long asbestos fibers completely. However, short fibers (75% <1.0 µm) such as chrysotile, which will be completely phagocytosed, generates similar amount of H 2 O Asbestos and human health Inhalation of asbestos fibers causes progressive pulmonary fibrosis, pleural disease and malignancies. Despite the tight regulation globally, asbestosis still remains a huge challenges to the health, especially in the developing countries. Total worldwide production in 2011 was 2 million metric tons. Since Sweden became the first country in

24 9 the world to ban asbestos, so far, 55 countries across the world has prohibited the production and consumption of asbestos. United States, however, has not banned the usage due to legal liability issues 47. It is estimated that more than 27 million workers in the United States were exposed to asbestos fibers between 1940 and Worldwide, over 125 million people are subjected to occupational asbestos exposure 47. The median latency from the time of exposure to the disease onset is about 32 years 49. Asbestosis is the only disease that its mortality has continued to increase since 1960, compared to other pneumoconiosis 50. Globally, WHO estimated that over 100,000 people died from asbestos-related disease from occupational exposure 47. The pulmonary parenchyma includes a variety of cells such as alveolar macrophages, pulmonary epithelial cells, mesothelial cells, endothelial cells, and fibroblasts. All cell types are susceptible to the toxic effect of asbestos 51. Molecular targets of asbestos and secondary ROS include lipids, DNA, and proteins. The chemical basis of asbestos-induced lipid oxidation is the ability of ferrous iron to catalyze alkoxyl radical production from organic hydroperoxides (Reaction. (1) ). Fe 2+ + ROOH Fe 3+ + RO + OH - (1) All six types of asbestos have been identified to be carcinogenic by the International Agency for Research on Cancer (IARC) 47. Genotoxicity of asbestos includes alteration of DNA bases, DNA double strand breaks, chromosomal aberrations, and sister chromatid exchanges. By using a human-hamster hybrid cell system, Hei et al. showed that chrysotile is a potent mutagen at that S1 locus of human chromosomes 52. Aust et al. also showed that crocidolite caused a two-fold increase in mutation frequency at the gpt locus 53. The IARC has determined that asbestos is associated with an increasing risk of lung, laryngeal, mesothelioma and ovarian cancer 54. Studies suggest that genetic components play a pivotal role in the development of asbestos-related disease, especially in mesothelioma 55. A germline mutation of BRCA1

25 10 associated protein-1 (BAP1) is found in two clusters of mesothelioma patients in Turkey 49,56. Meanwhile, somatic truncating BAP1 mutations and aberrant BAP1 expression are observed in sporadic mesothelioma cases. Both underscore a genetic predilection of developing mesothelioma after asbestos exposure. Polymorphism of two genes involved redox biology: glutathione S-transferase Mu1 (gstm1) and Theta1 (gstt1) are shown to render the population more susceptible to developing mesothelioma after asbestos exposure However, limited data reveal a genetic susceptibility to asbestosis. Kukkonen et al. reported that genes involved in innate immunity are associated with the risk of developing interstitial fibrosis and pleural thickening, in particular nlrp3 rs polymorphism 60. Macrophages The origin Macrophages are long-lived innate immune cells of the mononuclear phagocyte system 61,62. Based on differential expression of the specific mouse macrophage marker F4/80, at least two distinct mechanisms of hematopoiesis contribute to adult tissue macrophages. They are derived either from embryonic macrophages that develop early in the embryonal yolk sac and proliferate in situ, or from blood monocytes that develop along a specific line of progenitors in the bone marrow. Yolk sac-derived macrophages expressed high levels of F4/80, whereas monocyte-derived macrophages expressed low levels of F4/80. Macrophage polarization At least two monocyte subpopulations are known in mouse and human blood, which are distinguished based on the expression of different cell surface markers and migratory patterns 63. In the murine system, inflammatory monocytes are Ly6C + CCR2 + and CX3CR1 -. These cells are short-lived and upon infection are rapidly recruited to the

26 11 site of inflammation, where they differentiate to the inflammatory macrophages. Resident Ly6C - CCR2 - and CX3CR1 + monocytes are long-lived cells that initially may not migrate to the sites of infection due to the absence of CCR2 expression. Macrophage polarization is defined as acquisition of different phenotypes. At least in vitro, macrophages are able to switch the phenotypes readily. In an analogy to the T-helper-cell nomenclature, where Th1 cells are associated with the response against bacteria or viruses, and Th2 cells are associated with the response to parasitic infection and tissue remodeling, macrophages can be denoted as M1 and M2 macrophages. M1 macrophages are pro-inflammatory and have microbicidal and tumoricidal activity, whereas the M2 macrophages are involved in tumor progression and tissue remodeling, and can be pro-fibrotic in certain conditions 64. Classical macrophage activation requires priming with IFN-, the canonical cytokines generated by Th1 cells, and the activating of the downstream transcription factors such as nuclear factor-kappa light-chain-enhancer of activated-b cells (NF- B) and signal transducer and activator of transcription 1 (STAT1), as well as interferon regulatory factor 5 (IRF-5) to induce inflammatory gene transcription. Classically activated macrophages produce a wide range of pro-inflammatory cytokines such as TNF-, IL-1, and IL-6, as well as inflammatory chemokines, and have augmented phagocytic ability. These mechanisms are believed to contributing to the antitumor ability of the classically activated macrophages. Alternatively activated macrophages (AAMs) are usually activated by Th2 cytokines, IL-4 or IL-13. Their main properties are attenuating the production of proinflammatory cytokines and involving in extracellular tissue repairing. Alternatively activated macrophages secrete immunosuppressive cytokines such as IL-10 or IL-1ra (IL- 1 receptor antagonist) and growth factors such as fibroblast growth factors and transforming growth factors. Transcription factors involved in M2 polarization include STAT6, IRF-4, and peroxisome proliferator-activated receptor (PPAR)-. Differential

27 12 metabolism of L-arginine is characteristic of M1 and M2 macrophages 65. NO generated by IFN- -induced inos is shut down in M2 macrophages, with a shift to Arg1. M2 phenotype macrophages and human diseases M2 macrophages are actively involved in many processes associated with parasitic infection, immune tolerance, wound healing and tumorigenesis. The generation and role of AAMs has been studied extensively in helminth-related diseases 66-68, underscoring the importance of Th2 cytokines-mediated M2 polarization. Helminth infection not only initiates M2 polarization, but is also capable of subverting the M1 polarization as shown in Francisella tularensis infection 69. Pathogen was able to reprogram the macrophage to acquire an AAM phenotype. LPS tolerance is a common scenario in patients with sepsis. Peripheral blood monocytes and macrophages from these patients often display features resembling alternative activation of monocytes, including reduced production of pro-inflammatory mediators and expression of genes involved in tissue remodeling 70. Both IL-4 and IL-13 receptors have been shown to be essential for fibrosis development in S. mansoni granuloma formation 71. Induction of Arg1 by IL-4 and IL-13 has been implicated in collagen deposition and fibrosis development 72,73. Delayed healing or excessive fibrosis occurs in mice with dysfunctional M2 macrophages or deficiency of signature M2 gene expression such as arg1 74. Wound macrophages are known to undergo alternative activation despite a deficiency of Th2 cytokines, such as IL-4, in the wound environment; and the macrophage phenotype was sustained in mice lacking IL-4R 75. Up-regulation of ornithine decarboxylase and sequential generation of hydroxyproline and polyamines have been implicated in pro-fibrotic properties of M2 macrophages. Tumor-associated macrophages (TAMs) have many properties of M2 macrophages and they contribute to tumor local invasion through secreting proteinase such as cathepsin 76. They also promote angiogenesis and tumor growth through VEGF, leading to chemo-resistance 77,78. M2 macrophages promote tumorigenesis by increasing

28 13 signature M2 markers such as CCL IL-13, along with its receptors, contributes to the suppression of tumor rejection by Th1 cell-type immune response. In contrast, blockage IL-13R 2 via sirna reduces metastasis and promotes survival 80. Molecular inhibitor targeting to M2 macrophage have shown to be good candidates for cancer treatment, include using proapoptotic peptide 81 or anti-vegf antibody 82. Alternatively activated macrophages are also implicated in various pulmonary disorders including COPD, asthma and pulmonary fibrosis. Plasma Chitinase-1, a signature M2 protein, has been used to quantitative stratification of disease severity in COPD patients 83. A remarkable example of a pathogenic role of IL-13 in chronic obstructive pulmonary disease (COPD) has been reported. The macrophages upregulate IL-13R 1 expression and become alternatively activated by an autocrine or paracrine mechanism. M2 macrophages are known to be prevalent in the lungs of patients with idiopathic pulmonary fibrosis, sarcoidosis, and systemic sclerosis 84. M2 macrophages have also been shown to contribute to the fibrotic development in gamma-herpes virusesinduced pulmonary fibrosis 85. Epigenetic regulation of macrophage polarization Epigenetics refers to the changes in gene expression without alternation of the genetic code. This developmentally- or environmentally-induced modifications control how information encoded in DNA is expressed in a tissue- and context-specific manner 86. There are three major epigenetic mechanisms: 1) posttranslational modifications (such as methylation, acetylation, and phosphorylation) of histones; 2) methylation of CpG DNA motifs, and 3) noncoding RNAs 87. Although epigenetic marks are dynamically regulated, they are typically more stable than the rapidly fluctuating posttranslational modifications (such as phosphorylation) of upstream conventional signaling proteins. Thus, epigenetic modifications that persist after the original stimulus has resolved provide a mechanism for extending transient short-lived signals into a more

29 14 stable and sustained cellular response that is pertinent for chronic disease condition including pulmonary fibrosis 88. Besides effectively packaging genetic material in the nucleus, the octamer of histone proteins in nucleosome also plays a critical role in regulating gene activity by controlling DNA accessibility. Histone modifications are a multifaceted epigenetic process, which can be broadly divided into positive and negative marks that promote or suppress transcription, respectively 89. These histone marks are written and erased by enzymes termed chromatin regulators. These different modifications interact in a concerted manner to form a histone code to regulate gene expression 90. Various post-translational modifications can take place at the histone tails including acetylation, methylation (lysines and arginines), methylation (arginines), phosphorylation, ubiquitylation, sumoylation, ADP ribosylation, deamination and Proline isomerization 89. Among them, lysine acetylation and methylation are the most wellstudied histone modification. Acetylation is generally associated with transcriptional activation, whereas methylation can activate or inhibit transcription depending on where this modification occurs. Both histone methylation and acetylation are implicated in M2 polarization. Histone deacetylase 3 (HDAC3) inhibits IL-4-induced M2 polarization by deacetylating putative enhancers of IL-4-induced M2 genes 91. By contrast, alternative activation of macrophages in response to helminth infection in vivo is mediated by histone demethylase Jmjd3 92. In the canonical Th2 cytokine-mediated M2 polarization, jmjd3 expression is induced by IL-4 in a STAT6-dependent manner; and transcriptional activation is initiated when Jmjd3 binds to promoter regions of signature M2 gene such as chi3l3, retnla, and arg1.

30 15 Figure 1-1: The disulfide relay in the mitochondrial IMS facilitates protein folding and localization. After being synthesized in the cytosol, substrate proteins are transported across the mitochondrial outer membrane in an unfolded, reduced conformation. The catalytic cysteine pair of Mia40 interacts with substrate proteins facilitating the protein folding by the introduction of disulfide bonds. Electrons move from the substrates to Mia40, and then to the Erv1, a flavin adenine dinucleotide (FAD) containing sulfhydryl oxidase. Erv1 mediates the shift from two-electron to one-electron transfer and electrons are passed to the heme factor of Cyt c, then to the COX, and finally to molecular oxygen to generate water. White arrow annotates the electron flow and black arrow indicates the disulfide transfer. Redox potential for each step is given. Adapted from 93.

31 16

32 Table 1-1: Classification and chemical structure of asbestos fiber. 17

33 18 Class Type Chemical Composition Serpentine Chrysotile Mg 6 Si 4 O 10 (OH) 8 Amphibole Crocidolite Na 2 (Fe 3+ ) 2 (Fe 2+ ) 3 Si 8 O 22 (OH) 2 Amosite (Fe,Mg) 7 Si 8 O 22 (OH) 2 Anthophyllite (Mg,Fe) 7 Si 8 O 22 (OH) 2 Tremolite Ca 2 Mg 5 Si 8 O 22 (OH) 2 Actinolite Ca 2 (Mg,Fe) 5 Si 8 O 22 (OH) 2

34 19 Figure 1-2: Proposed mechanism for the generation of superoxide at the surface of asbestos. In the presence of water molecule and dioxygen, the ferrous ion on asbestos surface will be oxidized to ferric ion, leading to the production of superoxide. Adapted from 43.

35 20

36 21 CHAPTER II MITOCHONDRIAL CU,ZN-SOD PROMOTES PULMONARY FIBROSIS BY INCREASING H 2 O 2 Abstract The release of H 2 O 2 from alveolar macrophages has been linked to the development pulmonary fibrosis, but little is known about its source or mechanism of production. We found that alveolar macrophages from asbestosis patients spontaneously produce high levels of H 2 O 2 and have high expression of Cu,Zn-SOD. Additionally, Cu,Zn-SOD concentrated within mitochondrial IMS in asbestosis patients. Because Cu,Zn-SOD is found in the mitochondrial intermembrane space (IMS), we hypothesized that mitochondrial Cu,Zn-SOD-mediated H 2 O 2 generation contributed to pulmonary fibrosis. Asbestos induced translocation of Cu,Zn-SOD to the IMS was unique to macrophages and was dependent on functional mitochondrial respiration and the presence of at least one of the conserved cysteines required for disulfide bond formation. These conserved cysteine residues were also necessary for enzyme activation and H 2 O 2 generation. Cu,Zn-SOD-mediated H 2 O 2 generation was inhibited by knockdown of the iron-sulfur protein, Rieske, in complex III. The role of Cu,Zn-SOD was biologically relevant in that Cu,Zn-SOD -/- mice generated significantly less H 2 O 2 and had less oxidant stress in bronchoalveolar lavage fluid and the lung parenchyma. Furthermore, Cu,Zn- SOD -/- mice did not develop pulmonary fibrosis, and knock-down of Cu,Zn-SOD in macrophages attenuated collagen I deposition by lung fibroblasts. These findings demonstrate a novel mechanism for the pathogenesis of pulmonary fibrosis whereby the antioxidant enzyme Cu,Zn-SOD translocates to the mitochondrial IMS to increase H 2 O 2 generation in alveolar macrophages.

37 22 Introduction Pulmonary fibrosis is a progressive disease characterized by aberrant repair that results in remodeling and destruction of the normal architecture of lung tissue. Asbestos exposure is a prototypical cause of pulmonary fibrosis. While asbestos remains an important cause of pulmonary fibrosis, the mechanism for asbestos-induced lung injury is poorly understood. Reactive oxygen species (ROS), including H 2 O 2, play an important role in the pathogenesis of asbestos-mediated pulmonary fibrosis. Generation of ROS can occur in a cell-free system by the reduction of oxygen on the surface of the asbestos fiber, but the production is amplified during phagocytosis of fibers by neutrophils, macrophages, and monocytes 45,94. We have demonstrated that administration of catalase to wild-type (WT) mice attenuated the development of fibrosis after exposure to asbestos 95, signifying that H 2 O 2 generation by alveolar macrophages is a critical factor in the pathogenesis of asbestosis; however, the source and molecular mechanism of asbestos-induced H 2 O 2 generation in alveolar macrophages is unknown. H 2 O 2 generation primarily results from dismutation of superoxide anion (O - 2 ), which occurs at a rapid rate constant ( M -1 s -1 ) nonenzymatically, and superoxide dismutase (SOD) catalyzes the dismutation reaction at a rate constant around M -1 s -1 5,96,97. There are three SOD enzymes: Cu,Zn-SOD (SOD1) is located in the cytosol and mitochondrial intermembrane space (IMS); Mn-SOD (SOD2) is located in the mitochondrial matrix; and EC-SOD (SOD3), which is an extracellular SOD 27,28. Alveolar macrophages obtained from patients with pulmonary fibrosis, including asbestosis, have been shown to resemble monocytes 98. These monocytes and young macrophages release reactive oxygen species (ROS), including H 2 O 2, which is associated with the persistent inflammatory response, cell injury, apoptosis, cell proliferation, and fibrogenesis In addition, monocytes have a high level of Cu,Zn-SOD expression, which decreases with cell differentiation 101.

38 23 Coupled with our previous observations showing the role of mitochondria in collagen deposition 102, these data demonstrate a novel pathway by which mitochondrial H 2 O 2 generation is augmented by import of Cu,Zn-SOD into the IMS in monocytic inflammatory cells. Increased mitochondrial Cu,Zn-SOD expression and activation in monocytic cells induces pulmonary fibrosis by increasing fibroblast collagen production. These results provide a potential target that could protect against the development of a prototypical form of pulmonary fibrosis. Methods and Materials Materials Chrysotile asbestos was provided by the NAIMA Fiber Repository. p- hydroxylphenyl acetic acid (phpa), horseradish peroxidase (HRP), N,N -Dimenthyl- 9,9 -biacridinium dinitrate (Lucigenin reagent), and reduced β-nad phosphate tetrasodium (NADPH) were purchased from Sigma Chemical Company (St. Louis,MO). Human subjects The Human Subjects Review Board of the University of Iowa Carver College of Medicine approved the protocol of obtaining alveolar macrophages from normal volunteers. Normal volunteers had to meet the following criteria: (1) age between 18 and 55 years; (2) no history of cardiopulmonary disease or other chronic disease; (3) no prescription or nonprescription medication except oral contraceptives; (4) no recent or current evidence of infection; and (5) lifetime nonsmoker. Alveolar macrophages were also obtained from patients with asbestosis. Patients with asbestosis had to meet the following criteria: (1) FVC and DLCO at least 50% predicted; (2) current nonsmoker; (3) no recent or current evidence of infection; and (4) evidence of restrictive physiology on pulmonary function tests and interstitial fibrosis on chest computed tomography. Fiberoptic bronchoscopy with bronchoalveolar lavage was performed after subjects

39 24 received intramuscular atropine, 0.6 mg, and local anesthesia. Each subsegment of the lung was lavaged with five 20-mL aliquots of normal saline, and the first aliquot in each was discarded. The percentage of alveolar macrophages was determined by Wright- Giemsa stain and varied from 90 to 98%. Mice Wild-type (WT) and Cu,Zn-SOD -/- C57BL/6 mice (a generous gift from Dr. Steven Lentz, University of Iowa, Iowa City, IA) were used in these studies, and all protocols were approved by the University of Iowa Institutional Animal Care and Use Committee., Mice were intratracheally administered a dose of 100 µg of chrysotile asbestos suspended in 50 µl 0.9% saline solution after being anesthetized with 3% isoflurane using a precision Fortec vaporizer (Cyprane, Keighley, UK). One or twentyone days after exposure, mice were euthanized with an overdose of isoflurane, and bronchoalveolar lavage (BAL) was performed. BAL cells were used for determination of total and differential cell number, and BAL fluid was used to determine lipid peroxidation. The lungs were removed and stained for collagen fibers using Masson s trichrome stain. Cell culture Human monocyte (THP-1), mouse type II alveolar epithelial (MLE-12), and human lung fibroblast (HFL-1) cell lines were obtained from American Type Culture Collection (Manassas,VA). Cells were maintained in RPMI-1640 or DMEM media with following supplements: 10% fetal bovine serum, and penicillin/streptomycin. All experiments were performed with 0.5% serum supplement.

40 25 SOD activity assay SOD activity assays were performed as described previously 103. Briefly, SOD activity was measured by separating samples on a 12% native polyacrylamide gel. The gel was stained by incubation with 2.43 mm nitroblue tetrazolium (NBT), 28 µm riboflavin, and 28 mm TEMED in the dark. Determination of H 2 O 2 generation Extracellular H 2 O 2 production was determined by using a fluorometric assay as described previously 104. Briefly, cells were incubated in phenol-red free Hanks balanced salt solution supplemented with 6.5 mm glucose, 1 mm HEPES, 6 mm sodium bicarbonate, 1.6 mm phpa, and 0.95 µg/ml HRP. Cells were exposed to chrysotile asbestos and fluorescence of phpa-dimer was measured by using a spectrofluorometer at excitation of 320 nm and emission of 400 nm, respectively. Isolation of cytoplasm, membrane, mitochondria, mitochondrial intermembrane space, mitoplasts and endoplasmic reticulum Cellular compartment separation was performed as described previously 34,105,106. The cytoplasm was isolated by suspending cells in 200 μl of lysis buffer (50 mm Tris, ph 8, 10 mm EDTA, protease inhibitor), sonicated for 10 s on ice, and centrifuge at 2,000 g for 5 min. The supernatant was centrifuged at 100,000 g for 10 min after which the supernatant containing the cytoplasmic fraction was collected. For membrane isolation, cells were lysed in a buffer containing 50 mm Tris, ph 8.0, 10 mm EDTA, and protease inhibitors. Lysates were homogenized using a Kontes Pellet Pestle Motor and centrifuged at 3,000 rpm for 3 min at 4 C. Supernatants were then centrifuged at 100,000 g for 1 h. After removal of the supernatant, the membrane pellet was resuspended in lysis buffer. Mitochondria were isolated by lysing the cells in a mitochondria buffer containing 10 mm Tris, ph 7.8, 0.2 mm EDTA, 320 mm sucrose, and protease inhibitors. Lysates were homogenized using a Kontes Pellet Pestle Motor

41 26 and centrifuged at 2,000 g for 8 min at 4 C. The supernatant was removed and kept at 4 C, and the pellet was lysed, homogenized, and centrifuged again. The two supernatants were pooled and centrifuged at 12,000 g for 15 min at 4 C. The pellet was then resuspended in mitochondria buffer without sucrose. For mitochondrial IMS isolation, the mitochondria fractions were treated with digitonin (0.1 mg digitonin/mg mitochondria) for 1 h at room temperature, centrifuged at 10,000 g for 10 min, and the supernatant was collected. 100 mm iodoacetamide was added to prevent SOD1 activation while disrupting the outer membrane. For mitoplast isolation, mitochondria were incubated in 5X volume cold hypotonic buffer (10 mm Tris, ph 7.4, 1 mm EDTA, and 1 mm dithiothreitol) for 10 min on ice. 150 mm NaCl was added to buffer for 10 min on ice. Samples were centrifuged at 18,000 g for 20 min at 4 C, and then the pellet was resuspended in sucrose-free mitochondria buffer. Endoplasmic reticulum was isolated by homogenizing cells in a lysis buffer containing 0.3 M sucrose, 10 mm HEPES, ph 7.0, 2 mm dithiothreitol, and protease inhibitors. The homogenate was first centrifuged at 500 g for 10 min and the supernatant then centrifuged at 12,000 g for 15 min. The supernatant was collected and centrifuged at 30,000 g for 20 min to obtain the crude endoplasmic reticulum fraction. Lucigenin assay Lucigenin assay was performed as previously described 107. Briefly, protein lysates (10 µg) were mixed with lucigenin (5 mm), NADPH (100 mm), and PBS to a total volume of 1 ml, and luminescence was recorded every 30 s for 10 min as relative light units (RLU) using a Sirius Luminometer (Berthold, Pforzheim, Germany) Plasmids and transfections Human Cu,Zn-SOD cdna (NM_000454) with no stop codon was amplified by PCR and inserted into pcdna3.1d/v5-his-topo vector (Invitrogen). Mutations of

42 27 cysteines in Cu,Zn-SOD-V5-His were generated using the QuikChange II Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). The following mutants were generated: Cu,Zn-SOD-V5-His C57S, Cu,Zn-SOD-V5-His C146S, and Cu,Zn-SOD-V5-His C57,146S. To generate the Cu,Zn-SOD-GFP construct, SOD1 cdna with no stop codon was amplified by PCR using a forward primer containing a Nhe1 (underlined) site 5 -GCT AGC ATG GCG ACG AAG GCC GTG T-3 and reverse primer having a EcoRV site 5 -GAT ATC TTG GGC GAT CCC AAT TAC ACC-3. The resulting PCR product was subcloned into pcr4-topo (Invitrogen). The pcr4-sod construct was digested with Nhe1 and EcoRV and the product ligated into Nhe1-EcoRV sites of phmgfp vector (Promega) using T4 DNA ligase. The correct reading frame and sequence of plasmids used in this study were verified by fluorescent automated DNA sequencing performed by the University of Iowa DNA facility. Cells were transfected with vectors by using Fugene transfection reagent (Roche, Indianapolis, IN) according to the manufacturer s instructions. Adenoviral vectors Macrophages were infected with replication-deficient adenovirus type 5 with the E1 region replaced with DNA containing the cytomegalovirus (CMV) promoter region alone (Ad5.CMV) or Ad5.Cu,Zn-SOD vector (Gene Transfer Vector Core, University of Iowa Carver College of Medicine, Iowa City, IA) at a multiplicity of infection (moi) of 500 in serum-free RPMI medium. After 5 h serum was added to the medium to a final concentration of 0.5%, and the cells were allowed to recover for 48 h. Immunoblot analysis Whole cells lysates were obtained as previously described 108 and separated by SDS-PAGE. Immunoblot analyses were performed with the designated antibodies followed by the appropriate secondary antibody cross-linked to HRP.

43 28 Confocal microscopy Macrophages were transfected with phmgfp-sod vector in a cover-glass chamber and allowed to recover for 24 h. After exposing to chrysotile asbestos for 3 hours, medium was changed to Hank s Balanced Salt Solution containing 500 nm Mitotracker Red (Molecular Probes, Eugene, OR) and incubated for 30 min at 37 ºC. The 488- and 579-nm lines of a krypton/agron laser were used for measuring the fluorescence excitation of GFP and Mitotracker Red, respectively. Small interfering RNA (sirna) Macrophages were transfected with 100 nm scrambled (Santa Cruz,CA), human Rieske, or Cu,Zn-SOD sirna duplex (IDT, Iowa City, IA) using DharmaFect 2 reagent (Dharmacon Research, Lafayette, CO) or together with Cu,Zn-SOD-V5-His or empty vector by using DharmaFect Duo reagent in antibiotic- and serum-free media, according to the manufacturer s instruction. After 4 hours, serum was added to a final concentration of 10%, and the cells were allowed to recover for 72 h. Quantitative real-time PCR Total RNA from homogenized lungs or isolated BAL cells were obtained using Trizol reagent (Sigma). After reverse transcription using iscript reverse transcription kit (Bio-Rad Laboratories, Hercules, CA), Collagen I, TGF-, and HPRT mrna expression were determined by quantitative real-time PCR using Sybr Green kit (Bio-Rad Laboratories) on an IQ5 Real-time PCR machine (Bio-Rad Laboratories). Data were calculated by the ΔΔ CT method. Collagen I and TGF- mrna were normalized to HPRT and are expressed as arbitrary units.

44 29 ELISA Active TGF- in cell media was measured by using a TGF- ELISA kit (R&D, Minneapolis, MN) according to manufacturer s instructions. Lipid peroxidation Lipid peroxidation in BAL fluid was measured by using a thiobarbituric acid reactive substances (TBARS) kit (Cayman, Ann Arbor, MI) according to manufacturer s instructions. Glutathione assay Lung tissue that had been perfused to remove red blood cells was homogenized directly into 5-sulfosalicylic acid (5% w/v), centrifuged, and the supernatant saved at -80 C overnight for the glutathione assay. The protein pellet was dissolved in NaOH and protein concentration was determined. Total glutathione content was measured. Reduced glutathione (GSH) and glutathione disulfide (GSSG) were distinguished by addition of 20 µl of a 1:1 mixture of 2-vinylpyridine and ethanol per 100 µl of sample, followed by incubation for 2 h and assayed as described previously 109. All glutathione determinations were normalized to the protein content the lung homogenates. Statistical analysis Statistical comparisons were performed as indicated in figure legend. Values in figures are expressed as means with standard errors and p < 0.05 was considered to be significant.

45 30 Results Alveolar macrophages from patients with asbestosis generate high levels of H 2 O 2 and have increased expression and activity of Cu,Zn-SOD Alveolar macrophages obtained from the lungs of patients with pulmonary fibrosis are known to generate ROS, including H 2 O To confirm that this phenomenon occurred in patients with asbestosis, we obtained alveolar macrophages from normal subjects and patients with asbestosis. We found that alveolar macrophages obtained from asbestosis patients had 10-fold greater level of H 2 O 2 generation compared to normal subjects (Figure 2-1). SOD catalyzes H 2 O 2 generation fold faster from the dismutation of O 2 - compared to spontaneous dismutation, so we next determined if there was a difference in SOD expression and activity between patients and normal subjects. Whole cell lysates were separated by SDS-PAGE to determine SOD expression or by native gel to determine SOD activity. We found that alveolar macrophages obtained from asbestosis patients had similar Mn-SOD expression (Figure 2-2A) and activity compared with the normal subjects (Figure 2-2B and 2-2C). In contrast, the asbestosis patients had significantly more Cu,Zn-SOD expression (Figure 2-2A) and Cu,Zn-SOD activity than normal subjects (Figure 2-2B and 2-2D). Based on these results, we formulated the hypothesis that Cu,Zn-SOD is the primary determinant for increasing production of H 2 O 2 in asbestosis patients. Cu,Zn-SOD in the mitochondrial IMS contributes to H 2 O 2 production In order to determine whether Cu,Zn-SOD was important in increasing H 2 O 2, we over expressed Cu,Zn-SOD and measured H 2 O 2 generation. Macrophages were infected with a replicative-deficient adenovirus containing either an empty vector (Ad5.CMV) or Cu,Zn-SOD (Ad5.Cu,Zn-SOD). Extracellular H 2 O 2 concentration was significantly increased in cells infected with Ad5.Cu,Zn-SOD compared to Ad5.CMV-infected cells

46 31 (Figure 2-3A). Due to the fact that Cu,Zn-SOD is known to be located in the cytoplasm and in the mitochondrial IMS, we isolated each cell compartment and performed an immunoblot analysis for Cu,Zn-SOD. Cu,Zn-SOD was distributed in both mitochondria and cytoplasm. In mitochondria, Cu,Zn-SOD was concentrated in the IMS (Figure 2-3B). Because we found Cu,Zn-SOD expression in different cellular fractions, we next determined the site and effect of Cu,Zn-SOD over expression on the steady state level of O - 2. Cells were transfected with either an empty vector or Cu,Zn-SOD-V5-His, and cytoplasmic and mitochondrial fractions were isolated. O - 2 level progressively increased in the mitochondrial fraction, and over expression of Cu,Zn-SOD-V5-His significantly inhibited the generation (Figure 2-3C). In contrast, minimal O - 2 was generated in the - cytoplasm, and over expression of Cu,Zn-SOD-V5-His had no effect on cytoplasmic O 2 - levels. Over expression of Cu,Zn-SOD also reduced the rate of mitochondrial O 2 generation (Figure 2-3D). In aggregate, these data demonstrate that Cu,Zn-SOD is highly expressed in the mitochondria of monocytes, and it enhances the dismutation of O - 2 to H 2 O 2. We further investigated if Cu,Zn-SOD was localized in the mitochondria in alveolar macrophages from patients. Isolated mitochondria from alveolar macrophages obtained from patients with asbestosis demonstrated significantly greater immunoreactive Cu,Zn-SOD compared to normal subjects (Figure 2-4A). Fractionation controls of the mitochondria are shown by immunoblot analysis (Figure 2-4B). In contrast, Cu,Zn-SOD in the cytoplasm was much greater in the alveolar macrophages obtained from normal subjects (Figure 2-4C). To verify that mitochondria is the predominant source of ROS production in - alveolar macrophages isolated from asbestosis patients, we measured O 2 production rate in mitochondria and membrane fractions isolated from alveolar macrophages obtained from patients and normal subjects. In asbestosis patients, mitochondrial O - 2 production rate was significantly higher compared with that of the membrane fraction, as well as

47 32 greater than those of the membrane and mitochondrial fractions from normal subjects (Figure 2-4D). In contrast, there was no significant difference in the O - 2 production rate in the mitochondrial and the membrane fractions isolated from normal subjects. Combined with our prior data showing that overexpression of Cu,Zn-SOD decreased mitochondrial O - 2 levels, these results suggest that the majority of Cu,Zn-SOD-mediated H 2 O 2 generation in macrophages is from the mitochondria. Asbestos induced the translocation and activation of Cu,Zn-SOD in the IMS of mitochondria To further address the role of Cu,Zn-SOD in the mitochondria after asbestos exposure, macrophages were transfected with Cu,Zn-SOD-V5-His vector and exposed to chrysotile for 3 h. Mitochondrial fractions were isolated and an immunoblot assay was performed for the tagged protein. Cu,Zn-SOD-V5-His increased dramatically in the mitochondria obtained from cells exposed to asbestos (Figure 2-5A). Observation of translocation was confirmed with confocal microscopy (Figure 2-5B). Cells were transfected with phmgfp-sod vector. After 24 h, cells were exposed to chrysotile asbestos for 3 h, and Mitotracker Red was used to stain the mitochondria. In the absence of asbestos, the GFP expression had a diffuse distribution (upper left panel), while the Mitotracker Red distinguished the mitochondria (upper middle panel). The merged image of GFP and Mitotracker Red (Figure 2-5B, upper right panel) demonstrated that there were no aggregation of Cu,Zn-SOD in the mitochondria. In contrast, in cells exposed to chrysotile for 3 h, GFP (bottom left panel) was concentrated in mitochondria as seen with the Mitotracker Red (bottom middle panel), and the merged panel demonstrated that Cu,Zn-SOD localized to the mitochondria (Figure 2-5B, bottom right panel). Alveolar epithelial cells are known to generate mitochondrial ROS after exposure to asbestos and other environmental toxins 111,112, so we questioned whether asbestos increased translocation of Cu,Zn-SOD in epithelial cells. MLE-12 cells were transfected with the Cu,Zn-SOD-V5-His vector. After 24 h the cells were exposed to chrysotile for 3

48 33 h, and mitochondrial fractions were isolated. An immunoblot for the tagged protein demonstrated that Cu,Zn-SOD levels in mitochondria decreased after asbestos exposure (Figure 2-5C), suggesting that the phenomenon of Cu,Zn-SOD translocation is unique to monocytes. Translocation and activation of Cu,Zn-SOD requires critical cysteine residues Only apo-cu,zn-sod, an immature and inactive form, translocates to mitochondria 113. The import of proteins, such as Cox17 and TIM13 (translocase of the inner membrane), into the IMS requires the presence of conserved cysteine motifs 32. Cysteine-57 and cysteine-146 comprise the conserved cysteine motif in Cu,Zn-SOD that form an intramolecular disulfide bond, which is necessary for enzyme activation 114. To determine if the cysteines involved in forming the disulfide bond in Cu,Zn-SOD have a role in mitochondrial translocation and activation, we generated Cu,Zn-SOD constructs with mutations in either cysteine-57 (Cu,Zn-SOD-V5-His C57S ) or cysteine-146 (Cu,Zn- SOD-V5-His C146S ) and a construct containing mutations in both cysteine residues (Cu,Zn- SOD-V5-His C57,146S ). To examine the impact of cysteine mutation in mitochondrial translocation, we first determined whether these mutants were stably expressed in the cytoplasmic compartment. Cells were transfected with the mutant Cu,Zn-SOD expression vectors. After 24 h, cytoplasmic fractions were isolated, and an immunoblot assay was performed for the tagged protein. All Cu,Zn-SOD mutants presented in the cytoplasma, whereas the C57S and C146S mutants had much lower cytoplasmic expression compared to the C57,146S mutant (Figure 2-6A). This observation confirms that all Cu,Zn-SOD mutants are stably expressed. We next examined whether cysteine residues are critical for mitochondrial translocation. Macrophages were transfected with Cu,Zn-SOD-V5-His mutants and exposed to chrysotile asbestos for 3 h. Mitochondrial fractions were isolated and an

49 34 immunoblot assay was performed for the tagged protein. The single cysteine mutations in Cu,Zn-SOD did not alter translocation to the mitochondria, but the construct containing mutations in both cysteines was not detected in the mitochondria in either the presence or absence of asbestos (Figure 2-6B). In aggregate, these data demonstrate that Cu,Zn-SOD mitochondrial translocation requires the presence of at least one conserved cysteine residue; however, both cysteine residues are necessary for enzyme activation and subsequent H 2 O 2 generation after exposure to asbestos. To determine if these cysteine residues are important for Cu,Zn-SOD activity and mitochondrial H 2 O 2 production, we transfected macrophages with an empty vector, Cu,Zn-SOD-V5-His (WT) or the cysteine mutants (C57S, C146S, or C57,146S). After 24 h, cells were exposed to chrysotile asbestos for 3 h. H 2 O 2 generation in cells expressing the WT Cu,Zn-SOD was significantly higher than cells expressing the empty vector (Figure 2-6C). Cells expressing either one of the single mutants (C57S, C146S) had a significant reduction in H 2 O 2 generation to near control levels. Cells expressing C57,146S had a similar reduction in H 2 O 2 generation compared to cells expressing the WT Cu,Zn-SOD (Figure 2-6C). These observations highlight the importance of these critical cysteine residues in the formation of functionally active enzyme. Translocation and activation of Cu,Zn-SOD was redox sensitive The mitochondrial electron transport chain (ETC) is a major site of O 2 - production in both quiescent and activated cells. Because Cu,Zn-SOD translocation required a conserved cysteine motif and cysteines are targets of oxidation, we questioned whether increased mitochondrial ROS generation after asbestos exposure regulated translocation. Macrophages were transfected with the empty or Cu,Zn-SOD-V5-His vector in combination with either scrambled or Rieske sirna to determine the effect on H 2 O 2 generation as a function of Cu,Zn-SOD activity. Rieske is the iron-sulfur protein component of cytochrome bc1 complex (complex III). Overexpression of Cu,Zn-SOD

50 35 increased H 2 O 2 production in cells transfected with either Cu,Zn-SOD-V5-His alone or with the scrambled sirna (Figure 2-7A). In contrast, cells transfected with the Rieske sirna had a significant inhibition of H 2 O 2 production. Over expression of Cu,Zn-SOD in cells expressing the Rieske sirna resulted in marked inhibition of H 2 O 2 generation compared to the cells expressing the empty vector alone (Figure 2-7A). Rieske knockdown also significantly abrogated the rate of H 2 O 2 generation (Figure 2-7B). To confirm that the mitochondrial-derived ROS was required for mitochondrial Cu,Zn-SOD translocation and activation, macrophages were transfected with scrambled or Rieske sirna. After 72 h, cells were exposed to chrysotile asbestos for 3 h. Mitochondrial fractions were isolated and separated on a polyacrylamide gel in the presence or absence of β-me. In the presence of -ME, the disulfide bond-linked Cu,Zn- SOD was not present. In the absence of -ME, cells expressing the scrambled sirna had the activated, disulfide bond-linked in Cu,Zn-SOD. In contrast, cells expressing the Rieske sirna had a significant reduction in the activated Cu,Zn-SOD in the mitochondria (Figure 2-7C). In aggregate, these data demonstrate Cu,Zn-SOD-mediated H 2 O 2 generation is strikingly reduced with knockdown of a critical redox center of complex III. These data also show that translocation and activation the Cu,Zn-SOD in the IMS are coupled to the activity of mitochondrial ETC. Cu,Zn-SOD regulates asbestos-induced oxidative stress in vivo To better define the potential biological relevance of Cu,Zn-SOD in the pathogenesis of pulmonary fibrosis, we exposed WT and Cu,Zn-SOD -/- C57BL/6 mice to chrysotile asbestos. The predominant cells seen in the BAL fluid at 21 days were alveolar macrophages. To demonstrate that Cu,Zn-SOD had a role in the generation of H 2 O 2 ex vivo after asbestos exposure, we isolated monocytes and macrophages from the bone marrow from WT and Cu,Zn-SOD -/- mice. Cells were cultured in the presence or absence of asbestos, and H 2 O 2 generation was measured. Cells taken from WT animals

51 36 demonstrated significantly higher production of H 2 O 2 when compared to cells obtained from Cu,Zn-SOD -/- mice. H 2 O 2 generation was also significantly enhanced in cells from WT mice after asbestos exposure (Figure 2-8A). In contrast, asbestos exposure had no effect on H 2 O 2 production in Cu,Zn-SOD -/- monocytes/macrophages indicating that Cu,Zn-SOD is crucial for the H 2 O 2 generation after asbestos exposure ex vivo. Furthermore, Cu,Zn-SOD also augmented the rate of H 2 O 2 generation (Figure 2-8B). Due to the significant differences in H 2 O 2 levels between WT and Cu,Zn-SOD -/- cells, we questioned whether Cu,Zn-SOD had a role in mediating oxidative stress in the lungs after asbestos exposure. Unsaturated lipids are significant components of surfactant and are major targets for lipid peroxidation during inflammation and lung injury 115. WT and Cu,Zn-SOD -/- mice were exposed to chrysotile asbestos, and lipid peroxidation in BAL fluid was determined at 21 days utilizing the thiobarbituric acid reactive substances (TBARS) assay. WT mice had greater than 6-fold higher levels of lipid peroxidation in BAL fluid compared to Cu,Zn-SOD -/- mice (Figure 2-8C). In addition, when lung tissue from similarly treated animals was assayed for the percentage of total glutathione (GSH) in the disulfide form (%GSSG), which signifies an increase in oxidation of GSH, the lungs from WT mice again demonstrated significantly higher %GSSG relative to the Cu,Zn-SOD -/- (Figure 2-8D). Taken together, these data are completely consistent with the hypothesis that monocyte/macrophages from the lungs of WT mice exposed to asbestos have significantly greater H 2 O 2 production that increased oxidative stress in both the BAL fluid and the lung parenchyma. Furthermore, given that asbestos induced high levels of H 2 O 2 and that the levels of oxidative stress detected in the lungs were reduced in the lungs of Cu,Zn-SOD -/- mice strongly supports the conclusion that Cu,Zn-SOD was a significant source of H 2 O 2 following exposure to asbestos.

52 37 Cu,Zn-SOD -/- mice are protected from developing pulmonary fibrosis after asbestos exposure To determine whether the relationship between Cu,Zn-SOD and H 2 O 2 generation and oxidative stress in the lung had an effect on the development of pulmonary fibrosis, WT and Cu,Zn-SOD -/- mice were exposed to chrysotile asbestos. After 21 days, the animals were euthanized and lungs were excised and stained with Masson s trichrome to visualize collagen deposition. The lungs of WT mice had widespread collagen deposition in both peribronchial and parenchymal portions of the lung (Figure 2-9A). In contrast, the collagen deposition in Cu,Zn-SOD -/- mice was significantly attenuated (Figure 2-9B). To verify the histopathological observations, we determined the extent of pulmonary fibrosis biochemically. Total mrna was isolated from homogenized lungs obtained from mice 21 days after asbestos exposure. Collagen I mrna expression was greater than 5-fold higher in the lungs of WT mice compared to Cu,Zn-SOD -/- mice (Figure 2-9C). Hydroxyproline assay was also performed to measure collagen content. WT mice had significantly higher hydroxyproline levels in their lungs compared to Cu,Zn-SOD -/- mice (Figure 2-9D). These data further confirm our histological findings that Cu,Zn-SOD -/- mice are protected from developing pulmonary fibrosis. Because TGF- is a pro-fibrotic cytokine produced by macrophages, we determined the role of Cu,Zn-SOD in regulating its expression in vivo and in vitro. In WT and Cu,Zn-SOD -/- mice exposed to asbestos, BAL cells were obtained and TGF- mrna expression was quantified. Cells collected from asbestos-exposed WT mice expressed greater than 4-fold more TGF- mrna compared to Cu,Zn-SOD -/- mice (Figure 2-10A). To examined this observation in vitro, macrophages were infected with a replicative-deficient adenovirus containing either an empty vector or Cu,Zn-SOD. After 48 h the supernatants were harvested, and active TGF- was determined by ELISA. Cells expressing Cu,Zn-SOD produced significantly more active TGF- than cells expressing the empty vector (Figure 2-10B). These data suggest that mitochondrial

53 38 Cu,Zn-SOD regulates TGF- production in macrophages, which might contribute to fibrosis development. Because fibroblasts are the primary cell that produces collagen and in order to provide a direct link between Cu,Zn-SOD and pulmonary fibrosis, we transfected macrophages with either a scrambled or Cu,Zn-SOD sirna. After 72 h, the conditioned medium was collected. Human lung fibroblasts (HLF-1) were cultured for 24 h in the conditioned media obtained from the transfected cells in the presence or absence of chrysotile asbestos. The conditioned medium from the fibroblast cultures was used to measure collagen I secretion. HLF-1 cells exposed to conditioned medium from macrophages transfected with the scrambled sirna had significantly more procollagen I and collagen I compared to the cells exposed to the conditioned medium from the Cu,Zn- SOD sirna-transfected cells (Figure 2-11). Taken together, these data demonstrate that the antioxidant enzyme Cu,Zn-SOD induces pulmonary fibrosis via translocation and activation in the mitochondrial IMS where it enhances the generation of H 2 O 2. Discussion Although H 2 O 2 generation has been linked to pulmonary fibrosis, little is known about its source or mechanism of production. In this study, we demonstrate that Cu,Zn- SOD translocation and activation in the IMS is unique to monocytes and macrophages and is dependent on a conserved cysteine motif and mitochondrial ETC function. These data also demonstrate that H 2 O 2 generation is regulated by the presence of Cu,Zn-SOD in the IMS, which, in part, modulates the development of pulmonary fibrosis after asbestos exposure. Evidence to support this pathway include i) asbestos increased mitochondrial ROS generation and increases translocation of Cu,Zn-SOD to the IMS; ii) a conserved cysteine motif was necessary for translocation and activation; iii) knockdown of the ironsulfur protein, Rieske, decreased Cu,Zn-SOD translocation and activation; iv) mitochondrial H 2 O 2 generation is dependent on complex III O - 2 generation as

54 39 knockdown of Rieske inhibited H 2 O 2 production by Cu,Zn-SOD over-expression; v) Cu,Zn-SOD -/- mice had reduced H 2 O 2 generation, decreased oxidative stress in the BAL fluid and the lung parenchyma, and were protected from developing pulmonary fibrosis; and vi) knock-down of Cu,Zn-SOD in monocytes inhibits collagen I deposition by lung fibroblasts. Taken together, these observations provide novel insight into the mechanism linking H 2 O 2 generation to pulmonary fibrosis and delineate the role of Cu,Zn-SOD in regulating mitochondrial H 2 O 2 production. We have shown that mitochondrial H 2 O 2 production promotes the development of asbestos-induced pulmonary fibrosis by increasing collagen production in fibroblasts, and intratracheal administration of catalase attenuates asbestos-induced pulmonary fibrosis 95,102. The use of other antioxidant enzymes, such as PEG-SOD, have not been effective in preventing pulmonary fibrosis 116. It is unclear in this study, however, if PEG-SOD exacerbated the lung injury. Moreover, the effect of SOD on the development of pulmonary fibrosis is not known. Alveolar macrophages obtained from the lung of patients with chronic lung disease, such as pulmonary fibrosis, are known to resemble monocytes This is likely secondary to persistent recruitment of monocytes to the site of injury. Cu,Zn-SOD is highly expressed in monocytes, and the expression decreases with differentiation to mature macrophages 101. Studies have shown Mn-SOD to be increased in animal models after exposure to asbestos 118,119, whereas Cu,Zn-SOD expression was not altered with asbestos exposure. These studies, however, measured expression in whole lung homogenates, while we have focused on the role of inflammatory cells in the pathogenesis of pulmonary fibrosis. Our data suggest a new conceptual framework for understanding asbestosis, and potentially other forms of pulmonary fibrosis, in that the increased Cu,Zn-SOD in the alveolar macrophages from asbestosis patients is secondary to the presence of monocytes and young macrophages in the lung.

55 40 An observation in our study regarding the mechanism of Cu,Zn-SOD-mediated H 2 O 2 generation is that asbestos triggers translocation of Cu,Zn-SOD into the IMS, and this translocation was regulated by the presence of at least one cysteine in the conserved cysteine motif and mitochondrial ETC ROS production. Only apo-cu,zn-sod, an immature and inactive form, translocates to mitochondria 113. Activation of apo-cu,zn- SOD requires insertion of copper and zinc and the formation of an intramolecular disulfide bond between cysteine-57 and cysteine-146. While the mechanism of zinc insertion remains unknown, the copper chaperone for SOD1 (CCS) controls the insertion of copper, which is also necessary to activate Cu,Zn-SOD 28. However, in mammalian cells, a CCS-independent copper insertion and Cu,Zn-SOD activation pathway also exists 120. We found that there was no alteration of CCS in the mitochondria in cells exposed to asbestos (data not shown), suggesting activation of Cu,Zn-SOD was CCS-independent. Intramolecular disulfide bond formation between the thiol groups of the Cu,Zn- SOD monomer is also necessary for the activation of Cu,Zn-SOD 26, and our data demonstrate that the disulfide bond formation is regulated by the redox environment of the IMS. Complex III is the major ROS production site among all mitochondrial ETC complexes, and it is the only complex that generates O - 2 in the IMS 121. The Rieske protein is one of the four redox centers in complex III that plays an important role of transferring electrons from ubiquinol in cytochrome b to the heme group in cytochrome c 1. Inhibition of Rieske protein by myxothiazol has been shown to reduce the extracellular H 2 O 2 level in intact heart mitochondria 122. Our data, however, demonstrate that knockdown of the Rieske protein inhibits Cu,Zn-SOD translocation and activation, which underscores a critical functional role of the mitochondrial ETC and redox environment for Cu,Zn-SOD uptake and subsequent activation in the IMS. In addition, although at least one of the conserved cysteine residues is required for translocation, both Cys 57 and Cys 146 are necessary for Cu,Zn-SOD activation.

56 41 Other disorders, such as Down s syndrome and amyotrophic lateral sclerosis, which have either increased or altered Cu,Zn-SOD activity, exhibit oxidative stress that is linked to disease development 123,124. Our data demonstrate that WT mice have greater lipid peroxidation in BAL fluid and oxidized GSH in lung tissue than Cu,Zn-SOD -/- mice, suggesting H 2 O 2 generated by Cu,Zn-SOD results in increased oxidative stress in the lung. Our data demonstrate that decreased mitochondrial H 2 O 2 generation results in decreased collagen deposition and supports our notion that Cu,Zn-SOD-mediated H 2 O 2 generation is, in part, responsible for the development of pulmonary fibrosis after exposure to asbestos. These observations provide a potential target that could protect against the development of a prototypical form of pulmonary fibrosis.

57 42 Figure 2-1: Alveolar macrophages from asbestos patients produce high levels of H 2 O 2. Alveolar macrophages from asbestosis patients and normal volunteers were incubated in HBSS supplemented with phpa and HRP. Extracellular H 2 O 2 concentrations were measured spectrofluorometrically after 15 min. *p< vs. normal subjects.by twotailed Mann-Whitney test (n=6 per each group).

58 Subjects 43

59 44 Figure 2-2: Asbestos patients have increased expression and activity of Cu,Zn-SOD in alveolar macrophages. A, SOD expression from asbestosis patients (n=3) and normal subjects (n=4) were measured by immunoblot analysis. B Mn-SOD activity and Cu,Zn-SOD activity of alveolar macrophages from asbestosis patients (n = 3) and normal subjects (n =4) were measured by native gel with nitroblue tetrazolium staining. C, Mn-SOD activity and D, Cu,Zn-SOD activity were expressed as densitometry. Results were expressed as the mean ± SEM. *p < 0.03 vs. normal subjects by two-tailed Mann-Whitney test.

60 45 A Normal Asbestosis Cu,Zn-SOD Mn-SOD -actin B Subjects Normal Asbestosis Mn-SOD Cu,Zn-SOD -actin C D

61 46 Figure 2-3: Cu,Zn-SOD in the mitochondrial intermembrane spaces contributes to the increased level of H 2 O 2 production. A, THP-1 monocytes were infected with a replication-deficient adenovirus vector expressing either an empty vector (Ad5. CMV) or Cu,Zn-SOD vector (Ad5.Cu,Zn-SOD) for 48 h. Extracellular H 2 O 2 levels were measured by applying phpa method with a kinetic protocol at indicated time. *p<0.05 Ad5.CMV vs.ad5.cu,zn-sod. Determinations were performed in quadruplicate and expressed as the mean ± SEM. Results were representative of three separate experiments. B, Different cellular fractions were isolated and immunoblot analysis for Cu,Zn-SOD was performed. In mitochondria, Cu,Zn-SOD is localized in the intermembrane space. C, THP-1 monocytes were transfected with either an empty vector or pcdna3.1cu,zn-sod-v5-his vector. After 24 h, 10 µg of mitochondrial (mito) and cytoplasmic (cyto) protein lysates were used for measuring superoxide anion generation utilizing Lucigenin assay. Determinations were performed in triplicate and expressed as the mean ± SEM. Results were representative of three separate experiments. *p<0.05 Empty vs. Cu,Zn-SOD-V5-His. D, Superoxide anion generation expressed as RLU/min. Results were expressed as the mean ± SEM. *p < 0.05 vs. Empty Vector by two-tailed student t test.

62 A 47 * * * B Cu,Zn-SOD Cell Lysate Mitochondria Cytoplasm IMS Mitoplast C * * * * * * D

63 48 Figure 2-4: Alveolar macrophages from asbestosis patients have increased mitochondrial Cu,Zn-SOD. Mitochondria and cytoplasm were isolated from alveolar macrophages obtained from normal subjects (n = 3) and asbestosis patients (n = 3). A, A representative immunoblot analysis for Cu,Zn-SOD in mitochondria. B, A representative immunoblot analysis for BiP and VDAC in the mitochondrial fractionations. C, A representative immunoblot analysis for Cu,Zn-SOD in cytoplasm in alveolar macrophages. D, Membrane and mitochondria were isolated from alveolar macrophages from asbestosis patients and normal subjects. Membrane and mitochondrial protein lysates were used for measuring superoxide anion generation utilizing lucigenin assay. The superoxide generation rate (RLU s -1 ) was measured in both mitochondrial and membrane fractions. n = 3, Results were expressed as the mean ± SEM. ** p < 0.05 vs. asbestosis membrane. ** p < 0.05 vs. asbestosis mitochondria by one-way ANOVA with Tukey post-analysis test.

64 A Subjects Normal Asbestosis 49 Cu,Zn-SOD VDAC mitochondria B Subjects Normal Asbestosis BiP VDAC mitochondria ER C Subjects Normal Asbestosis Cu,Zn-SOD GAPDH cytoplasm D

65 50 Figure 2-5: Asbestos induced translocation of Cu,Zn-SOD into the mitochondrial IMS in macrophages. A, THP-1 monocytes were transfected with Cu,Zn-SOD-V5-His vector for 24 h and then incubated in the presence or absence of chrysotile asbestos. Mitochondrial fractions were isolated and immunoblot analysis was performed for V5. B, THP-1 monocytes were transfected with phmgfp-cu,zn-sod vector for 24 h and then incubated in the presence or absence of chrysotile asbestos. Cells were then suspended in HBSS with 500 nm Mitotracker Red at 37 C for 30 minutes and confocal microscopy was performed. C, MLE-12 alveolar type II epithelial cells were transfected with Cu,Zn-SOD-V5-His vector for 24 h and then incubated in the presence or absence of chrysotile. Mitochondrial fractions were isolated and immunoblot analysis was performed for V5. Exposure to chrysotile asbestos was for 3 h in (A-C).

66 51 A THP-1 cells - + Chrysotile V5 VDAC B Control GFP Mitotracker Red Merge Chrysotile C MLE cells - + Chrysotile V5 VDAC

67 52 Figure 2-6: Mitochondrial Cu,Zn-SOD translocation and activation requires conserved cysteines. A, THP-1 monocytes were transfected with Cu,Zn-SOD-V5-His mutants (C57S, C146S, or C57,146S) for 24 h and then exposed to chrysotile asbestos. Cytoplasmic fractions were isolated and separated by SDS-PAGE. B, THP-1 monocytes were transfected with Cu,Zn-SOD-V5-His mutants (C57S, C146S, or C57,146S). Mitochondrial fractions were isolated and immunoblot analysis was performed for V5. C, THP-1 monocytes were transfected with either an empty vector, Cu,Zn-SOD-V5-His (Wildtype), or Cu,Zn-SOD- V5-His mutants (C57S, C146S, and C57,146S) for 24 h and then exposed to chrysotile asbestos. H 2 O 2 generation was measured with phpa assay. Results were expressed as the mean ± SEM and were representative of three separate experiments. *p<0.05 Wildtype vs. Empty, **p<0.05 Wildtype vs. C57S, C146S, or C57,146S by one-way ANOVA with Tukey post-analysis test. Exposure to chrysotile asbestos was for 3 h in (A- C).

68 53 A C57S Cytoplasm C146S C57, 146S V5 -actin B Mitochondria C57, C57S C146S 146S _ + _ + _ + Chrysotile V5 Ponceau C

69 54 Figure 2-7: Translocation and activation of Cu,Zn-SOD is redox sensitive. A, THP-1 monocytes were transfected with either Rieske sirna or a scrambled sirna together with either an empty or Cu,Zn-SOD-V5-His vector for 72 hours and then exposed to chrysotile asbestos. H 2 O 2 generation was measured utilizing phpa method with a kinetic protocol at indicated time. Determinations were performed in quadruplicate and expressed as the mean ± SEM. Results were representative of three separate experiments. B, Rate of H 2 O 2 generation expressed as nmoles/10 6 cells/min. n=3. Results were expressed as the mean ± SEM, *p<0.01 vs. Empty, **p<0.01 vs. Empty by one-way ANOVA with Tukey post-analysis test. C, THP-1 monocytes were transfected with either Rieske sirna or a scrambled sirna for 72 hours and then incubated with chrysotile asbestos for 3 hours. Mitochondrial fraction were isolated and separated by SDS-PAGE with or without β-me. Ox=activated (oxidized) disulfide bond-linked Cu,Zn-SOD. Red=reduced Cu,Zn-SOD monomer.

70 A ** ** 55 ** * * * B sirna Scr R Rieske (R) -actin C Ox Cu,Zn-SOD Red sirna Scr Rieske Scr Rieske -ME VDAC

71 56 Figure 2-8: Cu,Zn-SOD modulates asbestos-induced oxidative stress in vivo. A, Macrophages and monocytes isolated from WT mice (n=7) and Cu,Zn-SOD -/- mice (n=7) bone marrow. Cells were cultured in the presence or absence of chrysotile asbestos. H 2 O 2 generation was measured by phpa assay with a kinetic protocol at indicated time. Determinations were expressed as the mean ± SEM. B, Rate of hydrogen peroxide generation is expressed in nmoles/10 6 cells/min. *p < 0.05 vs. Cu,Zn-SOD -/- control, **p < 0.01 vs. Cu,Zn-SOD -/- mice exposed to chrysotile asbestos by one-way ANOVA with Tukey post-analysis test. C, WT mice and Cu,Zn-SOD -/- mice were exposed to 100 g chrysotile asbestos intratracheally. Animals were euthanized after 21 days, and BAL fluid was obtained. Oxidized lipid is expressed as M of malondialdehyde (MDA)/ g protein in BAL fluid. WT (n=4) and Cu,Zn-SOD -/- (n=4), results were expressed as the mean ± SEM, *p < vs. WT mice by two-tailed student t test. D, Mice were exposed to chrysotile asbestos as in C. After 21 days the mice were euthanized and lungs were removed and homogenized for glutathione assay. Total GSH in disulfide form expressed as % GSH as GSSG. WT (n=3) and Cu,Zn-SOD -/- (n=3); results were expressed as the mean ± SEM, *p < 0.04 vs. WT by two-tailed student t test.

72 57 A B C D

73 58 Figure 2-9: Cu,Zn-SOD -/- mice were protected from developing pulmonary fibrosis after asbestos exposure. A, WT and B, Cu,Zn-SOD -/- mice were exposed to 100 µg chrysotile asbestos intratracheally. Twenty-one days later the animals were euthanized and lungs were removed and processed for collagen deposition using Masson s trichome staining. WT mice have peribronchial and parenchymal collagen deposition compared to Cu,Zn-SOD -/- mice. Representative micrographs of one out of 7 animals are shown. Bar indicates 200 µm. C, Total RNA was obtained from lung homogenates of mice exposed to chrysotile asbestos (100 g). Results show arbitrary units of collagen I mrna normalized to HPRT mrna. Cu,Zn-SOD -/- (n=6) and WT (n=4). Results were expressed as the mean ± SEM, *p < 0.01 vs. WT mice by two-tailed student t test. Inset, whole lung homogenates from WT (n=2) and Cu,Zn-SOD -/- (n=2) mice were separated by SDS-PAGE and immunoblot analysis for Mn-SOD and Cu,Zn-SOD was performed. D, Extracellular collagen deposition in lung of WT and Cu,Zn-SOD -/- mice after initial asbestos exposure was determined using hydroxyproline assay. Results were expressed as the mean ± SEM, *p < 0.03 vs. WT by two-tailed Mann-Whitney test. (n = 4 per each group).

74 59 A B WT C Cu,Zn-SOD -/- -/- WT Cu,Zn-SOD Mn-SOD Cu,Zn-SOD D

75 60 Figure 2-10: Cu,Zn-SOD alters pro-fibrotic cytokine TGF- production. A, Mice were exposed to chrysotile asbestos (100 g) intracheally. Twenty-one day later the animals were euthanized and BAL was performed. BAL cells were collected, and total RNA was obtained. Results show arbitrary units of TGF- mrna normalized to HPRT mrna. n=3 per strain. Results are expressed as the mean ± SEM. B, THP-1 cells were infected with either Ad5.CMV or Ad5.Cu,Zn-SOD. After 48 h, cell supernatants were collected and ELISA was performed. n=3, Results are expressed as the mean ± SEM, *p < 0.03 vs. WT by two-tailed student t test

76 61 A B

77 62 Figure 2-11: Knocking down Cu,Zn-SOD in macrophages decreases collagen production by fibroblasts. THP-1 cells were transfected with either a scrambled or Cu,Zn-SOD sirna (100 nm). After 72 h, conditioned medium was removed and placed on HLF-1 cells. HLF-1 cells were cultured for 24 h in the presence or absence of chrysotile asbestos. Procollagen I and collagen I in the medium was determined by immunoblot analysis.

78 63 Scrambled sirna Cu,Zn-SOD sirna THP-1 Conditioned Media Cu,Zn-SOD -Actin Procollagen I Chrysotile Collagen I

79 64 CHAPTER III CU,ZN-SOD-MEDIATED H 2 O 2 PROMOTES MACROPHAGE ALTERNATIVE ACTIVATION Abstract Macrophages not only initiate and accentuate inflammation after tissue injury, but they are also involved in resolution and repair. This difference in macrophage activity is the result of a differentiation process to either M1 or M2 phenotypes. M1 macrophages are pro-inflammatory and have microbicidal and tumoricidal activity, whereas the M2 macrophages are involved in tumor progression and tissue remodeling, and can be profibrotic in certain conditions. Because mitochondrial Cu,Zn-SOD-mediated H 2 O 2 is crucial for development of pulmonary fibrosis, we hypothesized that Cu,Zn-SOD modulated the macrophage phenotype. In these studies, we demonstrate that Cu,Zn-SOD polarized macrophages to an M2 phenotype. Furthermore, overexpression of Cu,Zn- SOD in mice resulted in a pro-fibrotic environment and accelerated the development of pulmonary fibrosis, while polarization of macrophages to the M1 phenotype attenuated pulmonary fibrosis. Taken together, these observations provide a novel mechanism of Cu,Zn-SOD-mediated M2 polarization and provide a potential therapeutic target for attenuating the accelerated development of pulmonary fibrosis. Introduction Pulmonary fibrosis is characterized by aberrant wound healing that leads to progressive deposition of collagen in extracellular spaces. Macrophages play an integral role in the pathogenesis of fibrosis by initiating an immune response and by generating reactive oxygen species 125. Macrophage plasticity is an important feature of these innate immune cells. Macrophage phenotypes are divided into two categories, the classically activated macrophages (CAM, M1 phenotype) and the alternatively activated

80 65 macrophages (AAM, M2 phenotype), depending on stimulation by Th1 or Th2 cytokines, respectively 126. M1 macrophages are commonly associated with the generation of proinflammatory cytokines, whereas M2 macrophages are anti-inflammatory and often associated with fibrotic conditions, including pulmonary fibrosis 127. M2 macrophages harbor a repertoire of pro-fibrotic signature changes, such as 1) increased expression of anti-inflammatory and pro-fibrotic immune effectors, including TGF- and C-C motif ligand 18 (CCL-18); 2) up-regulated L-arginine metabolism by arginase I instead of inos to generate polyamines and proline, an important precursor for collagen synthesis; and 3) increased resistin-like secreted protein, found in inflammatory zone (FIZZ1), and chitinase-like secretory lectin, Ym1, which are actively involved in extracellular matrix dynamics. Macrophages produce high levels of reactive oxygen species (ROS), including H 2 O 2, which has a critical role in the pathogenesis of pulmonary fibrosis. Superoxide anion (O - 2 ) reacts with superoxide dismutase (SOD) at a rate constant of 10 9 M -1 s -1, which is associated with H 2 O 2 generation. Among the three SOD enzymes, we have shown that the Cu,Zn-SOD (SOD1), which is located in the cytosol and mitochondrial intermembrane space (IMS), is increased in asbestosis patients and contributes to the increased mitochondrial H 2 O 2 production by alveolar macrophages. Furthermore, the lungs of Cu,Zn-SOD -/- mice have reduced oxidative stress and do not develop pulmonary fibrosis 46. These data suggest that Cu,Zn-SOD-mediated H 2 O 2 generation in alveolar macrophages is a critical factor in the pathogenesis of pulmonary fibrosis. We also had demonstrated that Cu,Zn-SOD overexpression increases TGF-. Cu,Zn-SOD -/- mice, which were protected from developing pulmonary fibrosis, had less TGF- compared with WT mice. Thus, understanding the effect of Cu,Zn-SOD-mediated H 2 O 2 generation in regulating alveolar macrophage polarization may uncover a mechanism by which H 2 O 2 levels mediate pulmonary fibrosis.

81 66 Methods and Materials Materials Chrysotile asbestos was provided Dr. Peter S. Thorne, University of Iowa College of Public Health. p-hydroxylphenyl acetic acid (phpa), polyethylene glycol-conjugated catalase (PEG-CAT), PEG-SOD, rotenone, antimycin A, and horseradish peroxidase (HRP) were purchased from Sigma-Aldrich (St. Louis, MO). Mice Wild-type (WT), Cu,Zn-SOD -/-, and Cu,Zn-SOD Tg (a generous gift from Dr. Donald Heistad, University of Iowa, Iowa City, IA) C57BL/6 mice were used in these studies, and all protocols were approved by the University of Iowa Institutional Animal Care and Use Committee. Mice were intratracheally administered 100 µg of chrysotile asbestos suspended in 50 µl 0.9% saline solution after being anesthetized with 3% isoflurane using a precision Fortec vaporizer (Cyprane, Keighley, UK). Mice were euthanized at the designated day after exposure with an overdose of isoflurane, and bronchoalveolar lavage (BAL) was performed. BAL cells were used for differential cell number. Alveolar macrophages were isolated from BAL fluid and cultured in RPMI medium supplemented with 10% fetal bovine serum. Bone marrow cells were isolated and incubated in L929 cell-conditioned media for 7 days to generate monocytes/macrophages. Cell culture Human monocyte (THP-1) and mouse alveolar macrophage (MH-S) cell lines were obtained from American Type Culture Collection (Manassas, VA). Cells were maintained in RPMI-1640 media with following supplements: 10% fetal bovine serum and penicillin/streptomycin. All experiments were performed with 0.5% serum supplement.

82 67 Hydroxyproline Determination Lung tissue was digested for 24 h at 112 C with 6 N hydrochloric acid. Hydroxyproline concentration was determined as previously described 95. Quantitative real-time PCR Total RNA were obtained using Trizol reagent (Sigma). After reverse transcription using iscript reverse transcription kit (Bio-Rad Laboratories, Hercules, CA), M1 and M2 marker gene mrna expression was determined by quantitative real-time PCR using SYBR Green kit. Immunohistochemistry Lung sections were de-paraffinized and rehydrated. Sections were H 2 O 2 quenched, blocked, and incubated overnight with goat anti-arginase I (Santa Cruz Biotechnology, CA) at 4 C. Tissues were sequentially incubated with the donkey antigoat IgG biotinylated secondary antibody, streptavidin-hrp, and 3,3-diaminobenzidine tetrahydrochloride (DAB) at room temperature. ELISA Active TGF-, TNF-, IL-1, IL-4, IL-13, and MIP-2 in BAL fluid and cell media was measured by ELISA (R&D, Minneapolis, MN), according to manufacturer s instructions. Arginase activity assay Arginase activity was measured by using a QuantiChrom Arginase Assay Kit (BioAssay System, Hayward, CA), according to manufacturer s instructions.

83 68 NO measurement Cellular generation of NO was determined from the accumulation of nitrite in culture media. Nitrite is the main oxidation product found in vitro due to the extracellular oxidation of NO by O 2. The accumulated nitrite in the cell culture media was determined with a Sievers 280i Nitric Oxide Analyzer (GE Analytical Instruments, Boulder, CO) using 1% potassium iodide in glacial acetic acid to reduce NO - 2 to NO. Concentration of NO was determined by chemiluminescence. Statistical analysis Statistical comparisons were performed as indicated in figure legend. Values in figures are expressed as means with standard errors and p < 0.05 was considered to be significant. Results Alveolar macrophages from Cu,Zn-SOD -/- mice have an M1 phenotype To investigate the role of Cu,Zn-SOD in modulating macrophage phenotype, we questioned whether alveolar macrophages from Cu,Zn-SOD -/- mice, which do not develop pulmonary fibrosis and have decreased TGF- in BAL fluid, had a predominant M1 phenotype compared to WT mice. WT and Cu,Zn-SOD -/- mice were euthanized 21 days after asbestos exposure, and bronchoalveolar lavage was performed. Proinflammatory cytokine levels in BAL fluid were measured. Cu,Zn-SOD -/- mice had significantly higher levels of M1 markers, such as IL-1, MIP-2, and TNF-, in BAL fluid compared to WT mice (Figure 3-1). These data suggest that alveolar macrophages from Cu,Zn-SOD -/- mice are primarily M1 macrophages, which may contribute to their protection from developing pulmonary fibrosis.

84 69 Cu,Zn-SOD provides additional signals to promote macrophage polarization Because alveolar macrophages from Cu,Zn-SOD -/- mice are primarily an M1 phenotype compared to WT mice, we investigated whether these changes were related to differences in Th2 cytokine levels in the lung. The concentrations of two prominent Th2 cytokines, IL-4 and IL-13, in BAL fluid were similar in WT and Cu,Zn-SOD -/- mice (Figure 3-2A). Taken together, these data demonstrate that alveolar macrophages from Cu,Zn-SOD -/- mice have a pro-inflammatory, or M1, phenotype and suggest that this phenotype is not due to an alteration in the level of Th2 cytokines. Furthermore, these data suggest that the absence of Cu,Zn-SOD has no effect on the expression of Th2 cytokines. Because macrophage polarization was different in WT and Cu,Zn-SOD -/- mice but both had similar levels of Th2 cytokines, we postulated that Cu,Zn-SOD might provide supplementary signals to initiate M2 polarization. WT and Cu,Zn-SOD Tg mice, which have more than three-fold increase of Cu,Zn-SOD protein expression and activity 128, were used to investigate the hypothesis that Cu,Zn-SOD mediates M2 polarization. To support the notion that Cu,Zn-SOD can induce alternative activation of macrophages, we incubated macrophages from WT and Cu,Zn-SOD Tg mice with BAL fluid collected from asbestos-exposed WT mice to investigate whether other factors contributed to macrophage polarization. Macrophages from Cu,Zn-SOD Tg mice had increased arginase I and FIZZ1 and decreased TNF- and IL-1 compared to those of WT mice in the presence of BAL fluid (Figure 3-2B), which suggests that Cu,Zn-SOD is responsible for modulating the macrophage phenotype. Cu,Zn-SOD overexpression induces macrophage M2 polarization To determine if M2 alveolar macrophages were the predominant macrophage phenotype in BAL fluid in Cu,Zn-SOD Tg mice, WT and Cu,Zn-SOD Tg mice were exposed to chrysotile asbestos. The lungs were excised and immunohistochemistry for

85 70 the M2 marker, arginase I, was performed. The majority of macrophages in the lungs from Cu,Zn-SOD Tg mice stained for arginase I compared to the limited number in WT mice (Figure 3-3A and 3-3B). Because Cu,Zn-SOD Tg mice had a predominant M2 phenotype in their lungs compared to WT mice, we investigated whether this difference in macrophage polarization was transient or sustained after asbestos exposure. WT and Cu,Zn-SOD Tg mice were exposed to chrysotile asbestos and euthanized on 10, 15, or 21 days after exposure. Alveolar macrophages were incubated in vitro with asbestos for 4 h, and M1 and M2 gene expression were measured. At each time point, Cu,Zn-SOD Tg mice had significantly greater expression of the M2 markers, arginase I and FIZZ1, and decreased expression of the M1 marker, TNF-, compared to WT mice, suggesting that Cu,Zn- SOD induced an M2 phenotype polarization (Figure 3-3C, 3-3D and 3-3F). The metabolic utilization of L-arginine differs in M1 and M2 macrophages, so we determined if Cu,Zn-SOD modulated this metabolic distinction. Macrophages were exposed to chrysotile asbestos, and nitrite production was measured. Asbestos increased nitrite concentration in macrophages from both WT and Cu,Zn-SOD Tg mice; however, the nitrite produced from Cu,Zn-SOD Tg macrophages was significantly less compared with that of WT mice in both the presence and absence of chrysotile (Figure 3-4A). In contrast, Cu,Zn-SOD overexpression dramatically altered L-arginine metabolism by significantly increasing arginase activity as measured by urea generation (Figure 3-4B). In aggregate, these data suggest that Cu,Zn-SOD modulates metabolism of L-arginine in a manner characteristic of the M2 phenotype. Cu,Zn-SOD-mediated H 2 O 2 modulates macrophage polarization Because the primary enzymatic activity of Cu,Zn-SOD is the dismutation of O 2 - into H 2 O 2, we questioned whether H 2 O 2 levels contributed to macrophage polarization. To examine the role of Cu,Zn-SOD-mediated H 2 O 2 on macrophage polarization, we

86 71 cultured macrophages from WT and Cu,Zn-SOD Tg mice in the presence or absence of PEG-CAT (100 U/mL) and measured H 2 O 2 generation. Macrophages treated with PEG- CAT had lower H 2 O 2 levels, similar to the level in WT macrophages. The rate of H 2 O 2 generation was nearly 5-fold less in cells cultured with PEG-CAT (Figure 3-5A). We next determined the effect of H 2 O 2 generation on M1/M2 gene expression. Cu,Zn-SOD Tg macrophages treated with PEG-CAT, which produced less H 2 O 2, had decreased Ym1 and FIZZ1 gene expression and augmented TNF- and inos gene expression (Figure 3-5B). To further investigate the effect of Cu,Zn-SOD on H 2 O 2 generation, alveolar macrophages from WT and Cu,Zn-SOD -/- mice incubated in the presence or absence of PEG-SOD (100 U/mL). PEG-SOD significantly increased H 2 O 2 levels compared to Cu,Zn-SOD -/- macrophages alone (Figure 3-5C). These data demonstrate the importance of Cu,Zn-SOD in regulating the redox state of macrophages. Since reducing H 2 O 2 levels decreased M2 gene expression, we investigated whether increasing H 2 O 2 would restore M2 gene expression. Cu,Zn-SOD -/- alveolar macrophages treated with PEG-SOD had significantly increased Ym1 and FIZZ1 gene expression, whereas it abolished M1 gene expression, as measured by TNF- and inos (Figure 3-5D). These observations suggest that Cu,Zn-SOD-mediated H 2 O 2 is a critical determinant of the macrophage phenotype. Cu,Zn-SOD-mediated M2 polarization accelerates the development of pulmonary fibrosis To determine the biological relevance of Cu,Zn-SOD-mediated alternative activation of macrophages in the pathogenesis of pulmonary fibrosis, we exposed WT and Cu,Zn-SOD Tg mice to chrysotile asbestos. The animals were euthanized 10, 15, and 21 days after exposure, and lungs were excised and processed for staining with Masson s trichrome to assess collagen deposition. The lungs of Cu,Zn-SOD Tg mice had collagen deposition in the peribronchial portions of the lung at day 10, whereas there was no

87 72 evidence of collagen deposition in the lungs of WT mice (Figure 3-6A). The fibrotic lesions progressed dramatically in a time-dependent manner in Cu,Zn-SOD Tg mice with parenchymal collagen deposition, while the lungs of WT mice showed small areas of peribronchial collagen at 15 days (Figure 3-6B). At 21 days after asbestos exposure, the lungs of Cu,Zn-SOD Tg mice revealed dense fibrosis in the majority of the lung (Figure 3-6C). In contrast, the WT mice had some peribronchial and parenchymal collagen deposition, but it was not nearly as wide-spread as seen in the Cu,Zn-SOD Tg mice. To further verify the histopathological observations, we performed a hydroxyproline assay to determine the extent of pulmonary fibrosis biochemically. Hydroxyproline levels in the lung were significantly higher in Cu,Zn-SOD Tg mice compared with WT mice, indicating increased collagen deposition and more extensive pulmonary fibrosis (Figure 3-7A). Because fibroblasts are the main source of collagen production and to provide a direct link between Cu,Zn-SOD and pulmonary fibrosis, we treated primary WT mouse lung fibroblasts with conditioned media from either WT or Cu,Zn-SOD Tg macrophages in the presence of asbestos overnight. Total RNA were isolated from mouse lung fibroblasts and collagen I mrna was measured. Mouse lung fibroblasts exposed to conditioned media from Cu,Zn-SOD Tg macrophages had a significant increase in collagen I gene expression compared with fibroblasts treated with conditioned media from WT macrophages (Figure 3-7B). TGF- is an important marker of M2-phenotype macrophages, and its expression in the activated form is linked to the pathogenesis of fibrosis. We measured the level of activated TGF- in BAL fluid from WT and Cu,Zn-SOD Tg mice. Active TGF- was significantly higher in Cu,Zn-SOD Tg compared to WT mice, indicating a dominant profibrotic environment and M2 macrophage polarization (Figure 3-7C). In aggregate, these data demonstrate that Cu,Zn-SOD induced macrophage M2 polarization and accelerated the development pulmonary fibrosis by increasing fibroblast collagen production.

88 73 Alveolar macrophages from asbestosis patients have an M2 phenotype Because M2 macrophages are associated with fibrogenesis, we investigated whether alveolar macrophages from asbestosis patients have a predominant M2 phenotype. Alveolar macrophages were obtained from normal subjects and patients with asbestosis, and mrna was isolated. The M2 genes, CCL-18 (Figure 3-8A) and surface mannose receptor (Figure 3-8B), were significantly increased in macrophages from asbestosis patients compared to normal volunteers, suggesting M2 polarization in alveolar macrophages obtained from the patients. Based on these novel observations, we formulated the hypothesis that Cu,Zn-SOD induces pulmonary fibrosis, in part, by promoting the early and sustained alternative activation of macrophages, which accelerates pro-fibrotic cytokine generation and collagen synthesis by modulation of H 2 O 2 generation. Discussion Macrophages have a critical role in both innate and adaptive immune responses. Macrophages not only initiate and accentuate inflammatory responses after tissue injury, but they are also involved in resolution and repair of the inflammation and injury. This distinction in macrophage activity is the result of a differentiation process that leads to a predominant pro-inflammatory M1 phenotype (classically activated) or an antiinflammatory M2 (alternatively activated) phenotype. The M2 macrophages are also considered pro-fibrotic in certain conditions. Aberrant healing often occurs when an imbalance in the macrophage phenotype is present with a dominant M2 polarization. This imbalance can occur in multiple tissues, including the lung during the development of pulmonary fibrosis. Because Cu,Zn-SOD mediated H 2 O 2 is crucial for the fibrogenic process in asbestos-induced pulmonary injury, we hypothesized that Cu,Zn-SOD modulated the macrophage phenotype. In this study, we demonstrate that overexpression of Cu,Zn-SOD polarized macrophages to an M2 phenotype. Furthermore, modulating the

89 74 redox level of the cell altered the macrophage phenotype. Evidence to support this mechanism include i) Cu,Zn-SOD -/- macrophages have increased M1 markers compared to WT macrophages; ii) Cu,Zn-SOD Tg macrophages polarized toward an M2 phenotype compared to WT macrophages; iii) an increase in H 2 O 2 levels by mitochondrial-localized Cu,Zn-SOD shifts the polarization pattern to an M2 phenotype; iv) overexpression of Cu,Zn-SOD leads to a pro-fibrotic environment and accelerates development of the fibrotic phenotype in vivo. These observations provide novel insight into the mechanism orchestrating macrophage activation and signify the importance of alternatively activated macrophages in pulmonary fibrosis. Furthermore, understanding the molecular mechanism(s) that result in and accelerate fibrotic repair offers a target for potential therapeutic intervention. Studies have linked the role of Th2 cytokines and alternatively activated macrophages in fibrotic disease development 129,130 ; however, it is not known whether any additional pathway can lead to the development of fibrosis. The development of granulomas from S. mansoni exposure was not impaired in IL-4-deficient mice 131,132, although other Th2 cytokines remained elevated. In addition, wound macrophages are known to undergo alternative activation despite a deficiency of Th2 cytokines in the wound environment, and the macrophage phenotype was sustained in mice lacking IL-4R 75. It is not clear from this study what induced the alternative activation. Our data are the first to demonstrate a molecular mechanism that mediates alternative activation of macrophages in a redox dependent manner, which is contingent on the redox state of the cell. Oxidative stress has long been known to play an important role in the development and progression of pulmonary fibrosis. Pro-inflammatory M1 genes, such as TNF-, IL-1, and inos, have all been shown to be regulated by redox proteins, including Cu,Zn-SOD Recently, Ym1 and FIZZ1 have emerged as oxidative stress proteins in pulmonary disease 136 ; however, the involvement of the redox status in

90 75 modulating the macrophage phenotype is poorly understood. Previous studies have shown that increases in the oxidative metabolic environment fuels alternative activation of macrophages 137, while others show that M2 macrophages generate low levels of ROS 138. No study has shown that the redox status of the macrophage can directly regulate the macrophage phenotype. Circulating M2 cells accelerate the pathological progression of amyotropic lateral sclerosis (ALS), a disease characterized with aberrant Cu,Zn-SOD function and excessive oxidative stress 139. One study demonstrated that increasing Mn- SOD activity in mitochondria enhanced electron transfer to oxygen to form O 2 - that is reduced to H 2 O ; however, the ability of increasing Cu,Zn-SOD levels to facilitate higher H 2 O 2 generation is not known. Our study is the first to demonstrate that Cu,Zn- SOD, the redox protein that catalyzes the generation of H 2 O 2, polarizes macrophages to an M2 phenotype. Moreover, Cu,Zn-SOD-mediated macrophage polarization can be altered by modulating H 2 O 2 generation, which suggests one mechanism by which oxidative stress is linked to pulmonary fibrosis. Differential metabolism of L-arginine is characteristic of M1 and M2 macrophages 65. We found that overexpression of Cu,Zn-SOD leads to a reduction of inos gene expression and NO synthesis, while arginase I expression and urea generation was enhanced. The L-arginine metabolic pattern is closely associated with collagen synthesis and fibrosis development. Overexpression of Cu,Zn-SOD has been shown to inhibit inos expression in endothelial cells in a subarachnoid hemorrhage model 141 ; however, arginase I expression and urea production were not determined. Furthermore, a steady state of NO greater than 30 nm will react with lipid peroxyl radicals near a diffusion limitation rate, and will have potent inhibitory effects on lipid peroxidation 142. We have shown that BAL fluid from Cu,Zn-SOD -/- mice have significantly less lipid oxidation, underscoring the potential role of NO in inducing lipid oxidation in vivo. These observations suggest that Cu,Zn-SOD Tg mice had early and sustained M2 gene expression and pro-fibrotic factor synthesis, which accelerated fibrosis development.

91 76 These observations provide potential therapeutic targets that are modulated by oxidative stress. M2 macrophages are known to be prevalent in the lungs of patients with idiopathic pulmonary fibrosis, sarcoidosis, and systemic sclerosis 84. We found that alveolar macrophages obtained from asbestosis patients have higher M2 macrophage markers compared with normal volunteers, and CCL-18 is known to induce lung fibroblast collagen production 143. In support of the fact that alveolar macrophages from asbestosis patients have increased Cu,Zn-SOD activity and generate higher levels of H 2 O 46 2, we also found in patient with asbestosis that the source of ROS generation was the mitochondria, that Cu,Zn-SOD was localized in the mitochondria of alveolar macrophages, and that these alveolar macrophages have an M2 phenotype. Furthermore, overexpression of Cu,Zn-SOD resulted in a dominant M2 phenotype, which accelerates fibrogenesis in a murine model of asbestos-induced pulmonary fibrosis. Cu,Zn-SOD increased expression of active TGF- and modulated intracellular L-arginine metabolism to generate proline, both of which facilitate development of pulmonary fibrosis. In aggregate, these observations provide evidence that Cu,Zn-SOD mediates pulmonary fibrosis, in part, via the early and sustained alternative activation of macrophages. Thus, intervening in this pathway may serve to prevent the development and/or progression of pulmonary fibrosis.

92 77 Figure 3-1: Cu,Zn-SOD -/- macrophages have increased M1 markers. WT and Cu,Zn-SOD -/- mice were exposed to 100 µg chrysotile asbestos intratracheally. IL-1 MIP-2, and TNF- cytokine levels were measured in BAL fluid of WT and Cu,Zn-SOD -/- mice 21 days after asbestos exposure. Results were expressed as the mean ± SEM, *p < 0.05 vs. WT. (n = 6 per each group) by two-tailed student t test

93 78

94 79 Figure 3-2: Cu,Zn-SOD induces macrophage M2 polarization A, WT and Cu,Zn-SOD -/- mice were exposed to 100 µg chrysotile asbestos intratracheally. IL-4 and IL-13 cytokine levels were measured in BAL fluid of WT and Cu,Zn-SOD -/- 21 days after initial asbestos exposure (n = 11 per each group). Results were expressed as the mean ± SEM and analyzed by two-tailed Mann-Whitney test. B, Macrophages isolated from WT and Cu,Zn-SOD Tg were cultured in BAL fluid from WT mice after asbestos exposure in the presence of chrysotile asbestos overnight. Total RNA was isolated, and TNF-, IL-1, arginase I, and FIZZ1 gene expression were measured. Results show arbitrary units normalized to -actin mrna. *p < 0.05 vs. WT. n = 3 per each group. Results were expressed as the mean ± SEM and analyzed by two-tailed student t test.

95 80 A BAL Fluid B WT BAL Fluid

96 81 Figure 3-3: Cu,Zn-SOD induces macrophage M2 polarization in vivo. WT and Cu,Zn-SOD Tg mice were exposed to 100 µg chrysotile asbestos intratracheally. Lung sections of both A, WT and B, Cu,Zn-SOD Tg were processed for arginase I immunohistochemistry staining. Representative micrographs of one out of 3 animals per genotype are shown. Magnification 10x, bar indicates 100 µm. Macrophages isolated from WT and Cu,Zn-SOD Tg were cultured in the presence or absence of chrysotile asbestos overnight. C, ten, D, fifteen, or E, twenty-one days after asbestos exposure the animals were euthanized and alveolar macrophages were isolated by BAL and exposed in vitro to chrysotile for 4 h. Total RNA was isolated, and TNF-, arginase I, and FIZZ1 gene expression were measured. Results show arbitrary units normalized to -actin mrna. *p < 0.05 vs. WT. (n = 4 per each group). Results were expressed as the mean ± SEM and analyzed by two-tailed student t test..

97 82 A WT B Cu,Zn-SOD Tg C D E

98 83 Figure 3-4: Cu,Zn-SOD induces macrophage M2-pattern metabolic changes. A, Nitrite concentration was measured in cell culture medium. B, Cell lysates were used to determine arginase activity by measuring urea synthesis and is expressed as U/L of sample. U represents 1 unit of arginase that converts 1 µmole of L-arginine to urea per minute. *p < 0.05 vs. Cu,Zn-SOD Tg. (n = 4 per each group). Results were expressed as the mean ± SEM and analyzed by one-way ANOVA with Tukey post-analysis test.

99 84 A B

100 85 Figure 3-5: Cu,Zn-SOD modulates M1/M2 polarization via H 2 O 2 levels. A, Macrophages isolated from WT and Cu,Zn-SOD Tg mice were cultured in the presence or absence of 100 U/mL PEG-CAT overnight and then treated with chrysotile asbestos. Extracellular H 2 O 2 level was measured by phpa assay at indicated time (left). Rate of H 2 O 2 generation is expressed in nmoles/10 6 cells/min (right). (n = 3) Determinations were performed in quadruplicate and expressed as the mean ± SEM analyzed by one-way ANOVA with Tukey post-analysis test. *p < 0.05 Cu,Zn-SOD Tg vs. WT, **p < 0.05 Cu,Zn-SOD Tg +PEG-CAT vs. Cu,Zn-SOD Tg. B, Total RNA from macrophages was isolated and TNF-, inos, FIZZ1, and Ym1 gene expression were measured. Results show arbitrary units normalized to -actin mrna. (n = 4) *p < 0.05 vs. Cu,Zn-SOD Tg. Results were expressed as the mean ± SEM by two-tailed student t test. C, macrophages isolated from WT and Cu,Zn-SOD -/- mice were cultured in the presence or absence of 100 U/mL PEG-SOD for 3 h and then treated with chrysotile asbestos. Extracellular H 2 O 2 level was measured by phpa assay at indicated time (left). Rate of H 2 O 2 generation is expressed in nmoles/10 6 cells/min (right). (n=3). Determinations were performed in quadruplicate and expressed as the mean ± SEM analyzed by one-way ANOVA with Tukey post-analysis test. *p < 0.05 Cu,Zn-SOD -/- vs. WT, **p < 0.05 Cu,Zn-SOD -/- +PEG-SOD vs. Cu,Zn-SOD -/-. D, Total RNA from macrophages was isolated and TNF-, inos, FIZZ1, and Ym1 gene expression were measured. Results show arbitrary units normalized to -actin mrna. (n=4) *p<0.05 vs. Cu,Zn-SOD -/-. Results were expressed as the mean ± SEM by two-tailed student t test.

101 86 A B C D

102 87 Figure 3-6: Cu,Zn-SOD-mediated M2 polarization accelerates pathogenesis of asbestosinduced pulmonary fibrosis in vivo. WT and Cu,Zn-SOD Tg mice were exposed to 100 µg chrysotile asbestos intratracheally. A, Ten, B, Fifteen, and C, Twenty-one days later the animals were euthanized and lungs were removed and processed for collagen deposition using Masson s trichrome staining. Representative micrographs of one out of 5 animals per genotype at each time point are shown. Magnification 4x, bar indicates 500 µm.

103 88 A 10 day WT Cu,Zn-SOD Tg B 15 day C 21 day

104 89 Figure 3-7: Cu,Zn-SOD-mediated M2 polarization promotes pulmonary fibrosis. A, Extracellular collagen deposition in lung of WT and Cu,Zn-SOD Tg mice twenty-one days after asbestos exposure were determined using hydroxyproline assay. *p < 0.05 vs. WT. (n = 3 per each group). Results were expressed as the mean ± SEM and analyzed by two-tailed student t test. B, WT mouse lung fibroblasts were treated with conditioned media from either WT or Cu,Zn-SOD Tg macrophages overnight in the presence of asbestos. Total RNA from macrophages was isolated, and collagen I gene expression was measured. Results show arbitrary units normalized to -actin mrna. (n = 3) *p < 0.01 vs. WT. Results were expressed as the mean ± SEM and analyzed by two-tailed student t test. C, Active TGF- in BAL fluid was measured. (n = 4 per each group) *p < 0.02 vs. WT. Results were expressed as the mean ± SEM by two-tailed student t test

105 90 A B C

106 91 Figure 3-8: Alveolar macrophages from asbestosis patients have an M2-phenotype. Total RNA of alveolar macrophages from asbestosis patients and normal volunteers were isolated and A, CCL-18 and B, Mannose receptor gene expression were measured. Results show arbitrary units normalized to HPRT mrna. *p < 0.05 vs. normal subjects. (n = 3). Results are expressed as the mean ± SEM and analyzed by two-tailed Mann- Whitney test.

107 92 A B

108 93 CHAPTER IV CU,ZN-SOD MEDIATES MACROPHAGE M2 POLARIZATION VIA REDOX REGULATION OF STAT6 Abstract M2 macrophages are implicated in the development of pulmonary fibrosis as they generate pro-fibrotic cytokines and growth factors. In this study, we demonstrate that Cu,Zn-SOD-mediated H 2 O 2 levels modulate macrophage M2 polarization by redox regulation of a critical cysteine in STAT6. The polarization process, at least in part, was regulated by epigenetic modulation. We show that STAT6 is indispensable for Cu,Zn- SOD-mediated M2 polarization. STAT6 upregulated the jumonji domain containing 3 (Jmjd3), a histone H3 lysine 27 demethylase, and initiated M2 gene transcriptional activation. Targeting STAT6 with leflunomide, which reduces cellular ROS production and inhibits STAT6 phosphorylation, abolished M2 polarization and ameliorated the fibrotic development. Taken together, these observations provide evidence of Cu,Zn- SOD-STAT6-Jmjd3 pathway as a novel regulatory mechanism for M2 polarization, and leflunomide may be used as a potential therapeutic agent in the treatment of pulmonary fibrosis. Introduction M2 phenotype macrophages contribute to the fibrotic pathology by increasing the generation of pro-fibrotic immune factors, collagen synthesis substrates, such as proline, and molecules implicated in extra-cellular matrix dynamics 144. We have shown that Cu,Zn-SOD-mediated H 2 O 2 signal can promote M2 polarization, providing a secondary pathway for macrophage alternative activation 145. The regulation of Th2 cytokine-induced M2 gene expression occurs, in part, at the transcriptional level by the activation of STAT DNA binding and transcriptional activity of STAT6 requires the Src homology domain 2 (SH2) after IL-4 stimulation 147.

109 94 The relationship between Cu,Zn-SOD and STAT6, however, is not known. Furthermore, redox regulation of STAT6 has not been investigated. Recent observations suggest that epigenetic modulations may be critical for the sustained phenotype of differentiated macrophages 148. Epigenetic regulations include 1) post-translational modifications of histones; 2) modifications of CpG islands; and 3) noncoding RNA 87. The methylation status of histones, dynamically regulated by sitespecific demethylases and methyltransferases, is important for M2 gene transcriptional initiation and suppression. The Jumonji C (JmjC) domain protein family is a group of histone demethylases that specifically demethylate their corresponding histone sites. In relation of macrophage polarization, the Jumonji domain containing 3 (Jmjd3) has been revealed to demethylate H3K27 and activate M2 gene transcription 149. Leflunomide is a disease-modifying anti-rheumatic drug that inhibits dihydroorotate dehydrogenase (DHODH) 150. DHODH is known to feed electrons into the mitochondrial ETC at several points in the region of CoQ 151 and therefore, could serve as the source of O - 2. Leflunomide has been shown to decrease cell oxygen consumption in various cell lines 152. Moreover, leflunomide functions as an inhibition of tyrosine kinases 153. The disease progression of pulmonary fibrosis shares many similarities with rheumatologic diseases with imbalances between inflammatory and antiinflammatory mediators 154. In this study, we demonstrate that STAT6 is activated by Cu,Zn-SOD-mediated redox signals and promotes M2 gene expression epigenetically by modulating histone methylation status via Jmjd3. Furthermore, targeting cellular ROS production and STAT6 activation via leflunomide, pulmonary fibrosis development is attenuated.

110 95 Methods and Materials Materials A , the bioactive compound of leflunomide, was purchased from EMD Millipore (Billerica, MA). Mice Wild-type (WT), Cu,Zn-SOD Tg, and STAT6 -/- (Jackson Lab, Bar Harbor, ME) C57BL/6 mice were used in these studies, and all protocols were approved by the University of Iowa Institutional Animal Care and Use Committee. Mice were intratracheally administered 100 µg of chrysotile asbestos suspended in 50 µl 0.9% saline solution after being anesthetized with 3% isoflurane using a precision Fortec vaporizer (Cyprane, Keighley, UK). Mice were euthanized at the designated day after exposure to an overdose of isoflurane, and bronchoalveolar lavage (BAL) was performed. Alveolar macrophages were isolated from BAL fluid for differential cell number, and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum. Bone marrow cells were isolated and incubated in L929 cell-conditioned media for 7 days to generate monocytes/macrophages. For leflunomide treatment, mice were subjected to intra-peritoneal injection of A (1.4 mg/kg) every other day starting from the day of chrysotile installation. Cell culture Human monocyte (THP-1) and mouse alveolar macrophage (MH-S) cell lines were obtained from American Type Culture Collection (Manassas, VA). Cells were maintained in RPMI-1640 media with following supplements: 10% fetal bovine serum and penicillin/streptomycin. All experiments were performed with 0.5% serum supplement.

111 96 Plasmids and transfections Human STAT6 cdna (NM_003153, GeneCopoeia, Rockville, MD) was cloned into pcdna3.1d/v5-his-topo vector (Invitrogen). Mutant pcdna3.1-stat6 C528S - V5-His was generated using the QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). DNA sequences of all plasmid constructs were verified by DNA sequencing (University of Iowa DNA facility). Plasmid vectors were transfected into cells using X-tremeGene 9 transfection reagent (Roche, Indianapolis, IN), according to the manufacturer s instructions. Small Interfering RNA (sirna) Macrophages were transfected with 100 nm scrambled or STAT6 sirna duplex (IDT, Coralville, IA) using DharmaFect 2 reagent (Dharmacon Research, Lafayette, CO) as previously described 46. Isolation of nucleus and cytoplasm Nuclear isolation was performed by resuspending cells in a lysis buffer (10 mm HEPES, 10 mm KCl, 2 mm MgCl 2, 2 mm EDTA) for 15 min on ice. Nonidet P-40 (NP- 40) (10%) was added to lyse the cells, and the cells were centrifuged at 4 C at 14,000 rpm. The nuclear pellet was resuspended in an extraction buffer (50 mm HEPES, 50 mm KCl, 300 mm NaCl, 0.1 mm EDTA, 10% glycerol) for 20 min on ice. After centrifuging at 4 C at 14,000 rpm, the supernatant was collected as nucleus extract. The cytoplasm was isolated by resuspending cells in a lysis buffer (50 mm Tris, ph 8, 10 mm EDTA, protease inhibitors) and sonicating for 10 s on ice. The lysate was centrifuged at 2,000 g for 5 min. The supernatant was centrifuged at 100,000 g for 10 min after which the supernatant containing the cytoplasmic fraction was collected.

112 97 Immunoblot analysis Whole cells lysates were obtained as described 108 and separated by SDS-PAGE. Immunoblot analyses were performed with the designated antibodies followed by the appropriate secondary antibodies cross-linked to HRP. Rabbit anti-stat6, rabbit antilamin A/C (Cell Signaling, MN), mouse anti- -actin (Sigma, MO), and sheep anti-cu,zn- SOD (Calbiochem, CA) were used for immunoblot analysis. Densitometry was determined utilizing ImageJ. ELISA Active TGF-, TNF-, and Ym-1 levels in BAL fluid and cell media was measured by ELISA (R&D, Minneapolis, MN), according to manufacturer s instructions. Oxygen consumption assay Oxygen consumption was measured by with an ESA BioStat multi-electrode system (ESA Products, Dionex Corp., Chelmsford, MA, USA) in conjunction with a YSI oxygen probe (5331) and glass reaction chamber vials in a YSI bath assembly (5301) (Yellow Springs Instruments, Yellow Springs, OH, USA), all at room temperature as previously described 155. Jmjd3 activity assay Jmjd3 activity was measured by using a Jmjd3/UTX activity assay from Epigentek (Farmingdale, NY), according to manufacturer s manual. Briefly, the nuclear fractions were isolated and incubated in the strip plate precoated with the trimethylated H3K27 substrate. After 90 min incubation, capture antibodies, detection antibodies and color reagents were added sequentially and the absorbance was measured. The activity of Jmjd3/UTX was calculated based on the standard curve formulated by a series dilution of monomethylated H3K27.

113 98 Statistical analysis Statistical comparisons were performed as indicated in figure legend. Values in figures are expressed as means with standard errors and p < 0.05 was considered to be significant. Results STAT6 is redox regulated by Cu,Zn-SOD-mediated H 2 O 2 generation STAT6 is a member of the signal transducers and activators of transcription (STAT) family of transcription factors that regulates Th2 cytokine-induced gene expression during M2 polarization. To investigate the effect of Cu,Zn-SOD on STAT6, macrophages were infected with a replicative-deficient adenovirus containing either an empty or Cu,Zn-SOD vector. Immunoblot analysis for STAT6 in the nuclear extract revealed that cells overexpressing Cu,Zn-SOD had a significant increase of STAT6 in the nucleus compared to the cells expressing an empty vector (Figure 4-1A). Because Cu,Zn- SOD regulates macrophage mitochondrial H 2 O 2 generation, we investigated if STAT6 nuclear translocation was modulated by Cu,Zn-SOD-mediated H 2 O 2. Macrophages were infected with a replication-deficient adenoviral vector expressing either an empty or Cu,Zn-SOD construct. After 48 h, cells were cultured in the presence or absence of PEG- CAT, and nuclear proteins were isolated. Cells expressing Cu,Zn-SOD cultured with PEG-CAT had a significant decrease in nuclear STAT6 localization to near control levels (Figure 4-1B). To demonstrate this difference in a quantitative manner, densitometry of STAT6 was compared to lamin A/C in three separate experiments and was expressed graphically as a ratio (Figure 4-1C). Because Cu,Zn-SOD activity in asbestos-exposed macrophages is localized to the mitochondria and requires two critical cysteine residues, Cys 57 and Cys , we determined if overexpression of a catalytically inactive mutant (C57S,C146S) Cu,Zn- SOD alters STAT6 nuclear translocation. Macrophages were transfected with STAT6 WT and either an empty vector, Cu,Zn-SOD WT, or Cu,Zn-SOD C57S,C146S. STAT6 WT was

114 99 significantly increased in cells expressing the active Cu,Zn-SOD WT, while STAT6 nuclear translocation was dramatically reduced below control levels in cells expressing the Cu,Zn-SOD C57S,C146S (Figure 4-2A). This difference is shown in a quantitative manner with densitometry of STAT6 compared to lamin A/C in three separate experiments and is expressed graphically as a ratio (Figure 4-2B). STAT6 is required for Cu,Zn-SOD-mediated alternatively activated macrophages Because over-expression of Cu,Zn-SOD induces STAT6 nuclear translocation, we investigated if loss of STAT6 in macrophages could skew macrophage polarization and if STAT6 was required for Cu,Zn-SOD-mediated macrophage M2 polarization. WT and STAT6 -/- macrophages were incubated in the presence or absence of PEG-SOD for 3 h and then treated with chrysotile asbestos. STAT6 -/- macrophages had an M1 dominant phenotype with significantly augmented TNF- and decreased Ym1 and FIZZ1 levels compared with WT macrophages. PEG-SOD treatment, as expected, promoted an M2 polarization in WT cells with increased Ym1 and FIZZ1 expression. However, PEG-SOD-treated STAT6 -/- macrophages had no effect on M2 genes expression. In fact, STAT6 -/- macrophages had a sustained an M1 phenotype in the presence or absence of PEG-SOD (Figure 4-3). These observations support the idea that STAT6 is crucial for Cu,Zn-SOD mediated M2 polarization. Redox activation of STAT6 requires critical cysteine residue Because STAT6 translocation was modulated by H 2 O 2 and cysteine residues are critical targets for H 2 O 46,156 2, we mutated Cys 528 in the SH2 domain, which is a region of STAT6 necessary for DNA binding and transcriptional activation 147 (Figure 4-4A). Macrophages were infected with either an empty (CMV) or Cu,Zn-SOD adenoviral vector. The cells were transfected the following day with either STAT6 WT or STAT6 C528S. After 24 h, FIZZ1 gene expression was determined. Overexpression of

115 100 STAT6 WT in cells expressing Cu,Zn-SOD significantly increased FIZZ1 gene expression compared to cells expressing the empty vector (Figure 4-4B). In contrast, FIZZ1 gene expression was significantly reduced in cells expressing STAT6 C528S and Cu,Zn-SOD to the level in cells expressing the empty vector. To verify that the STAT6 mutation did not alter nuclear translocation, cells were transfected with an empty vector, STAT6 WT, or STAT6 C528S, and nuclear extracts were isolated. WT and STAT6 C528S vectors were equally expressed in the nucleus as shown by immunoblot analysis for the tagged STAT6 vector (Figure 4-4C). To determine if the regulation of M2 gene required both Cu,Zn-SOD and STAT6, we transfected with either STAT6 WT or STAT6 C528S in combination with either Cu,Zn-SOD WT or the catalytically inactive Cu,Zn-SOD C57S,C146S vector. FIZZ1 gene expression was significantly reduced in cells expressing STAT6 C528S compared to the STAT6 WT in cells expressing the active Cu,Zn-SOD vector (Figure 4-4D). In contrast, FIZZ1 gene expression was completely abolished in all cells expressing the Cu,Zn-SOD C57S,C146S, which is catalytically inactive. These data suggest that STAT6 nuclear translocation and transcriptional activity is redox regulated, and STAT6-dependent gene expression requires a critical cysteine in the SH2 domain. STAT6 is activated by general redox signals To validate the importance of redox regulation of STAT6 nuclear translocation and activation, we treated macrophages with 250 mm tert-butyl hydroperoxide. STAT6 expression in the nuclear fraction was examined by immunoblot. t-booh treatment significantly increased STAT6 expression in the nuclear fraction. By contrast, STAT6 was dramatically decreased in the cytosolic fraction (Figure 4-5A). To determine if mitochondrial oxidative stress modulated STAT6-dependent M2 gene expression, we treated macrophages with mitochondrial electron transport chain inhibitors. Rotenone (10 µm) and antimycin A (10 µm) inhibit mitochondrial electron

116 101 transport chain complex I and III, respectively, which are the primary sites of mitochondrial O 2 - production 157,158. Antimycin A treatment significantly increased H 2 O 2 production compared to vehicle-treated cells (Figure 4-5B). In contrast, H 2 O 2 production was not altered in rotenone-treated cells. To determine the effect of these inhibitors on FIZZ1 gene expression, macrophages were transfected with either STAT6 WT or STAT6 C528S. The following day cells were cultured in the presence of either vehicle (DMSO), rotenone, or antimycin A. FIZZ1 gene expression was significantly inhibited in cells expressing the STAT6 mutant (C528S) compared to the WT in the presence of vehicle and antimycin A (Figure 4-5C). Similar to Cu,Zn-SOD overexpression, antimycin A significantly increased FIZZ1 expression compared to cells treated with vehicle. Taken together, these observations demonstrated that increased H 2 O 2 production in cells treated with antimycin A support our previous data that Cu,Zn-SOD-mediated H 2 O 2 production is closely linked to complex III activity 46. Furthermore, these data suggest that mitochondrial oxidative stress, in part, modulates M2 gene expression. STAT6 -/- macrophages maintain an M1 phenotype Given STAT6 is instrumental for Cu,Zn-SOD-mediated M2 polarization, we hypothesized that STAT6 -/- macrophage would polarize into the pro-inflammatory and anti-fibrotic M1 phenotype. Macrophages were transfected with either scrambled sirna or STAT6 sirna for 72 h and then treated with chrysotile asbestos for 4 h. CCL-18 gene expression was measured. Macrophages transfected with STAT6 sirna had decreased CCL-18 expression compared to the macrophages transfected with the scramble vector (Figure 4-6A), supporting our hypothesis that STAT6 -/- macrophages have a defective M2 polarization. To further test our hypothesis in vivo, WT and STAT6 -/- mice were exposed to 100 µg chrysotile asbestos intratracheally and euthanized on day 21. Bronchoalveolar

117 102 lavage was performed and signature M1 and M2 markers levels were measured in BAL fluid. STAT6 -/- mice have decreased Ym1 expression (Figure 4-6B) and an increase in TNF- (Figure 4-6C) in the BAL fluid compared with WT mice. Signature gene expressions in BAL cells were also measured. M2 genes, such as Ym1 and FIZZ1, were decreased in BAL cells collected from STAT6 -/- mice, and the M1 gene TNF- was increased compared with BAL cells collected from WT mice (Figure 4-6D), suggesting that STAT6 -/- macrophages have a dominant M1 and substandard M2 phenotype. STAT6 -/- mice are protected from developing pulmonary fibrosis Because STAT6 -/- had decreased M2 markers in vivo, we postulated that STAT6 -/- would have less fibrotic development compared with WT mice. The lungs of STAT6 -/- and WT mice were harvested and stained with Masson s Trichrome to assess collagen deposition. STAT6 -/- mice have less parenchymal and peribronchial collagen deposition compared with WT mice (Figure 4-7A). Furthermore, lung homogenates from STAT6 -/- mice have decreased hydroxyproline level compared with WT mice (Figure 4-7B). WT mice also had a higher level of active TGF- in the BAL fluid, suggesting an overall profibrotic environment in the alveolar spaces of WT mice (Figure 4-7C). Because fibroblasts are the primary cells responsible for collagen production and macrophages produce multiple growth factors to stimulate collagen synthesis of fibroblasts. We exposed primary WT mouse lung fibroblasts with BAL fluid from either WT mice or STAT6 -/- mice. Collagen I mrna level and secreted collagen I protein in the media were measured. Collagen I gene expression in fibroblasts cultured with BAL fluid from STAT6 -/- mice was significantly reduced compared to fibroblast incubated with BAL fluid from WT mice (Figure 4-8A). Collagen I secretion from WT fibroblasts was also significantly decreased when cultured with BAL fluid from STAT6 -/- mice (Figure 4-8B, C). Taken together, these data supported our hypothesis that STAT6 is important for

118 103 macrophages to polarize into the pro-fibrotic M2 phenotype, and the ablation of STAT6 genetically can ameliorate the fibrotic progression after chrysotile exposure in vivo. Cu,Zn-SOD-STAT6 pathway regulates M2 gene expression by Jmjd3 In the canonical Th2 cytokine-induced M2 polarization, IL-4/IL-13 activates STAT6 and increases jmjd3 expression. Because we showed that Cu,Zn-SOD can promote M2 polarization utilizing the STAT6 pathway, we hypothesized that Cu,Zn-SOD can increase jmjd3 expression and the enzyme activity, which lead to the initiation of M2 gene transcription and macrophage alternative activation. The jmjd3 promoter has five putative binding sites for STAT6 (Figure 4-9A), and region 4, which is the site closest to the transcription start site, has been previously reported to the dominant site for STAT6 binding 149. To validate that STAT6 binds to jmjd3 promoter region 4, we performed a ChIP assay in WT and STAT6 -/- macrophages. STAT6 -/- macrophages, which were used as a negative control, had no STAT6 binding to the jmjd3 promoter region 4 compared with WT macrophages, which have normal amount of STAT6 binding to jmjd3 promoter (Figure 4-9B). When we normalized to % input relative to the IgG negative control, STAT6 -/- macrophages had half of jmjd3 promoter region 4 expression, indicating a dramatically decreased jmjd3 expression (Figure 4-9C). To test this observation in vivo, we isolated mrna from WT and STAT6 -/- mice BAL macrophages after chrysotile exposure and measure jmjd3 expression. jmjd3 gene expression is significantly decreased in BAL cells from STAT6 -/- mice (Figure 4-9D), which indicates that STAT6 is crucial for jmjd3 expression. These observations further consolidate our hypothesis that in the absence of STAT6, macrophages will fail to polarize into the M2 phenotype. To evaluate the effect of Cu,Zn-SOD on jmjd3 expression, we first measured jmjd3 gene expression in WT and Cu,Zn-SOD Tg macrophages. Cu,Zn-SOD Tg macrophages, which have a predominant M2 phenotype, have two-fold increase in jmjd3

119 104 gene expression compared with WT macrophages (Figure 4-10A). We further isolated the nuclear fractions from both WT and Cu,Zn-SOD Tg macrophage and measured Jmjd3 activity. Cu,Zn-SOD Tg macrophages have increased Jmjd3 activity compared with WT macrophages (Figure 4-10B). Taken together, these data suggested that Cu,Zn-SOD increases Jmjd3 levels in macrophages, which contributes to the increasing chromatin accessibility and M2 gene transcriptional activation. We showed that Cu,Zn-SOD-mediated STAT6 activation requires the redox sensitive cysteine 528 residues within the SH2 domain of STAT6. Macrophages were transfected with either an empty vector or adenovirus expressing Cu,Zn-SOD (Ad5.CMV.Cu,Zn-SOD) for 24 h. After 24 h, the cells were transfected with either STAT6 WT or STAT6 C528S mutant for another 24 h. Cells were harvested and mrna level of jmjd3 was measured. Cells overexpressing Cu,Zn-SOD have increased jmjd3 mrna as expected. However, cells expressing the STAT6 C528S mutant had decreased level of jmjd3 to control levels (Figure 4-10C). We showed that alveolar macrophages isolated from asbestosis patients have an M2 phenotype and have increased Cu,Zn-SOD activity. Based on these data, we hypothesized that alveolar macrophages from asbestosis patients would have increased Jmjd3 levels. Nuclear fractions of alveolar macrophages from normal volunteers and asbestosis patients were isolated and Jmjd3 activity was measured. Alveolar macrophages from asbestosis patients have increased Jmjd3 activity, implying a status for M2 gene transcriptional activation (Figure 4-11). Taken together, these data suggest that Cu,Zn-SOD and STAT6 regulate M2 polarization by increasing jmjd3 gene expression and activity. The increase in demethylase activity mediates M2 gene expression by demethylating trimethylated and dimethylated histone H3 lysine 27.

120 105 Leflunomide inhibits M2 polarization Because Cu,Zn-SOD-mediated macrophage M2 polarization is dependent on the increased H 2 O 2 levels and STAT6 activation, we aimed to find a potential pharmacological agent which would abort these pathways. Leflunomide, which has been used for rheumatoid arthritis, harbors the function as a general tyrosine kinase inhibitor. Moreover, leflunomide has been shown to decrease the oxygen consumption and the production of ROS in various cancerous cell lines. Tyrosine kinase inhibitors, such as BIBF 1120, have been used in clinical trials for the treatment of idiopathic pulmonary fibrosis 159. We postulated that leflunomide could prevent the fibrotic progression in mice after asbestos exposure by inhibiting macrophage M2 polarization. Cu,Zn-SOD Tg macrophages, which has a primary M2 phenotype, consume twice as much as oxygen compared with the WT macrophages (Figure 4-12A). Macrophages were treated with either DMSO vehicle or leflunomide (200 µm) for 3 h and oxygen consumption was measured. Cells treated with leflunomide have decreased oxygen consumption compared with cells treated with DMSO vehicle (Figure 4-12B). Since oxygen consumption is closely correlated with cellular ROS production, we further investigated whether leflunomide abolished Cu,Zn-SOD-mediated H 2 O 2 generation. Macrophages were infected with either an empty or Cu,Zn-SOD adenovirus and after 48 h incubation, cells were incubated in the presence or absence of leflunomide for 3 h. Leflunomide treatment significantly reduced H 2 O 2 production in cells infected with empty vector. Moreover, leflunomide decreased H 2 O 2 generation to basal level even in cells overexpressing Cu,Zn-SOD (Figure 4-12C). These observations highlighted the role of leflunomide as a modulator of the cellular redox levels. To investigate whether leflunomide can abolish STAT6 phosphorylation, macrophages were incubated in the presence or absence of leflunomide (200 µm) for 2 h and then treated with either vehicle or IL-4 (20 ng/ml) for 30 min. IL-4 treatment increases STAT6 phosphorylation on the tyrosine 641. However,

121 106 leflunomide treatment inhibits IL-4 induced tyrosine phosphorylation of STAT6 (Figure 4-12D). Based on these observations, we hypothesized that leflunomide would alter M2 polarization via its dual function of inhibiting ROS and STAT6 activation. To test whether leflunomide can alter macrophage polarization, we measured gene profiles in macrophages treated with either DMSO vehicle or leflunomide for 3 h. Cells treated with leflunomide had decreased M2 markers, Ym1 and FIZZ1, and increased M1 marker, TNF-, indicating a primary M1 phenotype compared with cells incubated with DMSO vehicle (Figure 4-13A). Similar observations were recapitulated in vivo. We exposed WT mice to chrysotile asbestos and administered leflunomide (1.4 mg/kg) intraperitoneally every other day until day 21 when the mice were sacrificed. Signature gene expressions in BAL cells were measured. M2 genes such as Ym1 and FIZZ1 were decreased in BAL cells collected from mice treated with leflunomide and the M1 gene TNF- was increased compared with BAL cells collected from mice treated with DMSO control (Figure 4-13B). Taken together, these data suggest that macrophage polarization can be modulated in vivo pharmacologically. Leflunomide attenuates pulmonary fibrosis Since leflunomide treatment can inhibit macrophage M2 polarization, we further examined the potential therapeutic role of leflunomide in pulmonary fibrosis in vivo. We exposed Cu,Zn-SOD Tg mice to chrysotile asbestos and administered leflunomide (1.4 mg/kg) intraperitoneally every other day until day 21 when the mice were sacrificed. The lungs were harvested and stained using Masson s Trichrome method. Mice treated with DMSO vehicle had more severe parenchymal and peribronchial collagen deposition compared to mice received leflunomide treatment (Figure 4-14A). Lungs were harvested and collagen levels were measured by hydroxyproline assay. Compared with DMSO treated mice, leflunomide treated mice had a significant decrease in hydroxyproline

122 107 (Figure 4-14B), suggesting that leflunomide treatment prevented the fibrotic development after asbestos exposure. To summarize, these data suggested that leflunomide mitigates cellular ROS and inhibits STAT6 phosphorylation, both of which lead to the inhibition of M2 polarization and amelioration of the progression of pulmonary fibrosis. These data also showed that leflunomide is able to inhibit macrophage M2 polarization and attenuates pulmonary fibrosis after chrysotile asbestos exposure. BAL cells from DMSO vehicle or leflunomide treated WT mice were examined for jmjd3 gene expression. Macrophages from leflunomide treated WT mice had a significant decrease in jmjd3 gene expression compared with macrophages isolated from the vehicle treated mice (Figure 4-15). In aggregate, these data underscore the potential therapeutic benefits of leflunomide in pulmonary fibrosis. Discussion The function of macrophages was determined by their distinct differentiation and polarization process. Compared with the pro-inflammatory classically activated macrophages (M1 phenotype), the alternatively activated macrophages (M2 phenotype) are implicated in inflammation attenuation and, sometimes, fibrosis. We have shown that Cu,Zn-SOD-induced H 2 O 2 can polarize macrophages into the M2 phenotype and promote an accelerated development of pulmonary fibrosis. Epigenetic regulations play an important part in the acquisition and maintenance of macrophage phenotype. STAT6 has been shown to bind to the promoter region of the histone demethylase, Jmjd3, which regulates M2 gene transcriptional activation. Based on these data, we hypothesized that STAT6 is crucial for M2 polarization and is involved in the epigenetic modulation of the macrophage phenotype. In this study, we showed that Cu,Zn-SOD-mediated M2 polarization is closely linked to the activation of STAT6. STAT6 activation can be modulated by cellular ROS levels and relies on the cysteine 528 residue in the SH2 domain. STAT6 is indispensable for Cu,Zn-SOD-mediated M2 polarization. STAT6

123 108 upregulates Jmjd3, a histone H3 lysine 27 demethylase, and initiates M2 gene transcriptional activation. Targeting STAT6 with leflunomide, which can reduce cellular ROS production and inhibit STAT6 phosphorylation, abolishes M2 polarization and ameliorates the fibrotic development. Evidence to support this mechanism include the following: 1) Cu,Zn-SOD mediates M2 polarization by modulation of STAT6 nuclear translocation and transcriptional activity in a redox-dependent manner; 2) STAT6 -/- macrophages fail to polarize into the M2 phenotype even when Cu,Zn-SOD is over expressed; 3) STAT6 -/- mice are protected from developing pulmonary fibrosis; 4) alveolar macrophages isolated from STAT6 -/- after asbestos exposure showed a skewed M1 phenotype; 5) leflunomide treatment prohibits macrophage M2 polarization and attenuates pulmonary fibrosis in vivo; 6) Jmjd3 expression and activity is increased in Cu,Zn-SOD Tg macrophages; and 7) alveolar macrophages isolated from asbestosis patients have increased Cu,Zn-SOD levels, increased M2 gene expression, and increased Jmjd3 activity; 8) Jmjd3 expression and activity can be modulated by STAT6 activity and leflunomide treatment. ROS have emerged as an important regulator of macrophage polarization 160. However, most current research focuses on NO signal on the M1 differentiation. An increasing level of NO, which itself relates to the increase of inos during M1 polarization, leads to the accumulation of nitrosating species. Intracellular targets of S- nitrosation include multiple pro-inflammatory transcription factors such as NF- B 161 and HIF Limited data are available regarding ROS and M2 polarization. It has been proposed that similar to the M1/2 nomenclature, macrophages can be denoted as reductive or oxidative macrophages based on their intracellular glutathione levels 163. The M1 phenotype macrophages have more reduced glutathione, whereas the M2 phenotype macrophages have more oxidized glutathione. This observation is in consistent with our data that Cu,Zn-SOD macrophages, which produce more H 2 O 2 and have increased oxidative stress, manifest a dominant M2 phenotype.

124 109 In these studies, we showed that Cu,Zn-SOD-mediated M2 polarization requires STAT6, and STAT6 -/- mice are protected from developing pulmonary fibrosis, partially due to the inhibition of M2 gene expression. STAT6 is critical for M2 cytokine production and granuloma formation in S. mansoni-induced hepatic injuries, as STAT6 -/- mice fail to generate Th2 cytokines and have attenuated liver damage ROS generation is causally linked to STAT6 activation in certain cell lines; however, controversy remains whether STAT6 activation promotes ROS generation or STAT6 activation is downstream of ROS production 166,167. We found that overexpression of Cu,Zn-SOD modulates STAT6 translocation to the nucleus to initiate M2 gene transcription, which is dependent on the redox status of Cys528 in the SH2 domain of STAT6. Our data showed that despite similar Th2 cytokine levels in WT and Cu,Zn- SOD -/- mice, alveolar macrophage polarization patterns are differentially regulated. Increased levels of Cu,Zn-SOD resulted in a predominant M2-phenotype, whereas its absence was associated with an M1 phenotype. The redox regulation of STAT6 activation and M2 cytokine expression by Cu,Zn-SOD provide a novel mechanism of macrophage alternative activation. The protective effects of STAT6 ablation in pulmonary fibrosis might be multifactorial, and the specificity regarding macrophage polarization might warrant further investigation. STAT6 up-regulates Muc5ac in lung epithelium cells, which plays a pivotal role in the development of pulmonary fibrosis 168. STAT6 -/- mice also had been used in bleomycin-induced fibrosis model to evaluate FIZZ1 expression in alveolar type II epithelial cells 169. This study showed that the protective effect of STAT6 ablation was related to silencing FIZZ1 in alveolar epithelial cells. Given that STAT family proteins all harbor multiple cysteine residues and are common targets for redox regulation 46, it is also possible that Cu,Zn-SOD-mediated H 2 O 2 signal regulates macrophage phenotype by repressing STAT1, which is required for M1 polarization, or STAT3, which is implicated in regulatory macrophage differentiation 170.

125 110 Studies indicate that, in contrast to the rapidly fluctuating post-translational modifications by upstream conventional signaling proteins (kinase, phosphatase, etc), epigenetic modulations are generally more stable 87. This mechanism of sustained gene expression is particularly pertinent with the development of pulmonary fibrosis, whereas most cases are sterile inflammation or idiopathic which lack the classic microbial stimulus 171. Recent advances in cardiovascular and cancer research have suggested a potential link between ROS and epigenetics. ROS can recruit DNA methyltransferases (DNMTs) to chromatin and lead to hypomethylation in atherosclerosis 172. H 2 O 2 has been shown to induce histone H4 acetylation and decrease histone deacetylase (HDAC) activity 173. ROS promotes histone acetylation by increasing recruitment of histone acetylase (HAT) co-activators such as CBP/p However, whether ROS can dynamically regulate histone methylation remains largely unknown. Macrophage differentiation is rigorously regulated epigenetically 148, especially through the histone modifications. Methylation of histone H3 at lysine-4, -36, and -79 is implicated in activation of transcription, while methylation of histone H3 at lysine-9 and -27, and histone H4 at lysine-20 is correlated with repression of transcription. Jmjd3 promotes the anti-inflammatory phenotype in LPS-tolerant macrophages and 175. Small-molecule inhibitors named GSK-J1 and GSK-J4 have been shown to specifically inhibit Jmjd3 and modulate macrophage function 176. STAT6 binds to the jmjd3 gene loci immediately upstream of the macrophage transcriptional start site (MF- TSS) upon IL-4 stimulation and promotes transcription activation of multiple signature M2 genes 149. Here we showed that in parallel with up-regulation of STAT6 in Cu,Zn- SOD-induced H 2 O 2 signaling, jmjd3 expression is increased. This suggests that despite the different initial stimulus, ROS may utilize a similar pathway like Th2 cytokines in inducing M2 polarization. However, Jmjd3 -/- macrophages have significant, but not complete, reduction of M2 genes 149, implying other regulatory mechanism may exist and requires further exploration.

126 111 Epigenetic regulation in the development of pulmonary fibrosis is a growing area of research 177. All three mechanisms, DNA methylation, histone modification and mirnas, are involved in the disease progression 88. However, no study has, so far, investigated epigenetic regulation of macrophage phenotype in the context of pulmonary fibrosis. It is known that epigenetic regulation is highly cell type specific. The data we present here indicate that alveolar macrophages are also the subjects of epigenetic regulation during the disease development of pulmonary fibrosis. Future study should also be directed to other possible mechanisms of epigenetic regulation such as DNA methylation and mirna in alveolar macrophages to avoid over simplify the epigenetic modulations. Leflunomide was approved by FDA for the treatment of rheumatoid arthritis in 1998, and reports of interstitial pneumonitis were first reported in Japan in Controversy remains whether leflunomide is the culprit for these interstitial lung diseases in rheumatologic patients. In a study using 62,734 patients from the Medicare and Medicaid datebase, there was no increase in the relative risk of interstitial lung diseases (ILDs) in patients on leflunomide if they had no previous methotrexate use and no history of ILD 178. The correlation between leflunomide and ILD is likely due to channeling bias because leflunomide is more likely to be prescribed to patients have previous history of ILD, who cannot tolerate methotrexate. It is well-known that alternatively activated macrophages are involved in the hepatic fibrotic process and contribute to the pro-fibrotic environment. Leflunomide has been shown to suppress hepatic fibrosis in CCl 4 and leptin-induced animal models 179,180. Fibrotic markers such as collagen I, hydroxyproline and TGF- all decreased after systemic leflunomide treatment. Despite these reports on the potential efficacy of leflunomide in treating fibrosis in vivo, little is known about the biological mechanism of how leflunomide works. Multiple studies have shown that leflunomide treatment actively regulates matrix metalloproteinases with, however, conflicting data in the context of fibrosis One study has suggested that leflunomide,

127 112 through its inhibition of DHODH and de novo pyrimidine synthesis, promoted epithelialmesenchymal transition (EMT) 184. Nevertheless, the exact role of EMT in pulmonary fibrosis in vivo remains controversial. The observations we presented in this manuscript are the first to investigate the biological mechanism of leflunomide on macrophage phenotype and prevention of pulmonary fibrosis.

128 113 Figure 4-1: Cu,Zn-SOD-mediated H 2 O 2 activates STAT6 nuclear translocation. A, Macrophages were infected with a replication-deficient adenovirus vector expressing either an empty vector (CMV) or Cu,Zn-SOD vector (Cu,Zn-SOD) for 48 h. Nuclear fractions were isolated and immunoblot analysis for STAT6 was performed (right). B, Macrophages were infected with a replication-deficient adenovirus vector expressing either an empty vector (CMV) or Cu,Zn-SOD vector (Cu,Zn-SOD) for 24 h. Cells were cultured for an additional day in the presence or absence of PEG-CAT. Nuclear fractions were isolated and immunoblot analysis for STAT6 was performed. C, Ratio of densitometry of nuclear STAT6 expression to lamin A/C. (n=4), *p < 0.05 vs. CMV and Cu,Zn-SOD+PEG-CAT. Results were expressed as the mean ± SEM and analyzed by one-way ANOVA with Tukey post-analysis test.

129 114 A CMV Cu,Zn-SOD STAT6 Lamin A/C nucleus B CMV Cu,Zn-SOD Cu,Zn-SOD + PEG-CAT STAT6 Lamin A/C nucleus C

130 115 Figure 4-2: STAT6 nuclear translocation requires functional active Cu,Zn-SOD A, Macrophages were transfected with STAT6 WT vector with either an empty, Cu,Zn- SOD WT, or Cu,Zn-SOD C57S,C146S vector, and nuclear fractions were isolated. B, Ratio of densitometry of nuclear STAT6 expression to lamin A/C was performed. (n=3), *p < 0.05 vs. Empty and Cu,Zn-SOD C57S,C146S. Results were expressed as the mean ± SEM and analyzed by one-way ANOVA with Tukey post-analysis test.

131 116 A Cu,Zn-SOD Empty WT C57S,C146S STAT6 Lamin A/C nucleus B

132 117 Figure 4-3: STAT6 is indispensable for Cu,Zn-SOD-mediated M2 polarization. A, WT and STAT6 -/- macrophages were treated in the presence or absence of PEG-SOD for 3 h and then treated with chrysotile asbestos. Total RNA from macrophages was isolated and TNF-, FIZZ1, and Ym1 gene expression were measured. Results show arbitrary units normalized to β-actin mrna. (n = 4) *p< 0.05 WT vs. STAT6 -/-, ** p< 0.05 WT vs. WT+PEG-SOD, # p< 0.05 STAT6 -/- +PEG-SOD vs. STAT6 -/-. Results were expressed as the mean ± SEM and analyzed by one-way ANOVA with Tukey postanalysis test..

133 118

134 119 Figure 4-4: Cys 528 in STAT6 is critical for redox activation of STAT6. A, Schematic of STAT6 gene. B, Macrophages were infected with a replication-deficient adenovirus vector expressing either an empty vector (CMV) or Cu,Zn-SOD vector (Cu,Zn-SOD) for 24 h. After 24 h, cells were transfected with either STAT6 WT or STAT6 C528S vectors for 24 h. Total RNA from macrophages was isolated, and FIZZ1 gene expression was measured. Results show arbitrary units normalized to -actin mrna. (n = 4). *p < 0.05 Cu,Zn-SOD with STAT6 WT vs. all other groups. Results were expressed as the mean ± SEM and analyzed by one-way ANOVA with Tukey postanalysis test. C, Macrophages were transfected with STAT6 WT vector with either an empty, Cu,Zn-SOD WT, or Cu,Zn-SOD C57S,C146S vector, and nuclear fractions were isolated. Immunoblot analysis for STAT6 and lamin A/C was performed. D, Macrophages were co-transfected with either Cu,Zn-SOD-V5-His WT or Cu,Zn-SOD-V5-His C57S,C147S vector with either STAT6 WT or STAT6 C528S vector for 24 h. Total RNA was isolated, and FIZZ1 gene expression was measured. Results show arbitrary units normalized to -actin mrna. (n=4). * p < 0.05 Cu,Zn-SOD WT +STAT6 WT vs. Cu,Zn-SOD WT +STAT6 C528S. **p < 0.05 Cu,Zn-SOD C57S,C147S +STAT6 WT vs. Cu,Zn-SOD WT +STAT6 WT. Results were expressed as the mean ± SEM and analyzed by one-way ANOVA with Tukey post-analysis test.

135 120 A B C STAT6 Empty WT C528S V5-His Lamin A/C nucleus D

136 121 Figure 4-5: STAT6 and M2 gene transcription can be activated by general redox signals. A, Macrophages were treated with 250 mm tert-butyl hydroperoxide. Levels of STAT6 expression in the nuclear and cytoplasmic fractions were examined by immunoblot. B, Macrophages were treated with DMSO vehicle, 10 um rotenone, or 10 um antimycin A overnight. H 2 O 2 production rate was measured by phpa assay. * p < 0.05, antimycin A vs. DMSO. Results were expressed as the mean ± SEM and analyzed by one-way ANOVA with Tukey post-analysis test. C, Macrophages were transfected with either STAT6 WT or STAT6 C528S vector. 12 h later, cells were treated with either vehicle (DMSO), 10 µm rotenone, or antimycin A for another 12 h. Total RNA from macrophages was isolated and FIZZ1 gene expression was measured. Results show arbitrary units normalized to -actin mrna. *p < 0.05 vs. STAT6 WT in DMSO- and antimycin A-treated cells (n=3). **p < 0.05 vs. STAT6 WT +DMSO (n=3). Results were expressed as the mean ± SEM and analyzed by one-way ANOVA with Tukey postanalysis test.

137 122 A STAT6 t-booh Nuclear Cytoplasmic Lamin B C DMSO Rotenone Antimycin A

138 123 Figure 4-6: STAT6 depletion leads to an M1 dominant phenotype. A, THP-1 cells were transfected with either scramble or STAT6 sirna for 72 h. Total mrna is isolated and CCL-18 gene expression were measured. Results show arbitrary units normalized to HPRT mrna. *p<0.05 STAT6 sirna vs. scramble sirna. Results were expressed as the mean ± SEM and analyzed by two-tailed student t test. WT and STAT6 -/- mice were exposed to chrysotile (100 µg) intratracheally. B, Ym1, and C, TNF- in the BAL fluid were measured 21 days after asbestos exposure. n = 4; *p < Results were expressed as the mean ± SEM and analyzed by two-tailed student t test. D, Total RNA from alveolar macrophages was isolated and TNF-, Ym1, and FIZZ1 gene expression were measured. Results show arbitrary units normalized to β-actin mrna. (n = 4) *p< 0.05 vs. WT. Results were expressed as the mean ± SEM by two-tailed student t test.

139 124 A B C D

140 125 Figure 4-7: STAT6 -/- mice are protected from developing pulmonary fibrosis. WT and STAT6 -/- mice were exposed to chrysotile (100 µg) intratracheally. A, Lungs were harvested for Masson s trichrome from WT or STAT6 -/- mice. Representative micrographs of 1 of 6 animals. Magnification 4x, bar indicates 200 µm. Lungs were extracted 21 days after asbestos exposure, and B, Hydroxyproline was measured. n = 4; *p < Results were expressed as the mean ± SEM by two-tailed Mann-Whitney test.c, Active TGF- level in the BAL fluid were measured. n = 7; *p < Results were expressed as the mean ± SEM by two-tailed student t test.

141 126 A WT STAT6 -/- B C

142 127 Figure 4-8: Collagen production is decreased in WT fibroblasts treated with BAL fluid from STAT6 -/- mice. A, Lung fibroblasts were isolated from WT mice and cultured in BAL fluid from WT or STAT6 -/- mice. Collagen I mrna expression was determined by real-time PCR and normalized to -actin. n = 3; *p < Results were expressed as the mean ± SEM and analyzed by two-tailed student t test. B, Condition media was obtained from fibroblasts cultured as in A, and collagen protein expression was determined by immunoblot. C, Ratio of densitometry of collagen expression between fibroblasts cultured in BAL fluid from WT or STAT6 -/- mice. (n=3), *p < 0.05 vs. WT. Results were expressed as the mean ± SEM and analyzed by two-tailed student t test.

143 128 A BAL Fluid B C

144 129 Figure 4-9: STAT6 regulates jmjd3 gene expression. A, Schematic of putative STAT6 binding sites on jmjd3. Adapted from 149 B, Nuclear fractions were isolated from both WT and STAT6 -/- macrophages. Immunoprecipitation of STAT6 were performed from the DNA-protein complex, PCR amplification was performed using primers to detect putative STAT6 binding site on jmjd3 gene region 4. Histone 3 as positive control whereas IgG as negative control. Control DNA was amplified by using primers to detect mouse RPL30 intron2 provided by the manufacturer. C, Real-time PCR were performed using primers to detect putative STAT6 binding site on jmjd3 gene region 4 from STAT6 and IgG immunoprecipitated DNA-protein complex along with 2% input. Percentage of input for STAT6 and IgG immunoprecipitated DNA was calculated and presented as fold of increase by STAT6 versus IgG. n = 3; *p < Results were expressed as the mean ± SEM and analyzed by two-tailed student t test. D, WT and STAT -/- mice were exposed to chrysotile (100 g) intratracheally. Alveolar macrophages were isolate after 21 days. jmjd3 mrna expression was determined by real-time PCR, and normalized to -actin. n = 3; *p < Results were expressed as the mean ± SEM and analyzed by two-tailed student t test.

145 130 A B IP: STAT6 IP: Histone 3 WT STAT6-/- STAT6-binding site at Jmjd3 region 4 mouse RPL30 intron 2 IP: IgG mouse RPL30 intron 2 C D

146 131 Figure 4-10: Overexpression Cu,Zn-SOD up-regulates Jmjd3. A, Bone marrow-derived macrophages (BMDM) were obtained from WT and Cu,Zn- SOD Tg mice. Total RNA was isolated and jmjd3 mrna was measured. Results show arbitrary units normalized to -actin mrna. n = 3, * p < Results were expressed as the mean ± SEM and analyzed by two-tailed student t test. B, Nuclear fractions from WT and Cu,Zn-SOD Tg macrophages were isolated and Jmj3/UTX enzyme activity were measured. n = 3; *p < Results were expressed as the mean ± SEM and analyzed by two-tailed Mann-Whitney test. C, MH-S macrophages were infected with a replicationdeficient adenovirus vector expressing either an empty vector (CMV) or Cu,Zn-SOD vector (Cu,Zn-SOD) for 24 h. After 24 h, cells were transfected with either STAT6 WT or STAT6 C528S vectors for 24 h. Total RNA from macrophages was isolated and jmjd3 gene expression were measured. Results show arbitrary units normalized to -actin mrna. * p < 0.05 vs. STAT6 WT and **p < 0.05 vs. all other groups; (n = 3). Results were expressed as the mean ± SEM and analyzed by one-way ANOVA with Tukey post-analysis test.

147 132 A B C

148 133 Figure 4-11: Jmjd3 activity is increased in macrophages from asbestosis patients. Nuclear fractions from normal volunteer and asbestosis patients alveolar macrophages were isolated and Jmjd3/UTX enzyme activity were measured. n = 3; *p < Results were expressed as the mean ± SEM and analyzed by two-tailed student t test.

149 134

150 135 Figure 4-12: Leflunomide abolishes H 2 O 2 generation and inhibits STAT6 activation. A, O 2 consumption rate was determined in WT and Cu,Zn-SOD Tg macrophages. n=3, *p<0.05. Results were expressed as the mean ± SEM and analyzed by two-tailed student t test. B, Cells were treated with either vehicle control or leflunomide (LFN, 200 mm). Oxygen consumption rate was determine. n = 4, *p<0.05. Results were expressed as the mean ± SEM and analyzed by two-tailed student t test. C, Macrophages were infected with adenoviral vector containing either an empty or Cu,Zn-SOD construct. After 48 h, cells were incubated with DMSO or Leflunomide (200 mm). phpa assay was performed with results expressed as rate of H 2 O 2 generation. n =3, *, ** p<0.05 vs. DMSO (Empty) and LFN (Cu,Zn-SOD), respectively. Results were expressed as the mean ± SEM and analyzed by one-way ANOVA with Tukey post-analysis test. D, THP-1 cells were incubated in the presence or absence of leflunomide for 2 h and then treated with either vehicle or IL-4 (20 ng/ml) for 30 min. Cell lysate were isolated and immunoblot analysis for p-stat6 was performed.

151 136 A B C D p-stat6 -actin IL LFN +

152 137 Figure 4-13: Leflunomide treatment inhibits M2 polarization. A, Macrophages were treated with either vehicle or leflunomide (200 mm) overnight. Total RNA from macrophages was isolated and TNF-, Ym1, and FIZZ1gene expression were measured. Results show arbitrary units normalized to β-actin mrna. (n = 4) *p< 0.05 vs. DMSO. Results were expressed as the mean ± SEM and analyzed by two-tailed student t test. B, WT mice were exposed to chrysotile asbestos (100 mg intratracheally.). Vehicle (DMSO) or Leflunomide (1.4 mg/kg) was administered intraperitoneally every another day from day 10. Mice were euthanized 21 days after exposure. Total RNA from alveolar macrophages was isolated and TNF-, Ym1, and FIZZ1 gene expression were measured. Results show arbitrary units normalized to β-actin mrna. (n = 4) *p< 0.05 vs. DMSO. Results were expressed as the mean ± SEM and analyzed by two-tailed student t test.

153 138 A B

154 139 Figure 4-14: Leflunomide treatment attenuates pulmonary fibrosis. WT and Cu,Zn-SOD Tg mice were exposed to chrysotile asbestos (100 g intratracheally.). Vehicle (DMSO) or Leflunomide (1.4 mg/kg) was administered intraperitoneally every other day. Mice were euthanized 21 days after exposure. A, Lungs were harvested for Masson s trichrome. Representative micrographs of 1 of 6 animals. Magnification 4x, bar indicates 200 µm. B, Lungs were harvested from WT mice for hydroxyproline assay. (n=3), *p<0.05 vs. DMSO. Results were expressed as the mean ± SEM and analyzed by two-tailed student t test.

155 140 A DMSO Leflunomide Cu,Zn-SOD Tg B WT