The Role of Cyclooxygenase-2 in Newborn Hyperoxic Lung Injury

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Rodney D. Britt Jr.

Integrated Biomedical Science Program

The Ohio State University

2012

Dissertation Committee:

Dr. Lynette K. Rogers, PhD (Advisor)

Dr. Mark Hall, MD

Dr. Leif D. Nelin, MD

Dr. Douglas Kniss, PhD

Rodney D. Britt Jr.

2012

Abstract

Development of respiratory distress syndrome (RDS) adversely affects patient populations in neonatal and pediatric intensive care units. Patients with RDS require ventilation and oxygen therapy to maintain adequate tissue oxygenation. Preterm infants who develop RDS are at risk of developing the chronic lung disease, bronchopulmonary dysplasia (BPD). Lung ventilation and exposure to supraphysiological concentrations of oxygen contribute to the risk of developing BPD. Preterm infants with BPD have reduced alveolar and vascular development. Survivors of BPD have diminished lung function and are at risk of developing additional lung diseases such as asthma.

Dysregulation of the inflammatory response is a significant contributing factor to BPD.

Previous studies showed that cyclooxygenase-2 (COX-2) expression and leukocyte infiltration are increased in the lung during newborn hyperoxic exposure in mice. To determine the role of COX-2 in newborn hyperoxic lung injury, newborn pups were injected with aspirin (non-selective COX -2 inhibitor) and celecoxib (selective COX-2 inhibitor) during exposure to hyperoxia. We tested the hypothesis that COX-2 inhibition would (1) reduce macrophage infiltration and chemokine expression, (2) improve lung alveolarization, and (3) improve lung function in newborn mice exposed to hyperoxia.

Our data suggest that COX-2 has a pro-inflammatory role in macrophage infiltration but is not involved in lung alveolarization during hyperoxic lung injury. Understanding the

ii role of COX-2 in the developing lung during hyperoxic exposure may lead to therapeutic strategies to improve clinical outcomes and prevent development of BPD.

The role of nonciliated airway epithelial cells, or Clara cells, during inflammation remains poorly understood. Studies have suggested that Clara cells are critical for regeneration of the airway epithelium and produce mediators which regulate inflammation. Through immuohistochemical analysis, we have found that Clara cells express COX-2. Mouse transformed Clara cells (MTCC) were utilized as an in vitro model to assess Clara cell responses to pro-inflammatory stimuli. We tested the hypothesis that lipopolysaccharide (LPS) would increase COX-2 and chemokine expression in MTCC. Our data show that LPS stimulation increases COX-2 and chemokine mRNA and protein expression, while increasing prostanoid levels. LPS also stimulates phosphorylation of mitogen activating protein kinases: p38, JNK, and ERK.

Our data suggest that Clara cells may produce prostaglandins and chemotactic factors during inflammation. We speculate that Clara cells produce mediators to modulate leukocyte infiltration and the progression of inflammation during the pathogenesis of acute lung injury. Further characterization of Clara cell function may help identify therapeutic strategies to enhance regeneration of the airway epithelium in patients with chronic inflammatory lung diseases.

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This is dedicated to my great grandparents, grandparents, and parents, living and deceased. Thank you for the sacrifices you made to allow me to have an opportunity to pursue biomedical research.

Acknowledgments

There are many people who have significantly contributed to my development into a biomedical scientist. This process has been long and difficult but the support from family and friends have helped me achieve something that I never thought would be possible. First and foremost, I would like to thank Dr. Lynette K. Rogers for the opportunity to become her first graduate student. Under her guidance I have discovered a passion for science and developed a curiosity that will drive my future research efforts. I would also like to thank my family: my grandparents, parents, brother, cousins, aunts, uncles, and friends for their continuous support and encouragement. The faculty at Ohio

State who have helped me grow as a scientist including my committee members. Finally,

I would like to thank the classmates who helped me make it through my first year and beyond at The Ohio State University.

Vita

May 2001………………………..Atholton High School, Columbia MD

May 2006………………………..B.S. Chemistry, North Carolina A&T State University

2007 to Present…………………..Graduate Research Associate, College of Medicine,

The Ohio State University

Publications

Rogers LK, Tipple TE, Britt RD, Welty SE. Hyperoxia Exposure Alters Hepatic Eicosanoid Metabolism in Newborn Mice. Pediatric Research, Feb;67(2):144-9, 2010.

Rogers LK, Valentine CJ, Pennell M, Velten M, Britt RD, Dingess K, Zhao X, Welty SE, Tipple TE. Maternal DHA Supplementation Decreases Lung Inflammation in Hyperoxia-Exposed Newborn Mice. Journal of Nutrition, Feb;141(2):214-22, 2010.

Britt RD Jr, Locy ML, Tipple TE, Nelin LD, Rogers LK. Lipopolysaccharide-induced Cyclooxygenase-2 Expression in Mouse Transformed Clara Cells. Cellular Physiology and Biochemistry, 29(1-2):213-22, 2012.

Velten M, Britt RD Jr, Heyob KM, Welty SE, Tipple TE, Rogers LK. Perinatal Inflammation Exacerbates Hyperoxia Induced Functional and Structural Changes in Adult Mice. Am. J. Physiol Regul Integr Comp Physiol, Aug;303(3):R279-90, 2012.

Fields of Study

Major Field: Integrated Biomedical Science Program

Area of Emphasis: Molecular Basis of Disease

Table of Contents

Abstract ...... ii

Acknowledgments...... v

Vita ...... vi

Publications ...... vi

Fields of Study ...... vi

Table of Contents ...... vii

List of Tables ...... xi

List of Figures ...... xii

Chapter 1: Introduction ...... 1

Chronic lung disease in preterm infants ...... 1

Definition, Pathology, and Characteristics ...... 1

Inflammation ...... 2

Lung Function...... 4

Oxidative Stress during BPD ...... 5

Therapeutic Strategies ...... 6

Newborn Hyperoxic Lung Injury ...... 7

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Leukocyte Infiltration and Inflammatory Mediators ...... 9

Alveolarization and Lung Function ...... 10

Role of Cyclooxygenase-2 (COX-2) during Lung injury ...... 12

COX-2 Expression and Activity ...... 12

Role of COX-2 during lung inflammation ...... 14

Aspirin and its effect on inflammation ...... 15

COX-2 acetylation ...... 16

Lipoxins, Resolvins, and Protectins ...... 16

COX-independent effects of Salicylate and Aspirin ...... 19

Specific Aims, Objectives, and Rationale...... 20

Chapter 2: Role of COX-2 in >95% O2 mouse model of newborn hyperoxia ...... 22

Introduction ...... 22

Materials and Methods ...... 24

Results ...... 28

COX-2 Protein Expression ...... 28

Aspirin and salicylic acid plasma levels...... 28

Mortality and Body Weights ...... 28

Bronchoalveolar Lavage Fluid ...... 29

Prostanoid levels ...... 29

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Macrophage Counts ...... 29

Chemokine Expression ...... 30

Alveolarization...... 30

Lung Function...... 31

Discussion ...... 33

Chapter 3: Role of COX-2 in 85% O2 mouse model of newborn hyperoxia ...... 54

Introduction ...... 54

Material and Methods...... 56

Results ...... 59

COX-2 Protein Expression ...... 59

Mortality and Body weights ...... 59

BAL Protein Expression ...... 60

Chemokine Expression ...... 60

Macrophage counts ...... 60

Prostanoid levels ...... 61

Morphometric analysis ...... 62

Lung Function Assessments ...... 62

Discussion ...... 63

Chapter 4: COX-2 expression in mouse transformed Clara cells ...... 81

Introduction ...... 81

Materials and Methods ...... 85

Results ...... 89

COX-2 expression and activity ...... 89

Chemokine and cytokine expression ...... 90

Effect of COX inhibition on Chemokine Expression ...... 90

MAPK Phosphoylation and IκBα protein levels ...... 91

Discussion ...... 92

Chapter 5: Conclusions ...... 108

References ...... 115

List of Tables

Table 1. Pulmonary function following exposure to >95% O2...... 51

Table 2. Pulmonary function following exposure to 85% O2...... 79

List of Figures

Figure 1. COX-2 and COX-1 levels following >95% O2...... 40

Figure 2. Acetylsalicylic acid dissociation and plasma levels...... 41

Figure 3. Survival curve during >95% O2...... 42

Figure 4. Body weights...... 43

Figure 5. Bronchoalveolar lavage protein concentration...... 44

Figure 6. Prostanoid levels...... 45

Figure 7. Mac-3+ cells...... 46

Figure 8. Chemokine protein levels...... 47

Figure 9. Chemokine levels in BAL...... 48

Figure 10. Chemokine levels in lung homogenates...... 49

Figure 11. Morphometry on day 7 and 28...... 50

Figure 12. Pressure-Volume loop following >95% O2...... 52

Figure 13. Methacholine challenge...... 53

Figure 14. COX-2 and COX-1 expression following 85% O2...... 68

Figure 15. Survival during 85% O2...... 69

Figure 16. Body Weights...... 70

Figure 17. Bronchoalveolar lavage total cell count and protein concentration...... 71

Figure 18. Chemokines levels in BAL...... 72

Figure 19. F4/80+ cells...... 73 xii

Figure 20. Prostanoid levels...... 74

Figure 21. Hematopoeitic prostaglandin D synthase protein levels...... 75

Figure 22. Effect of aspirin on alveolarization...... 76

Figure 23. Effect of celecoxib on alveolarization...... 77

Figure 24. Effect of 15-epi-LXA4 on alveolarization...... 78

Figure 25. Pressure-Volume loop following 85% O2...... 80

Figure 26. COX-2 expression in nonciliated airway epithelial cells...... 97

Figure 27. COX-2 mRNA expression...... 98

Figure 28. COX-2 and COX-1 protein expression levels...... 99

Figure 29. LPS-induced prostanoid levels...... 100

Figure 30. Chemokine mRNA levels...... 101

Figure 31. Chemokine secretion...... 102

Figure 32. TNF-α and IL-6 expression levels...... 103

Figure 33. Effect of NS-398 on LPS-induced KC expression...... 104

Figure 34. LPS induces MAP kinase phosphorylation and IκB-α degradation...... 105

Figure 35. Regulation of COX-2 by SB203580 and SP600125...... 106

Figure 36. Regulation chemokines by SB203580 and SP600125...... 107

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Chapter 1: Introduction

Chronic lung disease in preterm infants

Definition, Pathology, and Characteristics

The rate of preterm birth continues to be a major health problem in the United

States and worldwide. Due to improvements in treatment over the past 30 years, mortality rates of infants born <36 weeks gestational age have dropped dramatically.

However, morbidities associated with preterm birth including lung injury continue to adversely affect preterm infants. Currently, there are no standard therapies to prevent the morbidities that influence the development of chronic lung disease in preterm infants.

Bronchopulmonary Dysplasia (BPD) is a chronic lung disease that was first described by Northway and colleagues in 1967 (1). Northway et al. observed that development of BPD most frequently occurs in infants who had previously developed

RDS and born between 30-36 weeks gestational age (2). These infants had over inflation of the lung, fibrosis, and reduced alveolar development (2). BPD was characterized by pulmonary inflammation, decreased alveolarization, pulmonary fibrosis, and scaring.

Preterm infants born <32 weeks gestation have underdeveloped lungs and cannot produce surfactant. They have difficulty breathing and are unable to effectively oxygenate resulting in development of respiratory distress syndrome (RDS). To prevent respiratory failure, these infants are provided with ventilation support and treated with supraphysiologic concentrations of supplemental oxygen. 1

Improvements in neonatal care have led to survival of preterm infants born <28 weeks gestational age. Upon introduction of surfactant, improved ventilation techniques, and use of lower oxygen concentrations in the 1990s, the etiology of BPD changed. In contrast to the infants with “old BPD”, these infants are born earlier and have mild inflammation, impaired alevolarization and vascularization, and diffuse fibrosis (3).

Currently, the diagnosis of BPD is based on the need for respiratory support for

>28 days of life and/or 36 weeks corrected gestational age (4, 5). Based on this definition, the mortality due to lung disease has decreased however the incidence of BPD following development of RDS remains relatively high among low birth weight infants

(6). The incidence of BPD is influenced by neonatal care and varies center to center, suggesting that clinical factors and treatment strategies influence the development (7).

Inflammation

Inflammation is a contributing factor to the development of BPD (8, 9). There are multiple sources of inflammation in preterm infants that include systemic maternal inflammation, in utero infection, hyperoxia exposure, ventilation, and postnatal infection.

Specifically, acute intraamniotic infection or chorioamnionitis, is highly associated with preterm birth (10, 11). Infection can be caused by pathogens such as Escherichia Coli, cytomegalovirus, and toxoplasma gondii creates a pro-inflammatory environment which can adversely affect the fetus (12). Systemic maternal inflammation due to chronic inflammatory diseases, such as diabetes, has been linked to preterm birth and development of BPD (13). Postnatally, preterm infants are at risk of nosocomial infection and development of sepsis while in the neonatal intensive care unit (NICU). 2

Exposure to supra-physiologic levels of oxygen induces oxidative stress and tissue damage that causes pro-inflammatory responses in the lung. Lung stretching due to mechanical ventilation is an additional source of lung injury and has been shown to increase pulmonary inflammation and contribute to development of BPD (14).

Pro-inflammatory responses are facilitated by increased levels of cytokines, chemokines, lipids, and infiltration of innate immune cells into the site of injury. Preterm infants who develop RDS and are at risk of developing BPD have increased expression of many pro-inflammatory mediators. These include interleukin (IL)-6, IL-8, IL-1β, and

IL-10 in whole blood (15), IL-1β and thromboxane (TX) B2 in tracheal aspirates (16), as well as increased activation of the transcription factor, nuclear factor kappa B (NFκB)

(17, 18).

Multiple studies have observed neutrophil and macrophage infiltration in the lungs of preterm infants who developed BPD. Expression of adhesion molecules, proteins that mediate neutrophil diapedesis, are increased in preterm infants who developed BPD (19, 20). Leukocyte chemoattractants such as complement 5a (C5a), leukotriene (LT) B4, and IL-8 are increased in infants who developed RDS compared to infants who did not (21, 22). Once in the lungs, neutrophil apoptosis causes degranulation and release of molecules and enzymes that further contribute to lung tissue injury. Conversely, apoptosis of neutrophils is an important process for resolution of lung injury (23). However, neutrophil apoptosis is altered in infants with BPD (24, 25).

The number of macrophages is also increased during the development of BPD (26).

Expression of monocyte chemoattractant protein-1 (MCP-1), a macrophage specific

3 chemoattractant, is increased in tracheal aspirates from preterm infants that developed

BPD (27). Inflammation is thought to be a significant contributor to lung injury and development of BPD and assessment of markers of inflammation may be a useful tool in identifying infants at risk.

Lung Function

Improved neonatal care in recent years has enabled younger and more fragile preterm infants to reach adulthood. Many studies have begun to investigate the long term effects of preterm birth and development of BPD on the lung. During the early years of life, infants who survive BPD exhibit persistent increases in compliance and lung volume due to deficits in alveolar development (28, 29). Lung function tests indicate that infants who were diagnosed with BPD have reduced force expiratory flow (FEF) and increased functional residual capacity (FRC), residual volume (RV), and bronchial responsiveness

(30). Also, infants who developed BPD are at higher risk of re-hospitalization during the first year of life (31).

Often children who survived BPD exhibit frequent wheezing and increased bronchial responsiveness, however, most do not take medication for their respiratory problems (32). Assessments of airway reactivity in children who were diagnosed with

BPD suggest that lung structure abnormalities contribute to airway responsiveness, rather than a persistence of inflammatory cells in the lung (33, 34). These findings support the idea that a diminished alveolar network may influence airway tethering to alveolar walls, resulting in increased airway reactivity (28). There is also evidence of the effects of chronic lung disease in adults with history of BPD (35, 36). Lung structure studies have 4 suggested that survivors of BPD have diminished alveolarization at 19-21 years of age

(37). The effect of preterm birth and chronic lung disease on the development of adult lung disease such as chronic obstruction pulmonary disease and emphysema remain unknown (38).

Oxidative Stress during BPD

Oxidative stress is a major component of tissue damage during hyperoxic lung injury. Increases in oxygen (O2) levels within the cell leads to an increase in reactive oxygen species (ROS) which include superoxide (O2˙), hydrogen peroxide (H2O2), and hydroxyl radical (OH˙). ROS can oxidize lipids, DNA, carbohydrates, and proteins resulting in alterations in cellular function. Hyperoxia exposure is the primary source of oxidative stress in preterm infants and markers of oxidation have been measured in infants who developed BPD (39, 40). Reduction in the concentration of oxygen used as therapy for preterm infants reduces the evidence of tissue oxidation and development of

BPD (41). Glutathione (GSH) is an important antioxidant system in the cell and levels are reduced in plasma of preterm infants (42) and infants who develop BPD (43). These studies suggest a reduced antioxidant capacity to respond to hyperoxia-induced oxidative stress in preterm infants. Further evidence of oxidation in preterm infants includes increased protein carbonyls (44), free iron levels in the blood (45, 46), and lipid peroxidation (47, 48).

Studies in transgenic mice have shown that antioxidant capacity is important for protection against hyperoxia. Mice overexpressing extracellular superoxide dismutase

(EC-SOD), an enzyme that converts O2˙ into H2O2, are protected from hyperoxia-induced 5 lung injury (49-51). While nuclear factor-erythroid 2 related factor 2 (Nrf2) deficient mice have are more susceptible to hyperoxia- induced lung injury compared to wild type mice (52). Reducing in ROS formation in newborn mice exposed to hyperoxia improved alveolarization (53, 54). Collectively, these data show that oxidative stress plays a critical role in hyperoxic lung injury.

Therapeutic Strategies

Antioxidant therapies have been a significant focus in neonatal care during the past 20 years. Treatment with exogenous bovine superoxide dismutase had modest improvements in infants who developed BPD (55). A more recent study evaluated recombinant CuZn-SOD supplementation and found no decrease in the incidence or severity of BPD (56). However, infants treated with CuZn-SOD had reduced rehospitalization and improved pulmonary outcomes (56). N-acetylcysteine, a GSH substrate, was given to preterm infants to enhance endogenous anti-oxidant capacities no benefit was observed (57). Thus far, the efficacy of antioxidant therapies has been relatively disappointing. Due to the complexity of BPD, additional therapeutic strategies may be required and used in combination with antioxidant therapies.

Antenatal administration of glucocorticoids is currently used to stimulate lung maturation and surfactant synthesis in utero if the fetus is at risk for preterm birth.

Postnatal steroids were used to reduce inflammation in infants at risk of developing BPD, but it is now rarely used because of its adverse neurological effects. Retinoic acid, a product from vitamin A metabolism, has also been assessed in newborn rodent models of lung development and has been shown to be an important mediator of alveolar septation 6

(58-60). Additionally, retinoic acid improves alveolar development during hyperoxia exposure to newborn mice (61-63). Interstitial cells within the alveolar epithelium secrete retinoic acids enabling alveolar septation (64, 65). Despite progress in the care of preterm infants, novel therapeutic strategies are required to reduce lung morbidities in infants at risk of developing BPD.

Newborn Hyperoxic Lung Injury

The pathogenesis of hyperoxic lung injury has been studied extensively in numerous animal models. The response to hyperoxia and degree of injury are dependent upon multiple factors including duration of exposure, oxygen concentration, and antioxidant capacity. Injury during exposure to lethal (can be 90-100% O2) levels of oxygen occurs in three phases: initiation, inflammatory, and destructive (66). Sublethal exposures (60%-80% O2) include additional fibrotic and proliferative phases which are focused on tissue repair (66).

Initially, hyperoxia exposure alters the metabolic rates of ROS production within a cell. Responses to hyperoxia are variable among different cell types. Continuous O2 exposure shifts the redox balance within the cell towards increased reactive oxygen species (ROS) levels. Increased ROS levels can lead to oxidation of proteins, lipids, and nucleic acids which results in abnormal cellular function (67). Lethal O2 exposure also alters the morphology of some cell types within the lung such as endothelial cells. In contrast, sublethal exposures increase ROS levels at lower rates and morphological changes are delayed. The activity of the enzymes, nicotinamide adenine dinucleotide phosphate quinone-oxidoreductase-1 (NQO1) and mitochondrial complex III, are 7 different in rats exposed to 60% versus 85% O2 (68). These data suggest that cells within the lung respond differentially to concentrations of oxygen and may influence tolerance to hyperoxia.

Oxidative stress leads to the initiation of cell death and pro-inflammatory pathways. Endothelial and epithelial permeability are increased allowing fluid to enter airspaces. Inflammatory cells such as platelets, macrophages, and neutrophils follow chemotactic gradients and enter the lung. Adult and newborn animals exposed to hyperoxia have increased levels of pro-inflammatory mediators and leukocyte infiltration into the lung (69-73). The timing of the inflammatory responses is delayed during sublethal exposure compared to lethal exposure.

As inflammation peaks the destructive phase begins. Alveolar epithelial cells and capillary endothelial cells undergo necrosis, while inflammatory cells proliferate. The destructive phase is characterized by significant lung tissue injury which ultimately becomes too severe to overcome and mortality can result.

The severity of the inflammatory and destructive phases is lessened by sublethal concentrations of O2. Decreased cellular injury allows resolution of pro-inflammatory responses and repair to begin to return the lung to homeostasis. Fibroblasts and macrophages coordinate tissue repair and restoration of the endothelium and alveolar epithelium. Prolonged exposure to sublethal hyperoxia can lead to impaired regulation of tissue repair and fibrosis can occur. Accumulation of fibrotic tissue diminishes lung function and structure.

Leukocyte Infiltration and Inflammatory Mediators

Pro-inflammatory cytokines, chemokines, and bioactive lipids have been measured and implicated in the pathogenesis of hyperoxic lung injury in newborn and adult models. Tumor necrosis factor-α, IL-6, MCP-1, and cytokine-induced neutrophil chemoattractant-1 (CINC-1) mRNA levels are increased in newborn rats (74), newborn mice (75), premature baboons (69), and term rabbits (70) exposed to hyperoxia.

Increased levels of transforming growth factor β (TGFβ), a profibrotic cytokine and

LTB4, a potent neutrophil chemoattractant, have also been observed in newborn mice exposed to hyperoxia (76-80). Cyclooxygenase (COX)-2 activity and subsequent metabolites, prostaglandin (PG) D2, PGE2, and TXB2 are increased in hyperoxia-exposed newborn mice compared to room air controls (81).

During inflammation, leukocytes infiltrate the lung which is guided by chemotactic gradients composed of chemokines. Newborn rats exposed to >95% O2 had increased levels of neutrophil chemoattractants, CINC-1 and macrophage inflammatory protein-2 (MIP-2) expression (82). Treatment with antibodies designed to neutralize

CINC-1 and MIP-2 during hyperoxia exposure was shown to decrease neutrophil infiltration, myeloperoxidase activity, and DNA damage compared to vehicle treated rats.

Further studies using neutralization of MCP-1 in newborn rats exposed to hyperoxia reduced neutrophil and macrophage lung infiltration, and oxidation (83). Improvement in lung function and alveolarization were also observed with anti-chemokine treatments (84,

85). Inhibition of 5-lipoxygenase (5-LO) results in reduction of LTB4 levels and

9 leukocyte infiltration, while preventing abnormal alveolarization (86-90). These studies suggest a key role for chemoattractants during hyperoxic lung injury.

Chemokine receptors, CXCR2 and CCR2, mediate neutrophil and macrophage infiltration during hyperoxia exposure (91, 92). Furthermore, inhibition of CXCR2, the receptor for CINC-1 and MIP-2 similarly reduced neutrophil infiltration (93) and improved lung alveolarization in rats exposed to hyperoxia (94). Depletion of macrophages reduces tissue oxidation and improves hyperoxia-induced pulmonary hypertension (95, 96). These studies support a role for neutrophils and macrophages in contributing to hyperoxia-induced inflammation and impairment of alveolar development.

Alveolarization and Lung Function

Lung development has 5 stages: embyonic, pseudoglandular, canalicular, saccular, and alveolar (38). Most of lung development occurs throughout embryogenesis, however, the alveolarization stage is initiated postnatally. Alveoli are required for exchange of O2 and carbon dioxide. Alveolar development is a complex process, but septation of saccules is an important process required to form alveoli (97). Postnatal vascular development is also thought to be a critical event for alveoli formation (98).

In humans, alveolarization begins after birth and continues for 3 years and potentially beyond (99). Alveolar development in rodents is estimated to occur between postnatal day 4 and day 13 (99). Similar to preterm infants at risk of developing BPD, newborn rodents are born into the saccular stage of lung development. Newborn rodents are utilized to model the effects of hyperoxia on alveolarization. Numerous studies have 10 shown that exposure to hyperoxia causes decreased alveolar septation and number (81,

87, 100, 101). Mechanical ventilation and hyperoxia exposure in preterm lamb and baboon models blunts alveolar developments (69, 102).

Airway disease is a common symptom for in survivors of BPD. Children who develop BPD also are more susceptible to developing asthma (103). Airway reactivity is a symptom of airway disease and characterized by increased sensitivity to methacholine, increased mucus production, airway smooth muscle cell hyperplasia, increased collagen deposition, and alterations in lung structure.

The effects of hyperoxia exposure on airway reactivity have been investigated in animal models. Exposure to 95% O2 for 8 days in 21-day old rats increased airway reactivity and airway smooth muscle cell proliferation compared to room air controls

(104-106). Airway reactivity continued following room air recovery for 16 days but was returned to normal by 48 days (107). Hyperoxia exposure also increased airway reactivity in lungs from newborn guinea pigs which was persistent following room air recovery for 9 days (108). These data suggest hyperoxia induced changes in the lung that result in increases in airway reactivity. However these changes are largely resolved over time.

Recent studies have provided insights into the mechanisms that contribute to hyperoxia induced airway reactivity. Exposure of newborn rats to 85% O2 for 14 days increases sensitivity to methacholine, while treatment to deplete the lung of mast cells reduced airway reactivity (109). Accumulation of mast cells in the lung may play an important role in hyperoxia induced airway reactivity. Hyperoxia increases expression of

11 neurotrophins in newborn mice and influence airways smooth muscle cell proliferation and contractility which may contribute to airway remodeling that occur following hyperoxia exposure (110).

In animal models, current studies have begun to investigate the long term consequences of hyperoxic exposure during the newborn period. Persistent reduction in alveolar surface area due to hyperoxia exposure leads to impairment of lung function as mice become adults (100, 111, 112). Interestingly, mice exposed to 100% O2 during postnatal day 1-4 displayed evidence of pulmonary hypertension at 67 weeks of age

(113). There is evidence that newborn hyperoxia exposure persistently alters inflammatory responses in the lung. Studies have also suggested that neonatal hyperoxia exposure in mice increase susceptibility to viral infection (114-116) and cigarette smoke

(117).

Role of Cyclooxygenase-2 (COX-2) during Lung injury

COX-2 Expression and Activity

COX isoforms, COX-1 and COX-2, are expressed in most tissues including the lung. COX-1 expression is primarily constitutive, while COX-2 expression is induced by various stimuli particularly during inflammation (118-120). Although this expression pattern is frequently seen in a variety of tissues, it is important to note that expression of

COX-1 and COX-2 varies by cell and tissue type. COX-2 expression is up-regulated in response to stress and pro-inflammatory stimuli. NFκB activation has been implicated as

12 a key mechanism that regulates COX-2 expression upon viral and bacterial infection in lung epithelial cells (121-123).

Eicosanoids are a class of arachidonic acid (20:4 ω-6) derived fatty acids that have many physiological properties. Phospholipase A2 (PLA2) cleaves arachidonic acid from the sn-2 position of phospholipids, making free arachidonic acid available for metabolism. COX both cyclooxygenase to and peroxidase activities to form the intermediate, PGH2. The membrane binding domain allows COX to associate with endoplasmic reticulum and nuclear envelope membranes. While the dimerization domain, mediated by hydrophobic interactions, modulates formation of COX homodimers. Dimerization is thought to be needed for COX activity to occur (124).

COX requires Fe from a heme moity to oxidize the Tyr385, located in the cyclooxygenase domian, into a radical. Formation of the Tyr385 radical is critical for cyclooxygenase activity (124). Cyclooxygenase activity results in the addition of O2 onto C-15 of arachidonic acid to form PGG2. Peroxidase activity reduces –OOH on C-15 into –OH to form the intermediate metabolite, PGH2.

PGH2 is a substrate for specific enzymes that form prostanoid products including

PGD2, PGE2, PGF2α, and TXB2. Additional enzymes metabolize PGH2 into specific prostanoids, for example, prostanglandin D synthase metabolizes PGH2 into PGD2.

Prostanoids induce intracellular signaling via binding specific G protein coupled receptors (GPCR) expressed on different cell types. Through receptor binding prostanoids regulate many cellular processes in the lung, including inflammation (125).

Role of COX-2 during lung inflammation

The role of COX-2 has been investigated in experimental models of acute inflammation. Early studies suggested that inhibition of COX-2 during the inflammatory response exacerbated inflammation (126, 127). These data support the hypothesis that

COX-2 could be important for the resolution of inflammation. During a model of acute peritoneal inflammation, COX-2 expression was found to increase during the early phase and the late phase of inflammatory response producing different products during the early and late phases (128). The early phase was characterized by high levels of PGE2 which was associated with increased inflammatory cell infiltration. The late phase had low levels of PGE2, while PGD2 levels were elevated. PGD2 is non-enzymatically converted into 15-deoxy-∆12, 14-PGJ2, which has been shown to have anti-inflammatory properties

(129). Elevation in 15-deoxy-∆12, 14-PGJ2 was associated with reduction in inflammatory cell infiltration in to peritoneal fluid (128). Together, these data suggest that COX-2 may be a key modulator for the initiation and resolution of inflammation.

Studies assessing the role of COX-2 in models of fibrosis, acute lung injury

(ALI), and allergic airway inflammation have produced conflicting results. COX-2-/- mice have increased airway reactivity but no changes in neutrophil infiltration upon LPS administration (130). Meanwhile, COX-2-/- mice have increased leukoctye infiltration and TNF-α expression compared to wild type mice following challenge with V2O5, a toxin that induces fibrosis (131). COX-2-/- mice have diminished lung function but no alterations in inflammation during bleomycin-induced fibrosis (132). Additional studies have suggested that PGE2 is an important negative mediator for attenuation of fibrosis

(133). Administration of PGE2 in bleomycin-treated mice reduced inflammation and improves lung function (134). PGE2 has also been suggested to mediate fibroblast apoptosis in idiopathic pulmonary fibrosis (IPF), which could reduce the severity of fibrosis (135).

COX-2 and its metabolites have important roles during allergic airway inflammation. PGD2 is a mediator that induces bronchoconstriction and stimulates mucus production during allergic airway inflammation (136). PGE2 induces bronchodilation and is considered bronchoprotective (137). Recent studies have suggested that COX-2 is important for T cell differentiation and expansion of the Th17 cell population during the progression of murine allergic airway inflammation (138).

Recent studies in models of ALI have suggested that COX-2 metabolites are important for mechanisms of resolution of inflammation (139). The anti-inflammatory metabolite 15-deoxy-∆12, 14-PGJ2 has been suggested to reduce carrageenan-induced

ALI in part through activation of Nrf2 and PPAR-γ (140). Together these studies suggest that the role of COX-2 and prostanoids is complex and can influence pulmonary inflammation as well as function. The role of COX-2 during lung injury remains unclear and the variability in responses could be attributed to the model and timing of assessments.

Aspirin and its effect on inflammation

Acetylsalicylic acid or aspirin is a non-selective COX inhibitor with unique anti- inflammatory properties. Aspirin was first synthesized in 1887 following its precursor, salicylic acid, was found to have undesirable side effects (141). Aspirin and salicylic 15 acid were used to reduce symptoms of inflammation including pain and swelling. The mechanism of action of aspirin was unknown until Vane and colleagues discovered that aspirin and salicylic acid inhibited COX and the production of prostaglandins (142, 143).

Aspirin has a higher selectivity for COX-1 than COX-2.

COX-2 acetylation

Following the isolation of COX enzymes, further investigation into the interaction between aspirin and COX revealed that aspirin acetylated a Serine residue of COX-1 and

COX-2 (144). Inhibition of COX-1 by aspirin prevents fatty acids from docking into the cyclooxygenase domain (145). In contrast, acetylation of COX-2 by aspirin induces an irreversible conformational change altering the orientation of fatty acids entering the cyclooxygenase domain (146). For example, acetylated COX-2 metabolizes arachidonic acid to form 15-hydroxyeicosatetraenoic acid (15-HETE) instead of PGH2 (147).

Additional studies demonstrated that 15-HETE formation by COX-2 is specific to aspirin

(146, 148-150). These studies lead to the discovery that aspirin increases the formation of 15-epi lipoxin A4 (15-epi-LXA4), a member of a class of lipids shown to enhance resolution of inflammation (151). In addition, recent studies have identified docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA) derived electrophilic lipids that are synthesized by COX-2 and have anti-inflammatory properties (152).

Lipoxins, Resolvins, and Protectins

Mechanisms that mediate the resolution of inflammation have emerged as an important focus of research. Lipoxin A4 (LXA4) was first discovered by Serhan et al. in

1984 and was shown to modulate neutrophil function (153). Further studies have shown that LXA4 reduces neutrophil chemotaxis and diapedesis (154-156). Additionally, LXA4 was shown to inhibit neutrophil-endothelial interactions and vascular permeability (157).

Additional lipid molecules have been discovered and mediate inflammatory resolution similar to lipoxins. E-resolvins (RvE1) and D-resolvins (RvD)/protectins (PD) are synthesized from eicosapentaenoic acid (EPA, 20:5 ω-3) and docosahexaenoic acid

(DHA, 22:6 ω-3), respectively, and have pro-resolution properties similar to lipoxins.

Formyl peptide receptor 2 (FPR2), a G-Protein Coupled Receptor, was discovered to be a receptor for LXA4 (158, 159). Expression of FPR2 or lipoxin A4 receptor

(ALXR) has been identified in humans and rodents (160, 161). The anti-inflammatory effects of LXA4 have been suggested to be through AXLR in a mouse model of acute inflammation (162). Lipoxins modulate neutrophils, at least in part, through regulation of polyisoprenyl phosphate (PIPP) and presqualene diphosphate (PSDP) signaling resulting in diminished neutrophil activation (163). Additionally, LXA4 has been shown to regulate chemotaxis of neutrophils through reducing expression of chemokines by epithelial cells (164, 165).

During inflammation, neutrophils consume cellular debris at the site of inflammation. Neutrophils undergo apoptosis and degranulate, releasing toxic molecules including proteases and ROS that can harm the surrounding tissue. LXA4 via ALXR stimulates neutrophil apoptosis (166). Removal of neutrophil apoptotic debris is a key process during the resolution of inflammation. LXA4 stimulates migration of

17 macrophages into sites of inflammation (167, 168) and mediates clearance of apoptotic neutrophils by macrophages (169, 170).

Lipoxins and resolvins have been shown to resolve lung inflammation during models of ALI, fibrosis, cystic fibrosis, and allergic airway inflammation. Increases in

15-epi-LXA4 via acetylation of COX-2 were associated with reduction in acid-induced lung inflammation (139). Treatment of mice with 15-epi-LXA4 during ALI caused decreases in myeloperoxidase activity and increases in neutrophil apoptosis (171) and treatment with a LXA4 analog reduced inflammation in mice challenged with LPS (172).

A recent study has shown that intravenous lovastatin administration increased the formation of 15-epi-LXA4 and attenuated acute mucosal inflammation in mice (173).

Furthermore, lipoxin analog was shown to reduce fibrosis including collagen deposition in mice treated with bleomycin (174). LXA4 levels were found to be reduced in cystic fibrosis patients compared to control patients with other inflammatory lung diseases

(175)

LXA4 and PD1 treatment reduces the allergy-induced airway inflammation and reactivity to methacholine challenge (176-178). In ex vivo studies, blood and BAL cells from patients with severe asthma display a limited ability to produce LXA4 compared to control patients (179, 180). RvE1 was protective for mice challenged with bacterial induced ALI (181) and was shown to increase resolution of allergic airway inflammation which was associated with reduced IL-23 expression (182). The pro-resolution properties of RvE1 during allergic airway inflammation may be through regulation of natural killer cells (183). RvD1 has similar pro-resolution properties in models of allergic airway

18 inflammation and acute lung injury (184, 185). These data suggest that lipoxins and resolvins enhance inflammatory resolution processes in mouse models of lung injury.

COX-independent effects of Salicylate and Aspirin

COX-independent mechanisms of action by aspirin have also been studied (186,

187). Treatment with aspirin and salicylate reduced carrageenan-induced cell infiltration while PGE2 levels were decreased by salicylate but not aspirin (188). These data suggested that aspirin may have effects independent of COX inhibition. At high concentrations, aspirin and salicylate inhibit NFκB activation (189-192). Further studies suggested that aspirin inhibited IκB kinase (IKKβ) activity preventing IκBα phosphorylation and subsequent degradation while MAP kinase activity was unaffected

(193). Aspirin, but not indomethacin and fluriprofen (other COX inhibitors), was shown to inhibit NFκB and IL-4 expression in stimulated T cells (194). Other studies have suggested that aspirin and salicylate inhibit Activating Protein-1 (AP-1) activation, a transcription factor that regulates pro-inflammatory genes (195, 196).

Aspirin has also been shown to stimulate adenosine release due to uncoupling of oxidative phosphorylation and depletion of adenosine triphosphate (ATP) from the cell

(197). Accumulation of adenosine was associated with inhibition of leukocyte adhesion to endothelial cells (198). Similar effects were observed in COX-2-/- and p105-/-

(precursor for the NFκB subunit, p50) mice, suggesting a COX independent effect of aspirin (198). Additional studies have suggested that aspirin could act as an antioxidant by scavenging the OH˙ radical (199, 200).

Specific Aims, Objectives, and Rationale

Preterm infants require oxygen therapy to maintain adequate oxygenation of the blood and tissues. Despite being a life saving measure, hyperoxia exposure causes lung injury and contributes to the impairment of alveolar development. Our objective was to assess the role of COX-2 activity in newborn hyperoxic lung injury. We hypothesized that COX-2 inhibition would reduce hyperoxia-induced lung injury. Further studies identified COX-2 expression and activity in a nonciliated airway epithelial cell line, mouse transformed Clara cells (MTCC).

Specific Aim I: Evaluate the role of COX-2 during hyperoxia-induced inflammation

 Sub Aim I: To test the hypothesis that pharmacologic inhibition of COX-2 will

decrease macrophage and chemokine expression in newborn pups exposed to

hyperoxia

Specific Aim II: Investigate the effect of COX-2 inhibition during alveolar development in newborn mice exposed to hyperoxia

 Sub Aim I: To test the hypothesis that inhibition of COX-2 will improve

alveolarization in newborn pups exposed to hyperoxia

 Sub Aim II: To test the hypothesis that inhibition of COX-2 will improve

pulmonary function in newborn pups exposed to hyperoxia

Specific Aim III: Evaluate the effect inflammation on COX-2 and expression in MTCC using the toll-like receptor 4 agonist, lipopolysaccharide (LPS).

 Sub Aim I: To test the hypothesis that LPS treatment will increase COX-2

expression and activity in MTCC

Chapter 2: Role of COX-2 in >95% O2 mouse model of newborn hyperoxia

Introduction

COX-1 and COX-2 expression in the lung varies by species during homeostasis and disease. Immunohistochemical analysis of the developing human lung found COX-2 expression during the progression of lung development (201). The fetal (16-32 weeks gestation), preterm (23-30 weeks gestation) and term (38-42 weeks gestation) lung demonstrated COX-2 staining in the alveolar epithelium. In preterm infants who developed BPD, there was expression of COX-2 in the bronchiolar epithelium but not the alveolar epithelium (201). In contrast to humans, sheep express only COX-1 during lung development (202). COX-1 expression was detected in the endothelium and airway epithelium during late gestation and postnatal life (202). In rodents, COX-1 and COX-2 are expressed in the lung particularly in the vasculature and bronchiolar epithelium (203).

COX-2 is also expressed in airway and alveolar epithelial cells, endothelial cells, and macrophages during the progression of lung infection (204-206).

During hyperoxia exposure in adult mice, COX-2 expression has been detected in alveolar type II cells, interstitial cells, and macrophages (207). Prostanoids including

TXB2 and PGE2 are increased in BAL of adult rabbits exposed to hyperoxia (208).

Recent studies have shown that COX-2 expression and activity is increased in lung tissues of newborn C3H/HEN pups exposed to 85% O2 or >95% O2 compared to room air 22 exposed mice (81). The subsequent levels of PGD2, PGE2, PGF2α, and TXB2 were similarly increased by hyperoxia exposure (81).

The role of COX-1 and COX-2 during newborn hyperoxic lung injury remains unknown. Acetylsalicylic acid, or aspirin, has different properties than other nonselective

COX inhibitors such as indomethacin. Through COX-2 acetylation, aspirin has been shown to reduce prostaglandin levels and stimulate production of lipid mediators with inflammatory resolving properties i.e. lipoxin A4. In the present studies, we evaluated the expression of COX-2 in the lungs of newborn pups and the effect of COX inhibition, via aspirin, during exposure to >95% O2 for 7 days. We hypothesized that acetylsalicylic acid would reduce hyperoxia-induced leukocyte infiltration. Secondly, we hypothesized that acetylsalicylic acid would improve hyperoxia-induced deficits in alveolarization.

Materials and Methods

Animal model. Within 16 hrs of birth, newborn C3H/HEN mice were exposed to room air or >95% O2. The following day mice began daily injections of phosphate buffered saline (PBS), 5, or 10 mg/kg aspirin. Stock solutions of aspirin were prepared daily by dissolving in PBS at 65-70 ºC. To avoid lung injury, dams were switched daily between room air and hyperoxia exposure. O2 levels in oxygen chambers were monitored daily using an oxygen sensor. On day 7, pups were anesthetized with 200 mg/kg ketamine and

20 mg/kg xylazine. Lungs were harvested or formalin fixed, while some hyperoxia exposed mice were placed in room air until day 28. Lung tissues and BAL supernatants were snap frozen and stored at -80ºC.

Salicylic and Aspirin Measurements. Quantitation of salicylic and aspirin was measured using protocol from Xu et al. (Xu 2009). Ten-day old pups were injected intramuscularly with vehicle or 10 mg/kg aspirin solution. At 0.5, 4, 24 hrs, blood was collected and stored at -80ºC. To prepare sample, 5 L 100 mg/mL potassium fluoride, 150 L 0.1 formic acid, and 15 L 6-methoxy-salicylic acid (internal standard) was added to 25-50

L aliquot of plasma. Cold acetonitrile was used to bring total final volume to 300 L.

Sample was centrifuged at 13000g at 4ºC for 5 min. Supernatant was collected and 5 L of supernatant was injected into LC-MS/MS. A 1 mg/mL stock of acetylsalicylic acid was prepared and heated at 65ºC for 10, 30, or 60 min to dissolve in sterile PBS for generation of a standard curve. Stock solutions were placed on ice, and then 5 uL was injected into LC-MS/MS.

Western blot. Protein concentrations of cell lysates were determined by Bradford assay.

Samples were separated by SDS-PAGE, and transferred to polyvinylidene fluoride

(PVDF) or nitrocellulose membranes at 25 V for 1 h. Following blocking for 1.5 h, blots were probed with primary antibodies for COX-1 (rabbit polyclonal, Cayman), or COX-2

(rabbit monoclonal, 1:200, Abcam, Cambridge, MA). For loading controls, α-tubulin

(rabbit polyclonal, 1:10000, Abcam) or β-actin (rabbit monoclonal, 1:10000, Abcam) primary antibodies were used. Horseradish peroxidase conjugated goat anti-rabbit or goat anti-mouse secondary antibodies (1:12000, BioRad Laboratories, Hercules, CA) were applied for 1 h. Immunoblots were developed using enhanced chemiluminescence western blotting detection (GE Healthcare, Buckinghamshire, UK) and band densities were quantified using Image Quant TL software, version 5.0 (GE Healthcare). During band quantification, background was subtracted.

Bronchoalveolar lavage (BAL) and cell counts. Lungs were flushed 3X with 300 µL sterile PBS. Aliquots were combined and BAL was centrifuged at 3000 rpm for 10 minutes and supernatant was recovered and stored at -80ºC. Prior to performing cell counts, cell pellets were resuspended in 50 µL ammonium-chloride-potassium lysis buffer (to lyse red blood cells) and 50 µL cold PBS, and then place on ice for 10 min.

Cells were again centrifuged at 3000 rpm for 10 minutes and resuspended in 50 µL PBS.

Cell counts were obtained with a hemacytometer using trypan blue exclusion.

25 photomicrographs at 100X magnification and manually counted. The number of cells per field was averaged.

Enzyme-linked immunosorbent assay (ELISA). KC, MIP-2, and MCP-1 levels in BAL samples were assessed using Duoset ELISA kits (R&D systems) by following the manufacturer’s protocols. A 96 well plate was coated with capture antibody and allowed to sit overnight at room temperature. Coated plates were washed with PBS-Tween.

Blocking buffer (1% bovine serum albumin) was added to plates for 1-2 h. Standard and samples were diluted and plated in duplicate. Then plates were placed in at 4ºC to incubate overnight. Then detection antibody was added to plate and allowed to incubate for 2 h followed by strepavidin for 20 min. Substrate, 3,3'-5,5'- tetramethylbenzidine

(TMB), was added to plate for 5-20 min. A 2 N H2SO4 solution was used to stop the reaction and concentrations were determined using a spectrophotometer. Standard curves were utilized to determine chemokine concentrations.

Morphometric Analysis. Formalin fixed lung sections were stained with H&E. Five representative microphotographs were taken at 100X magnification. Average number, area, perimeter, and septal thickness were quantified using Image Pro software.

Prostaglandin levels. Lung tissue was homogenized in 0.9 mL buffer containing 0.1 M

NaH2PO4, 0.9% NaCl, at pH 5 and 0.1 mL 0.1% butylated hydroxyltoluene. Internal standard containing 0.5 ng/µL of deuterated PGF2α, TXB2, PGD2, LTB4, and 5-HETE was added to each sample. To perform Bligh and Dyer lipid extraction, homogenized tissue was immediately added to 4X sample volume 2:1 chloroform/methanol and then centrifuged at 2000 rpm for 2 min. The organic phase was extracted and placed under a

26 stream of N2. The chloroform/methanol extraction step was repeated and the extraxts combined and dried under N2. Following evaporation of the organics, lipids were reconstituted in 100 µL ethanol. Samples were loaded on Shimadzu high performance liquid chromatography coupled with Applied Biosystems 4000 Q trap (HPLC/MS-MS).

Analytes were separated at a flow rate of 0.3 mL/min using 8.3 mM acetic acid, pH 5.7

(mobile phase A) and 1:1 acetonitrile/isopropanol (mobile B) on a Zorbax SB-C18 column. Standard curves were used to quantify concentrations (81).

Lung Function Assessments. Pulmonary function tests were performed the same a previous studies (100). On day 28, pups were anesthetized with 200 mg/kg ketamine and

20 mg/kg xylazine. The trachea was cannulated and pulmonary function was assessed using a SCIREQ flexivent. For each mouse, maneuvers were performed in triplicate and values were averaged. Pressure-Volume loop coordinates for each treatment group were averaged and graphed. To assess airway reactivity, vehicle (H2O), 5, 10, 15, 25, and 50 mg/mL methacholine was aerosolized by a nebulizer and total resistance was measured.

Statistics. Statistical analysis was performed using Graph Pad Prism. Data were analyzed by one way ANOVA followed by Newman-Keuls multiple comparison test to assess differences between groups.

Statistics. Statistical analysis was performed using Graph Pad Prism 6.0. Data were analyzed by two way ANOVA followed by Turkey’s multiple comparison test to assess differences between groups.

Results

COX-2 Protein Expression

COX-2 and COX-1 protein expression levels were measured in lung tissues by

Western blot (Figure 1). Compared to RA controls, COX-2 levels were significantly increased in pups exposed to >95% O2 for 7 days. COX-1 levels were not statistically different between O2 and room air (RA).

Aspirin and salicylic acid plasma levels

Aspirin and salicylic acid were measured in plasma by LC/MS-MS from 10-day old pups which had been injected with 10 mg/kg aspirin (Figure 2). At 30 minutes post injection, aspirin levels averaged 1 ng/mL, while salicylic acid levels averaged 30 ng/mL.

At 4 hours post-injection, salicylic acid levels averaged 2.75 ng/mL while aspirin levels were below limit of detection. Salicylic acid and aspirin were not detected 24 hrs following injection. Aspirin and salicylic acid were not detected in plasma from mice injected with PBS.

Mortality and Body Weights

Newborn pups were exposed to RA or >95% O2 for seven days. During exposures, pups were injected intramuscularly with vehicle, 5, or 10 mg/kg aspirin. After the seven day exposure, some litters of mice were placed back in RA until 21 days of life.

RA exposed pups had a 100% survival rate, while the survival rate of O2/vehicle , O2/5 aspirin, or O2/10 aspirin treated mice was 85%, 87.5%, or 90%, respectively. Mortality

28 was significantly decreased in all three hyperoxia exposed groups compared to

RA/vehicle mice (Figure 3).

Body weights were recorded following daily injections. Each litter had a gradual increase in weight gain each day during and after RA or >95% O2 exposure. On day 7 and day 28, the body weights of hyperoxia exposed mice were significantly lower than the RA/vehicle exposed pups (Figure 4).

Bronchoalveolar Lavage Fluid

Lungs were lavaged with PBS and collected from pups on day 7. BAL protein concentrations were measured by Bradford assay. Compared to room air controls, >95%

O2 exposure significantly increased BAL protein concentration in O2/vehicle and O2/5 aspirin mice (Figure 5).

Prostanoid levels

Prostanoid levels were measured in lung homogenates on day 7 (Figure 6). TXB2 and 15-deoxy-∆12, 14-PGJ2 levels were significantly increased in lung tissues from

O2/vehicle mice compared to RA/vehicle mice. Treatment with 5 mg/kg aspirin significantly lessened the increases in TXB2 levels induced by exposure to >95% O2.

PGE2, 6-keto PGF1α, PGJ2, and 13,14-dihydro-15-keto-PGD2 levels were not significantly different among the treatment groups.

Macrophage Counts

To assess macrophage infiltration, lung sections were immunohistochemically stained with Mac-3 antibody (Figure 7). Compared to RA controls, O2/vehicle pups had

29 significantly greater numbers of Mac-3+ cells in the lung. Meanwhile treatment with 5 mg/kg aspirin inhibited the macrophages infiltration induced by exposure to >95% O2.

Chemokine Expression

KC, MIP-2, and MCP-1 were measured in lung homogenates from mice exposed to RA or >95% O2 on day 1, 3, 5, 7, and 10 (Figure 8). On day 7, KC, MIP-2 and MCP-1 expression levels were significantly greater than RA exposed mice. On day 10, KC and

MIP-2 expression returned to levels similar to RA controls; however, MCP-1 expression levels remained significantly elevated. Interestingly, MIP-2 expression was elevated in

RA exposed mice on Day 5, 7 and 10.

KC and MCP-1 expression was measured in BAL and lung tissues by ELISA. In

BAL, O2/vehicle treated mice had significantly greater KC and MCP-1 levels than

RA/vehicle, RA/5 aspirin, and O2/5 aspirin (Figure 9). In contrast, KC levels were significantly higher in lung tissues of O2/vehicle and O2/10 aspirin pups than RA controls

(Figure 10). Interestingly, O2/10 aspirin had greater KC and MCP-1 levels than

O2/vehicle exposed mice.

Alveolarization

Alveolar development begins on postnatal days 3-4 (97). On day 7, pups exposed to >95% O2 did not exhibit significant differences in alveolar number, area, or perimeter than RA mice. RA/10 aspirin pups did however have significantly greater alveolar numbers compared to RA/vehicle treated pups (Figure 11).

On day 7, some litters were maintained in RA for 21 days after their exposure to

>95% O2. Lung alveolarization was assessed on day 28 at a time point where alveolar development is complete in mice (Figure 11). Pups exposed to >95% O2 had significantly reduced alveolar number and larger alveolar size than the RA/vehicle exposed pups and treatment with 5 and 10 mg/kg aspirin significantly reduced the alveolar area.

Lung Function

On day 7, mice exposed to >95% O2 were placed in RA for an additional 21 days.

Pulmonary function tests were performed using the SCIREQ flexivent. Mice exposed to hyperoxia had significantly greater lung compliance and significantly lower tissue elastance than RA exposed mice (Table 1). Airway resistance was elevated in mice exposed to hyperoxia compared to RA controls and the elevation was not lessened by aspirin treatment. Tissue damping, or tissue resistance, was modestly improved in O2/10 aspirin mice.

Pressure-Volume loop (PV loop) measurements were graphed and indicated that lungs of >95% O2 exposed mice were easier to inflate at a lower pressure compared to

RA controls (Figure 12). O2/10 aspirin mice had significantly reduced lung volume and lower volume following inflation with 30 cm H2O compared to O2/vehicle mice. Form of deflating, the curvature of deflating PV loop, was significantly decreased in hyperoxia exposed mice compared to RA/vehicle exposed mice (Table 1).

Airway reactivity was assessed following challenge with increasing concentrations of methacholine. Methacholine increased total resistance in mice from all 31 treatment groups (Figure 13). There were no significant effects of hyperoxia exposure or aspirin treatment on airway reactivity in response to methacholine.

Discussion

Analysis of plasma from pups that were injected with 10 mg/kg aspirin revealed that within 30 min there was a dissociation of aspirin into salicylic acid (Figure 2). The half-life of aspirin is 15-20 min and SA is 2-30 hrs but varies by the species studied

(209). Our data suggest that following intramuscular injection, there is rapid dissociation of aspirin into salicylic acid. The dissociation of aspirin in the blood is influenced by the environmet in the blood and interaction with protein present in the plasma. Following injection, aspirin and salicylic acid pass through the liver and are metabolized into gentisuric acid which is secreted in the urine (141). The short half-life of aspirin and salicylic acid are influenced by liver metabolism. Hyperoxia exposure alters lipid metabolism in the liver (210), therefore hyperoxia exposure could alter the metabolism of aspirin and salicylic acid. We did not assess the effects of hyperoxia exposure on aspirin and salicylic acid metabolism. Assessments of COX activity suggest that there was not an effect of hyperoxia on aspirin ability to inhibit COX activity.

To limit handling of newborn pups and injury following injection, pups were injected once per day. Based on our assessments of aspirin plasma levels, our effects on

COX-1 and COX-2 were transient. However, prostanoid level assessments indicated that treatment with 5 mg/kg aspirin reduced COX activity during room air and hyperoxia exposure on day 7.

Hyperoxia had a significant effect on the mortality and body weight of the pups.

During the 7-day exposure period, mice exposed to >95% O2 mice had a significantly lower survival rate of 85-90% compared to RA controls. Daily injections did not have an

33 effect on mortality because there was no mortality observed in RA/vehicle exposed mice

(Figure 3). Also, there was no mortality between day 8 and day 28. Overall, mortality during exposure to >95% O2 was relatively low.

Body weights increased daily in all treatment groups, regardless of exposure, which is an indication of effective feeding by the dams (Figure 4). On day 7 and 28, pups in >95% O2 had significantly reduced body weights compared to RA mice similar to that observed in other studies of neonatal hyperoxia exposure (81).

Previous studies have shown that COX-2 protein and activity levels are increased in lung tissues of newborn mice exposed to hyperoxia (81). Our data demonstrate that pups exposed to >95% O2 for 7 days had elevated COX-2 levels compared to RA controls but differences in COX-1 levels did not reach statistical significance (Figure 1).

The mechanisms responsible for the increases in COX-2 expression in our model are unknown. The expression of COX-2 is regulated by pro-inflammatory stimuli which activate NFκB-mediated pathways (118). Increased levels of reactive oxygen species

(ROS) within the cell also leads to activation NFκB-mediated pathways (211). Recent studies have shown that ROS can regulate COX-2 expression in an alveolar epithelial cell line, A549 cells (212). We speculate that COX-2 expression is influenced by hyperoxia- induced ROS levels and pro-inflammatory cytokines in the lung.

TXA2 levels increase when thromboxane A2 synthase enzymatically metabolizes

PGH2 into an unstable product, TXA2. Non-enzymatically TXA2 is converted to a stable metabolite, TXB2 (213). Studies have suggested that TXB2 mediates pro-inflammatory responses. Other models of have suggested that TXB2 regulates vasodilation during

34 allergic airway inflammation and vascular permeability during acute lung injury (214).

Recent studies have shown that inhibition of TXA2 synthase reduced oleic acid-induced lung injury (215, 216). Additional studies have shown that inhibition of thromboxane receptors improves blood oxygenation during oleic acid-induced lung injury in rabbits

(217). Ketoconazole, an inhibitor of TXA2 synthase, was evaluated a potential treatment for patients at risk of developing acute lung injury (218). However, it was proven to be ineffective in reducing duration of ventilation and mortality in patients with acute lung injury (219).

Hematopoietic and lipocalin-type prostaglandin D synthase metabolizes PGH2 into PGD2 (220). PGD2 is then non-enzymatically metabolized into 15-deoxy-∆12, 14-

PGJ2. Increases in PGD2 and 15-deoxy-∆12, 14-PGJ2 levels in lung tissues suggest that

PGD2 levels are increased by hyperoxia and are then metabolized into downstream metabolites. Our data indicate that COX inhibition altered the PGD2 metabolic pathway in both RA and hyperoxia exposed mice.

Models of allergic airway inflammation have suggested that PGD2 regulates bronchoconstriction and vasodilation (136). Recent studies have shown that 15-deoxy-

∆12, 14-PGJ2 has anti-inflammatory effects during lung injury (140, 221, 222). Multiple mechanisms of action has been suggested for the anti-inflammatory properties of 15- deoxy PGJ2 including functioning as a ligand for peroxisome proliferation activation receptor-γ (PPARγ) (223), activating Nrf2 (140, 222), and directly inhibiting the NFκB pathway (224-226). However, more recent studies have suggested that 15-deoxy-∆12,

14-PGJ2 is not an endogenous ligand for PPARγ (227). Our studies suggest that

35 hyperoxia increases levels of TXB2, PGD2 and its metabolites, which is associated with reduced macrophage infiltration into the lung (Figure 6). We speculate that TXB2, PGD2, and 15-deoxy-∆12, 14-PGJ2 could mediate macrophage infiltration in our model.

The measurement of multiple COX metabolites in one sample is an advantage for our studies (Figure 6). However, the cellular distribution of prostanoid synthases, particularly PGD2 and TXA2 synthases, in the lung remains undefined. Based on our data, it is difficult to determine which cell types are producing prostanoids. Our studies did not evaluate prostanoid levels at time points earlier than 7 days. There could be earlier changes in levels of other prostanoids such as PGE2 during hyperoxia exposure.

Further investigations were needed to understand the expression patterns, activity of prostaglandin synthases, and time course of prostanoid synthesis in newborn hyperoxia lung injury.

Chemokines, particularly KC and MCP-1, have been shown to play a role in leukocyte infiltration into the lung during newborn hyperoxic lung injury in rats (82-84).

Compared to RA controls, >95% O2 exposed mice had significantly increased protein levels of KC, MIP-2, and MCP-1 in lung tissues on day 7 (Figure 8). These increases correlated with the timing of influx of neutrophils and macrophages previously reported by Rogers et al. 2009 (81). Interestingly, MIP-2 levels in lung tissues of RA pups were elevated on day 5, 7, and 10 compared to day 1 and 3 (Figure 8). We speculate that MIP-

2 may have an undefined role in lung development.

KC and MCP-1 levels were increased in BAL from mice exposed to >95% O2, while O2/5 aspirin pups had KC and MCP-1 levels similar to RA controls (Figure 9). In

36 contrast, KC levels in lung tissues were significantly increased in O2/vehicle, while MCP-

1 was not significantly increased (Figure 10). Interestingly, O2/10 aspirin pups had 1- fold higher levels of KC and MCP-1 compared to O2/vehicle pups (Figure 10). The discrepancy between these two data sets could be due to differences in experiments or between BAL and lung tissue chemokine.

Treatment with 5 mg/kg aspirin also reduced >95% O2-induced macrophage infiltration into the lung (Figure 7). Our data suggest that reduced >95% O2-induced macrophage infiltration in 5 mg/kg aspirin treated mice may be due to, in part, reduced levels of PGD2, TXB2, KC, and MCP-1. Inhibition of thromboxane receptor leads to reduced MCP-1 levels in stimulated vascular endothelial cells (228). In additional studies, inhibition of TXA2 synthesis reduced MCP-1 and IL-8 mRNA in an animal model of acute lung injury (215). These data suggest that a reduction in macrophages infiltration could be due, in part, to a reduction in >95% O2-induced TXB2 levels.

Models of newborn hyperoxia lung injury suggest neutrophils and macrophages release pro-inflammatory mediators and ROS which can lead to a toxic environment for the developing lung. Treatment with antibodies and antagonists to neutralize chemokine action and receptor binding resulted in reduced lung injury and improved alveolarization

(82, 94-96). These data suggest that leukocytes contribute to injury and impaired alveolar development during newborn hyperoxia exposure.

Hyperoxia exposure impairs alveolar development in preterm infants and newborn mice (14, 81). On day 7, we observed a trend of reduced alveolar number and increased alveolar area in O2/vehicle pups (Figure 11). However, alveolar development

37 is still ongoing on day 7. Recent studies have shown that neonatal hyperoxia exposure induces persistent deficits in alveolarization and lung function weeks after the exposure period (100, 112). Therefore, we assessed alveolarization and lung function 3 weeks after exposure to >95% O2. On day 28, Alveolar number was not improved in O2/5 aspirin and O2/10 aspirin (Figure 11). However, alveolar area was significantly smaller in O2/5 aspirin and O2/10 aspirin mice compared to O2/vehicle mice. But these improvements did not result in an improvement in lung function (Table 1). Together, these data show that hyperoxia induces persistent deficits in alveolarization and COX inhibition does not lead to improved alveolar development.

The Scireq flexivent was used to assess lung function in mice. The first maneuver inflates the lung with a pressure of 30 cm H2O to measure the total lung capacity. Next the lung is ventilated with 3 inspiration/expiration cycles to determine dynamic compliance, elastance, and total resistance. Compliance and elastance are measurements that assess lung stiffness and recoil following inspiration/expiration. Total resistance measures the ability of air to flow through the lung. A constant pressure is applied to the lung to determine central airway resistance, tissue damping, and tissue elastance. Tissue damping and elastance measures how difficult it is for the lung tissue to inflate. Pressure- volume curves are determined by measuring lung volume during application of increasing and decreasing (0-30 cm H2O) pressure. Static compliance and elastance are determined when a constant pressure is applied to the lung.

Our data show that exposure to >95% O2 led to increased lung volume and compliance which correlated with reduced alveolar number and larger air spaces

(Table 1). Compared to O2/vehicle treated mice, treatment with O2/10 aspirin mice resulted in significantly lower lung volume and delivered volume but not compliance.

PV loops show that lungs from hyperoxia exposed mice were easier than RA exposed mice to inflate (Figure 12). Mice treated with 10 mg/kg aspirin and exposed to hyperoxia had lower lung volume at 30 cm H2O pressure than O2/vehicle mice.

Previous studies have shown that newborn hyperoxia exposure increased airway reactivity which was characterized by thickening of the airway smooth muscle layer (108,

109). To assess airway reactivity, we challenged mice on day 28 with increasing doses of methacholine and measure total resistance (Figure 13). We found that hyperoxia and aspirin treatment had no significant effect on airway reactivity in the lung. Due to the size of the mice we were unable to measure airway reactivity immediately following hyperoxia exposure. Immediately following exposure to 95% O2, 21 day old rats had increased airway reactivity. But after 16 days of recovery in RA, airway responsiveness returned to baseline levels (107). We speculate that hyperoxia exposure may alter air reactivity after 7 days, however these effects resolved by day 28.

In conclusion, treatment with 5 mg/kg aspirin reduced macrophage infiltration and levels of pro-inflammatory mediators in pups exposed to >95% O2. However, aspirin treatment, with doses of 5 and 10 mg/kg, did not improve deficits in alveolar development or lung function. COX-1 and COX-2 may have an important role in the macrophage infiltration to hyperoxia exposure.

Figure 1. COX-2 and COX-1 levels following >95% O2.

(A) COX-2 and (B) COX-1 protein expression levels were measured by Western blot. Data were analyzed by using unpaired t test. Data are expressed as mean ± SEM, p<0.05, (A) n=8, (B) n=4 per group. Significant differences are indicated with symbols: RA (*).

Figure 2. Acetylsalicylic acid dissociation and plasma levels.

(A) To prepare stock solution, 0.5 mg acetylsalicylic acid was heated in 10 mL PBS for 10, 30, or 60 min. (B) Ten day old pups were injected with vehicle or 10 mg/kg acetylsalicylic acid and blood was collected after 30 min, 4 h, and 24 h. Acetylsalicylic acid and salicylic acid levels were measured using LC-MS/MS.

Figure 3. Survival curve during >95% O2.

Data were analyzed by using log rank (Mantel-Cox) test. Data are expressed as mean ± SEM, p<0.05, n=23-44 per group. Significant differences are indicated with symbols: RA/vehicle (*).

Figure 4. Body weights.

Body weights were recorded on (A) day 7 and (B) day 28. Data were analyzed by using two-way ANOVA with Turkey’s multiple comparison test to assess differences between groups. Data are expressed as mean ± SEM, p<0.05, (A) n=26-48 per group, (B) n=27- 36 per group. For (A) day 7 and (B) day 28, there is a significant effect of exposure and injection. Significant differences are indicated with symbols: RA/vehicle (*).

Figure 5. Bronchoalveolar lavage protein concentration.

BAL was collected and protein concentration was measured by Bradford assay. Data were analyzed by using two-way ANOVA with Turkey’s multiple comparison test to assess differences between groups. Data are expressed as mean ± SEM, p<0.05, n=5-8 per group. There is a significant effect of exposure. Significant differences are indicated with symbols: RA/vehicle (*).

Figure 6. Prostanoid levels.

Prostanoid levels in lung homogenates were measured by LC-MS/MS. Data were analyzed by using two-way ANOVA with Turkey’s multiple comparison test to assess differences between groups. Data are expressed as mean ± SEM, p<0.05, n=4-10 per group. There are significant effects of injection for PGF2α, PGE2, 15-deoxy-∆12, 14- PGJ2, PGJ2, and 13, 14-dihydro-15-keto-PGD2. Significant differences are indicated with symbols: RA/vehicle (*), O2/vehicle ($).

Figure 7. Mac-3+ cells.

Five representative lung sections were stained for the macrophage surface marker, mac-3. Data were analyzed by using two-way ANOVA with Turkey’s multiple comparison test to assess differences between groups. Data are expressed as mean ± SEM, p<0.05, n=4-5 per group. There are significant effects of injection and exposure, as well as a significant interaction. Significant differences are indicated with symbols: RA/vehicle (*), O2/vehicle ($).

Figure 8. Chemokine protein levels.

KC, MIP-2, and MCP-1 protein levels were measured in lung homogenates by ELISA on day 1, 3, 5, 7, and 10. Data were analyzed by using two-way ANOVA with Turkey’s multiple comparison test to assess differences between groups. Data are expressed as mean ± SEM, p<0.05, n=3-5 per group. There are significant effects of exposure and day and a significant interaction. Significant differences are indicated with symbols: RA (*) on same day and RA day 1 (#).

Figure 9. Chemokine levels in BAL.

KC and MCP-1 levels were measure in BAL by ELISA. Data were analyzed by using two-way ANOVA with Turkey’s multiple comparison test to assess differences between groups. Data are expressed as mean ± SEM, p<0.05, n=4 per group. There is a significant effect of exposure for KC and MCP-1 and an effect of injection for KC. Significant differences are indicated with symbols: RA/vehicle (*).

Figure 10. Chemokine levels in lung homogenates.

KC and MCP-1 levels were measure in lung homogenates by ELISA. Data were analyzed by using two-way ANOVA with Turkey’s multiple comparison test to assess differences between groups. Data are expressed as mean ± SEM, p<0.05, n=3-4 per group. There is a significant effect of exposure for KC and MCP-1 and an effect of injection for KC. There is also a significant interaction for KC. Significant differences are indicated with symbols: RA/vehicle (*), O2/vehicle ($).

Figure 11. Morphometry on day 7 and 28.

Alveolar number, area and perimeter per high powered field were assessed in 5 representative photomicrographs on (A) day 7 and (B) day 28. Data were analyzed by using two-way ANOVA with Turkey’s multiple comparison test to assess differences between groups. Data are expressed as mean ± SEM, p<0.05, n=5-10 per group. There is a significant effect of exposure for (B) day 28. Significant differences are indicated with symbols: RA/vehicle (*), O2/vehicle ($).

RA + RA + 5 mg/kg RA + 10 O + 5 mg/kg O + 10 mg/kg O + Vehicle 2 2 Vehicle Aspirin mg/kg Aspirin 2 Aspirin Aspirin

Total Lung Capacity# 0.787±0.021 0.771±0.016 0.725±0.026 1.012±0.025* 1.048±0.029* 0.879±0.032*$ (mL)

Total Lung Capacity per 48.674±1.206 46.988±1.182 49.722±3.945 70.459±2.095* 69.104±1.850* 67.041±3.157* Body Weight# (mL/kg)

Delivered Volume#% 0.531±0.023 0.534±0.018 0.445±0.020 0.761±0.031* 0.809±0.034* 0.621±0.025*$ (mL)

Total Resistance# 0.995±0.030 1.006±0.032 1.460±0.309 1.359±0.216 1.141±0.064 1.294±0.107 (cmH O.s/mL) 2

Central Airway 0.270±0.037 0.319±0.031 0.492±0.102 0.706±0.204 0.518±0.059 0.462±0.075 Resistance#

(cmH O.s/mL) 2

Compliance# 0.025±0.001 0.024±0.001 0.022±0.001 0.034±0.001* 0.032±0.001* 0.029±0.001* (mL/cmH O) 2

Tissue Damping# 9.789±0.377 9.733±0.401 9.122±0.678 7.999±0.298* 8.389±0.208 9.496±0.414 (cmH2O/mL)

Tissue Elastance# 42.227±1.918 43.882±1.723 41.541±3.429 28.507±1.323* 30.806±0.035* 32.662±2.119* (cmH2O/mL)

Total Lung Capacity 0.521±0.025 0.529±0.017 0.629±0.200 0.736±0.033* 0.790±0.035* 0.594±0.025*$ in PV loop#%

(mL)

Inspiratory Capacity for 0.716±0.044 0.652±0.029 0.568±0.029$ 0.820±0.045 0.769±0.036 0.658±0.029 Zero Pressure#% (mL)

Form of Deflating PV 0.177±0.010 0.159±0.005 0.158±0.006 0.150±0.007* 0.139±0.004* 0.138±0.005* Loop#

(1/cmH O) 2

Static Compliance#% 0.050±0.003 0.047±0.002 0.040±0.002$ 0.057±0.003 0.053±0.003 0.045±0.002

Table 1. Pulmonary function following exposure to >95% O2.

Pulmonary function was assessed in mice exposed to >95% O2 for 7 days and RA recovery for 21 days. Data were analyzed by using two-way ANOVA with Turkey’s multiple comparison test to assess differences between groups. Data are expressed as mean ± SEM, p<0.05, n=12-25 per group. Significant effects of exposure are indicated by (#) and injection is indicated by (%). Significant differences are indicated with symbols: Different from RA/vehicle (*), O2/vehicle ($).

Figure 12. Pressure-Volume loop following >95% O2.

Lung volumes were measured during increasing and decreasing pressure. Data are expressed as mean, n=17-25 per group.

Figure 13. Methacholine challenge.

Mice were challenged with water (baseline), 5, 10, 15, 25, 50 mg/mL methacholine. Following treatment with each dose of methacholine, total resistance was measure. Data are expressed as mean ± SEM, p<0.05, n=5-8 per group.

Chapter 3: Role of COX-2 in 85% O2 mouse model of newborn hyperoxia

Introduction

The severity of morbidities in preterm infants has reduced over the past 20 years, and this has resulted in improved survival. Reducing the concentration of supplemental oxygen has been a key factor to the improved survival of preterm infants receiving care in the neonatal intensive care unit (NICU). Exposure to lower concentrations of oxygen reduces lung injury, oxidation, and the incidence of BPD (41). Therefore, oxygen exposure is closely monitored and oxygen concentrations are reduced as soon as it is physiologically possible. Currently in the NICU, preterm infants are exposed to 30%-

90% O2 based on their blood oxygenation requirements.

Preterm infants at risk of developing BPD still have evidence of inflammation and impaired alveolar and vascular development (14, 15). The severity of BPD is influenced by the duration and concentration of oxygen exposure. Infants exposed to moderate levels of oxygen but no longer require respiratory support at 28 days of life develop mild

BPD. Infants exposed to <30% O2 for at least 28 days develop moderate BPD and infants exposed to higher levels of oxygen (>30% O2) for at greater than 28 days develop severe BPD (5). Yee et al. demonstrated that the reduced alveolarization during hyperoxia exposure is dependent upon the oxygen concentration (112). Overall, hyperoxia exposure plays a key role in the development of chronic lung disease in preterm infants. 54

Our studies have shown that exposure to 85% O2 was similar to the >95% O2 model in increased COX-2 expression and activity, increased neutrophil infiltration, and reduced alveolar development (81). However, the number of infiltrating neutrophils in the lung were significantly reduced in pups exposed to 85% O2 compared to >95% O2 mice (81). On day 7, 85% O2 and >95% O2 exposed pups had the same number of infiltrating neutrophils. By day 14, the number of neutrophils increased by 2-fold in

>95% O2 exposed pups while the number of neutrophils in 85% O2 exposed pups remained about the same. These data suggest that oxygen concentration may influence the magnitude of the neutrophil infiltration.

Our data from chapter 2 suggest that aspirin treatment reduces inflammation but does not improve alveolarization during exposure to >95% O2. However, we observed trends toward improvement in alveolar area and lung function with 10 mg/kg aspirin treatment. We reasoned that exposure to a lower concentration of oxygen would help better define the role COX inhibition on hyperoxia-induced impairment in alveolarization and lung function. Therefore, we assessed the role of COX-2 in the developing lung during exposure to 85% O2. We evaluated the effects of COX-2 pharmacologic inhibition during exposure to 85% O2. We hypothesized that COX inhibition would reduce leukocyte infiltration and improve alveolar development in newborn mice exposed to 85% O2. In the present studies, the effects of aspirin were compared to celecoxib, a selective COX-2 inhibitor, and 15-epi-LXA4, a product of COX-2 acetylation.

Material and Methods

Animal model. Within 16 hrs of birth, newborn C3H/HEN pups were exposed to room air (RA) or hyperoxia (85% O2). The following day, mice received daily injections of vehicle (PBS), 10 or 40 mg/kg aspirin, 5 mg/kg celecoxib, or 40 µg/kg 15-epi-LXA4.

Dams were switched daily between RA and hyperoxia exposure. During exposures, O2 levels in chambers were monitored daily using an oxygen sensor. On day 14, lungs were lavaged with PBS or formalin fixed, while some hyperoxia exposed litters were placed in

RA until day 28. Lung tissues and BAL supernatants were stored at -80ºC.

Cells were again centrifuged at 3000 rpm for 10 minutes and resuspended in 50 µL PBS.

Cell counts were attained with a hemacytometer using trypan blue exclusion.

Leukocyte assessments. Lungs were formalin fixed at 25 mmH2O and embedded in paraffin. Lung sections were stained, immunohistochemically, with antibodies specific for macrophages (F4/80+). Leuckocytes were quantified by taking five representative microphotographs at 100X magnification and manually counted. The number of cells per field was averaged. Airspace and interstitial F4/80+ cells were quantified separately then summed together to determine total macrophage number.

ELISA. KC and MCP-1 levels in BAL samples were assessed using Duoset ELISA kits

(R&D systems) by following the manufacturer’s protocols. A 96 well plate was coated with capture antibody and allowed to sit overnight at room temperature. Coated plates were washed with PBST. Blocking buffer (1% BSA) was added to plates for 1-2 h.

Standard and samples were diluted and plated in duplicate. Then plates were placed in at

4ºC to incubate overnight. Then detection antibody was added to plate and allowed to incubate for 2 h followed by strepavidin for 20 min. Substrate, 3,3'-5,5'- tetramethylbenzidine (TMB), was added to plate for 5-20 min. A 2 N H2SO4 solution was used to stop the reaction and concentrations were determined using a spectrophotometer. Standard curves were utilized to determine chemokine concentrations.

Prostaglandin levels. Lung tissue was homogenized in 0.9 mL buffer containing 0.1 M

57 reconstituted in 100 µL ethanol. Samples were loaded on Shimadzu high performance liquid chromatography coupled with Applied Biosystems 4000 Q trap (HPLC/MS-MS).

Analytes were separated at a flow rate of 0.3 mL/min using 8.3 mM acetic acid, pH 5.7

(mobile phase A) and 1:1 acetonitrile/isopropanol (mobile B) on a Zorbax SB-C18 column. Standard curves were used to quantify concentrations (81).

Lung Function Assessments. Pulmonary function tests were performed the same a previous studies (100). On day 28, pups were anesthetized with 200 mg/kg ketamine and

20 mg/kg xylazine. The trachea was cannulated and pulmonary function was assessed using a SCIREQ Flexivent. For each mouse, maneuvers were performed in triplicate and values were averaged. Pressure-Volume loop coordinates for each treatment group were averaged and graphed. To assess airway reactivity, vehicle (H2O), 5, 10, 15, 25, and 50 mg/mL methacholine was aerosolized by a nebulizer and the snapshot maneuver was performed.

Statistics. Statistical analysis was performed using Graph Pad Prism. Data were analyzed by one way ANOVA followed by Newman-Keuls multiple comparison test to assess differences between groups.

Results

COX-2 Protein Expression

Newborn pups were exposed to RA or 85% O2 for 14 days. COX-2 and COX-1 protein expression levels in lung tissues were measured by Western blot (Figure 14).

Compared to RA controls, pups exposed to 85% O2 for 14 days had increased COX-2 protein levels but COX-1 protein levels remained similar to mice exposed to RA.

Immunohistochemical analysis show COX-2 expression in the lung in RA and 85% O2 exposed pups (Figure 14C). COX-2 was expressed in airway epithelials cells, macrophages, and cells within the alveolar walls.

Mortality and Body weights

Mortality during exposure to the 85% O2 for 14 days was similar to the >95% O2 exposure for 7 days model. Pups injected with vehicle or aspirin while exposed to RA had 100% survival (Figure 15). The survival rate was 85% in RA/5 celecoxib pups and

92% in RA/15-epi-LXA4. Death was also observed in vehicle, aspirin, and celecoxib injected pups while exposed to 85% O2. The survival rates were 93%, 83%, and 96% in

O2/vehicle, O2/10 aspirin, and O2/5 celecoxib, respectively.

On day 14 and 28, 10 and 40 mg/kg aspirin-treated pups had 5-10% lower body weights than RA/vehicle treated mice (Figure 16A and 16B). The body weights of

O2/vehicle and O2/5 Celecoxib pups were not different from RA controls on day 14, but were significantly lower on day 28 (Figure 16C and 16D). Pups treated with 15-epi-

LXA4 did not have different body weights from vehicle treated pups (Figure 16E).

BAL Protein Expression

Hyperoxia exposure significantly increased BAL protein concentration and cells recovered from BAL (Figure 17). Treatment with aspirin reduced hyperoxia induced

BAL protein concentration compared to vehicle injected mice. Celecoxib treatment reduced hyperoxia-induced increases in total cell number and BAL protein concentration

(Figure 17B). 15-epi-LXA4 treatment during hyperoxia exposure did not significantly affect BAL protein or cell number (Figure 17C).

Chemokine Expression

Hyperoxia exposed, vehicle injected pups had significantly increased KC and

MCP-1 BAL levels. Treatment with 10 and 40 mg/kg aspirin significantly reduced MCP-

1 expression, but not KC, in BAL of mice exposed to hyperoxia (Figure 18A). Celecoxib and 15-epi-LXA4 treatment hyperoxia-induced KC and MCP-1 expression (Figure 18B).

Interestingly, KC levels were greater in O2/40 aspirin and O2/15-epi-LXA4 treated mice compared to 10 mg/kg aspirin treated mice (Figure 18C).

Macrophage counts

Lung sections were stained with the macrophage surface marker, F4/80.

O2/vehicle pups had significantly increased F4/80+ cells per high power field (hpf) in the airspaces and alveolar interstitial walls (Figure 19). Injection with 40 mg/kg aspirin

(Figure 19A), 5 mg/kg celecoxib (Figure 19B), and 40 µg/kg 15-epi-LXA4 (Figure 20C) during hyperoxia exposure reduced the total number of F4/80+ cells compared to vehicle injected mice.

Prostanoid levels

We assessed the effect 40 mg/kg aspirin and 5 mg/kg celecoxib on prostanoid levels in lung tissues of RA and hyperoxia exposed mice on day 14 (Figure 20).

Compared to tissues obtained from RA/vehicle exposed pups, O2/vehicle pups had significantly greater PGE2, 15-deoxy-∆12, 14-PGJ2, and 13,14-dihydro-15-keto-PGD2.

Treatment with 40 mg/kg aspirin and 5 mg/kg celecoxib significantly inhibited hyperoxia-induced increases in PGE2, 15-deoxy-∆12, 14-PGJ2, and 13,14-dihydro-15- keto-PGD2. PGD2 and PGJ2 levels were lower in RA/40 aspirin and O2/40 aspirin pups compared to RA/vehicle pups. There were no significant changes in TXB2, PGF2α, and

8-iso-PGF2α levels. Studies have shown that aspirin treatment increases 15-epi-LXA4 levels (151). We were unable to detect LXA4/15-epi-LXA4 levels in lung homogenates.

Our data show that aspirin and celecoxib treatment reduced lung COX activity in our model.

Due to significant alterations the PGD2 pathway, we measured hematopoietic prostaglandin D synthase (HPGDS) protein levels in lung homogenates from pups exposed to RA or hyperoxia for 14 days by Western blot (Figure 21A). Pups exposed to hyperoxia had significantly higher HPGDS levels than RA controls (Figure 21B).

Immunohistochemical analysis shows that HPGDS was expressed within the airway epithelium. These data suggest that increased HPGDS protein levels may contribute to increased PGD2 metabolite levels from airway epithelial cells.

Morphometric analysis

Lung sections were stained with H&E and alveolar development was assessed on day 14 and day 28. On day 14 and 28, hyperoxia exposed, aspirin injected pups had similar deficits in alveolarization as O2/vehicle exposed mice (Figure 22). Celecoxib treatment did not significantly improve alveolar development on day 14 (Figure 23).

However on day 28, O2/5 celecoxib treated mice had significantly smaller alveolar area and perimeter compared to room air controls. Treatment with 15-epi-LXA4 did not improve 85% O2-induced deficits in alveolar development on day 14 (Figure 24).

Lung Function Assessments

Hyperoxia exposure significantly increased lung volume and compliance in vehicle and aspirin injected mice (Table 2). Pups treated with 5 mg/kg celecoxib had significantly reduced lung volume per g body weight, but lung compliance did not return to control levels. Pressure-Volume loops confirm that lungs exposed to hyperoxia had significantly increased lung compliance (Figure 25). Airway resistance and central airway resistance was not affected by hyperoxia exposure, aspirin, or celecoxib.

Discussion

Previous studies have shown that newborn pups exposed to 85% O2 experience reduced leukocyte infiltration compared to pups exposed to 95-100% O2 (81). However, the effect on postnatal lung development is similar, reduced alveolar development. Since preterm infants are exposed to lower levels of oxygen (30-90%) we assessed the effects of aspirin in a less inflammatory model exposure to 85% O2. Additional studies were performed with celecoxib and 15-epi-LXA4 to compare with the aspirin treatments.

Some RA exposed pups injected with celecoxib and 15-epi-LXA4 died during the first week of injection (Figure 15). This may be due to an effect of dam feeding or injury due to injections. Since the majority of pups that died in the RA/5 celecoxib treatment group were from one liter, we suspect that 5 mg/kg celecoxib did not have a significant effect on mortality. Hyperoxia-exposed pups exhibited an increase in mortality compared to RA-exposed pups. The survival of newborn pups exposed to 85% O2 was 85-95%

(Figure 15). During the experiments, pups in each treatment group experienced consistent weight gain each day. Hyperoxia exposed pups weighed significantly less than

RA/vehicle pups (Figure 16).

Exposure to 85% O2 increased epithelial and vascular permeability which is indicated by increased BAL protein concentration, total cell counts, and chemokine levels. Aspirin and celecoxib treatment reduced 85% O2-induced BAL protein concentration, while 15-epi-LXA4 showed no effect (Figure 17). Only 5 mg/kg celecoxib treatment reduced 85% O2-induced total cell counts (Figure 17). These data suggest that

COX-1 and COX-2 inhibition may affect vascular and alveolar permeability during 85%

O2 exposure.

Treatment with 10 or 40 mg/kg aspirin reduced hyperoxia-induced increases in

MCP-1, while celecoxib and 15-epi-LXA4 had no effect (Figure 18). Interestingly, treatment with 40 mg/kg aspirin and 40 µg/kg 15-epi-LXA4 increased BAL KC levels compared to O2/vehicle pups. Similar observations were made with aspirin treatment in chapter 2 (Figure 10). The mechanism responsible for this increase in KC levels by aspirin and 15-epi-LXA4 is unknown. The KC promoter includes DNA binding regions for NFκB, AP-1, and CAAT enhancer binding protein (C/EBP) (229). Aspirin and/or 15- epi-LXA4 may enhance a feedback loop for KC transcription. Another potential mechanism is an aspirin, prostanoid, or 15-epi-LXA4 metabolite could indirectly affect

KC expression.

Since this was also observed in 15-epi-LXA4 and aspirin-treated pups, aspirin may be stimulate endogenous LXA4 synthesis via COX-2 acetylation. Since this was not observed in celecoxib treated pups, 15-epi-LXA4 may enhance hyperoxia-induced KC expression in the lung. This effect could be unique to KC expression, since MCP-1 and

MIP-2 levels were not enhanced by aspirin or 15-epi-LXA4 treatment. Since KC is a neutrophil chemoattractant, future studies are required to determine if these increases in

KC levels result in neutrophil infiltration.

Immunohistochemical analysis for macrophages revealed that 85% O2 increased

F4/80+ cells in the lung compared to RA-exposed mice (Figure 19). O2/40 aspirin, O2/5 celecoxib, and O2/15-epi-LXA4 treatment reduced F4/80+ cells in the lung compared to

O2/vehicle pups (Figure 19). MCP-1 is a chemokine that mediates macrophage chemotaxis and has been implicated in the pathogenesis of newborn hyperoxic lung injury. In contrast to celecoxib and 15-epi-LXA4 treatment did not affect MCP-1 levels in BAL (Figure 18). Additionally, O2/10 aspirin pups also had significantly increased macrophage numbers despite reduced BAL levels of the macrophage chemokine, MCP-1.

Our data suggest that COX-2 inhibition, nonselective and selective, and 15-epi-LXA4 treatment reduces macrophage infiltration independently of elevated MCP-1 expression levels. Aspirin, celecoxib, and 15-epi-LXA4 may affect expression of other macrophage chemoattractants, such as MIP-1α, and may have a role in newborn hyperoxic lung injury. These treatments could also potentially affect expression of adhesion molecules expressed by macrophages that mediate their diapedesis into the lung.

Compared to RA/vehicle mice, O2/vehicle-exposed mice had significantly greater levels of PGE2 and PGD2 metabolites, 15-deoxy-∆12, 14-PGJ2 and 13,14-dihydro-15- keto-PGD2 in lung tissue (Figure 20). Treatment with 5 mg/kg aspirin and 5 mg/kg celecoxib significantly inhibited PGE2, 15-deoxy-∆12, 14-PGJ2, and 13,14-dihydro-15- keto-PGD2 in RA and hyperoxia exposed pups. LXA4 levels were below limit of detection for most lung tissues from aspirin treated pups.

PGD2 is enzymatically metabolized by 15-hydroxy-prostaglandin dehydrogenase to form 13,14-dihydro-15-keto-PGD2 (230). PGD2 and 13,14-dihydro-15-keto-PGD2 bind the chemoattractant receptor homologous-molecule expressed on T-helper-type-2 cells (CRTH2) on T cells, eosinophils, and monocytes and mediate chemotaxis (231-

233). Recent studies have suggested that PGD2 and 13,14-dihydro-15-keto-PGD2, via

65 activation of CRTH2, and PGE2, via the EP4 receptor, stimulate macrophage chemokinesis and/or chemotaxis (234). PGD2 has been shown to influence macrophage activation and ability to produce chemokines during allergic airway inflammation (235).

Inhibition of COX-2 reduces stimulated macrophage IL-6 production, suggesting that

COX-2 has a role in production of pro-inflammatory mediators by macrophages (236).

Our data suggest that COX activity contributes to macrophage infiltration induced by

85% O2.

Aspirin and celecoxib treatment reduced 15-deoxy-∆12, 14-PGJ2 levels in mice exposed to hyperoxia (Figure 20). 15-deoxy-∆12, 14-PGJ2 has been shown to have anti- inflammatory effects in models of lung injury (140, 221). In contrast, our studies show that increased levels of 15-deoxy-∆12, 14-PGJ2 is associated with increased chemokine expression during exposure to 85% O2. Recent studies have showed that15-deoxy-∆12,

14-PGJ2 binds to CRTH2 and stimulates lymphocyte chemotaxis (232). Since suppression of 15-deoxy-∆12, 14-PGJ2 levels were associated with reduced macrophage infiltration, we speculate that 15-deoxy-∆12, 14-PGJ2 may have a role in macrophage infiltration into the lung in our model.

The mechanism of action of 15-epi-LXA4 may be through activation of its receptor, ALXR. Previous studies have shown that LXA4 reduces connective tissue growth factor (CTGF) induced MCP-1 expression in cultured rat mesangial cells (164).

LXA4 was shown to inhibit NFκB and MAPK pathways stimulated by CTGF (164).

LXA4 has been shown to stimulate an inflammatory resolution macrophage phenotype

(237). In our model, 15-epi-LXA4 could alter the macrophage phenotype during

66 hyperoxia exposure thus affecting their infiltration. These changes could affect the manner in which macrophages respond to exposure to 85% O2. Although 15-epi-LXA4 does not affect MCP-1 expression, it may alter macrophage responses to chemokines such as MCP-1.

Although aspirin, celecoxib, and 15-epi-LXA4 administration reduced macrophage infiltration, they did not improve hyperoxia-induced deficits in alveolar development (Figures 22-24). Pups exposed to 85% O2 had significantly reduced alveolar number and larger alveolar area compared to RA exposed pups on day 14 and day 28. Impaired alveolar development persisted following 85% O2 exposure. Lung function assessments showed that deficits in alveolarization resulted in altered pulmonary function characterized by increased lung volume and compliance (Table 2). Aspirin or celecoxib treatment did not change 85% O2-induced increased lung compliance. In contrast to previous studies (104, 105), exposure to 85% O2 did not increase total resistance and central airway resistance. This could be due to the differences in response to hyperoxia previous rodent models.

Figure 14. COX-2 and COX-1 expression following 85% O2.

(A) COX-2 and (B) COX-1 protein expression levels were measured by Western blot. Data were analyzed by using unpaired t test. Data are expressed as mean ± SEM, p<0.05, n=6 per group. Significant differences are indicated with symbols: Different from RA (*). (C) Immunohistochemical staining of COX-2 in lung sections. COX-2 expression is found in airway epithelial cells, macrophages, and cells within the alveolar walls in pups exposed to RA and 85% O2. Arrows identify COX-2 positive cells.

Figure 15. Survival during 85% O2.

Data were analyzed by using log rank (Mantel-Cox) test. Data are expressed as mean ± SEM, p<0.05, n=12-31 per group. Significant differences are indicated with symbols: RA/vehicle (*).

Figure 16. Body Weights.

Body weights were recorded on (A), (C), (E) day 14 and (B), (D) day 28. Data were analyzed by using two-way ANOVA with Turkey’s multiple comparison test to assess differences between groups. Data are expressed as mean ± SEM, p<0.05, (A) n=12-31 per group. There is a significant effect of exposure for (C) and (D), effect of injection and interaction for (A) and (B). Significant differences are indicated with symbols: RA/vehicle (*).

Figure 17. Bronchoalveolar lavage total cell count and protein concentration.

On day 14, BAL was collected from lungs of mice treated with vehicle, (A) aspirin, (B) celecoxib, and (C) 15-epi-LXA4. BAL protein concentration was measured by Bradford assay, while total cells were counted using a hemacytometer and trypan blue exclusion. Data were analyzed by using two-way ANOVA with Turkey’s multiple comparison test to assess differences between groups. Data are expressed as mean ± SEM, p<0.05, (A) n=5-9 per group, (B) 4-9 per group, (C) 5-9. There is a significant effect on exposure for (A), (B), and (C). Significant differences are indicated with symbols: RA/vehicle (*).

Figure 18. Chemokines levels in BAL.

On day 14, KC and MCP-1 levels in BAL were measured by ELISA. Data were analyzed by using two-way ANOVA with Turkey’s multiple comparison test to assess differences between groups. Data are expressed as mean ± SEM, p<0.05, (A) n=5-8 per group, (B) n=3-6 per group, (C) n=5-6 per group. (A) There is a significant effect of exposure and injection for KC and a significant effect of exposure for MCP-1. (B) There is a significant effect of exposure for KC and MCP-1. (C) There is a significant effect of exposure and injection for KC and a significant effect of exposure for MCP-1. Significant differences are indicated with symbols: RA/vehicle (*), O2/vehicle ($).

Figure 19. F4/80+ cells.

Five representative photomicrographs of lung sections stained for F4/80+ cells were analyzed for macrophages indicated as F4/80+ cells. Data were analyzed by using one- way ANOVA with Newman-Keuls multiple comparison test to assess differences between groups. Data are expressed as mean ± SEM, p<0.05, (A) n=3-4 per group, (B) n=4 per group, (C) n=3-4 per group. There is a significant effect of exposure for (A), (B), and (C). Significant differences are indicated with symbols: RA/vehicle (*), O2/vehicle ($).

Figure 20. Prostanoid levels.

Prostanoid levels in lung homogenates were measured by LC-MS/MS. Data were analyzed by using two-way ANOVA with Turkey’s multiple comparison test to assess differences between groups. Data are expressed as mean ± SEM, p<0.05, n=4-7 per group. There is a significant effect of injection on all prostaglandin levels except for PGF2α. Significant differences are indicated with symbols: RA/vehicle (*), O2/vehicle ($).

Figure 21. Hematopoeitic prostaglandin D synthase protein levels.

(A) Hematopoeitic prostaglandin D synthase (HPGDS) protein expression levels were measured in lung tissues by Western blot on day 14. Data were analyzed by using unpaired t test. Data are expressed as mean ± SEM, p<0.05, n=3 per group. Significant differences are indicated with symbols: RA (*). (B) Immunohistochemical staining of HPGDS in lung sections. HPGDS expression is found in some airway epithelial cells in pups exposed to RA and 85% O2. Arrows identify HPGDS positive cells.

Figure 22. Effect of aspirin on alveolarization.

Alveolar number, area, and perimeter per high powered field were assessed in 5 representative photomicrographs stained with H&E on (A) day 14 and (B) day 28. Data were analyzed by using two-way ANOVA with Turkey’s multiple comparison test to assess differences between groups. Data are expressed as mean ± SEM, p<0.05, (A) n=3- 9 per group, (B) n=6-12. There is a significant effect of exposure for (A) day 14 and (B) day 28. Significant differences are indicated with symbols: RA/vehicle (*).

Figure 23. Effect of celecoxib on alveolarization.

Alveolar number, area, and perimeter per high powered field were assessed in 5 representative photomicrographs stained with H&E on (A) day 14 and (B) day 28. Data were analyzed by using one-way ANOVA with Newman-Keuls multiple comparison test to assess differences between groups. Data are expressed as mean ± SEM, p<0.05, (A) n=5-9 per group, (B) n=6-10. (A) There are significant effects of exposure and injection. (B) There is a significant effect of exposure. Significant differences are indicated with symbols: RA/vehicle (*).

Figure 24. Effect of 15-epi-LXA4 on alveolarization.

Alveolar number, area, and perimeter per high powered field were assessed in 5 representative photomicrographs stained with H&E on day 14. Data were analyzed by using one-way ANOVA with Newman-Keuls multiple comparison test to assess differences between groups. Data are expressed as mean ± SEM, p<0.05, (A) n=2-9 per group. There is a significant effect of exposure. Significant differences are indicated with symbols: RA/vehicle (*).

RA + 10 mg/kg RA + 40 mg/kg RA + 5 mg/kg O + 10 mg/kg O + 40 mg/kg O + 5 mg/kg RA + Vehicle O + Vehicle 2 2 2 Aspirin Aspirin Celecoxib 2 Aspirin Aspirin Celecoxib

Total Lung Capacity# 0.779±0.034 0.806±0.025 0.767±0.038 0.810±0.020 0.941±0.026* 1.008±0.038* 1.078±0.057* 0.905±0.035* (mL)

Total Lung Capacity per 45.891±1.173 49.346±1.251 48.614±2.416 46.789±1.731 63.441±1.912* 60.237±1.962* 65.818±3.251* 57.128±1.761*$ Body Weight# (mL/g)

Delivered Volume# 0.516±0.031 0.533±0.033 0.502±0.040 0.549±0.021 0.678±0.027* 0.744±0.038* 0.819±0.058* 0.649±0.034* (mL)

Total Resistance 1.070±0.038 1.137±0.081 1.336±0.154 1.127±0.136 1.259±0.081 1.267±0.097 1.133±0.086 1.250±0.092 (cmH2O.s/mL)

Central Airway 0.243±0.034 0.295±0.039 0.400±0.109 0.313±0.084 0.403±0.042 0.446±0.066 0.338±0.059 0.377±0.044 Resistance (cmH2O.s/mL)

Compliance# 0.023±0.001 0.026±0.002 0.021±0.001 0.026±0.001 0.028±0.001* 0.031±0.002* 0.035±0.001* 0.029±0.002 (mL/cmH2O)

Tissue Damping# 10.666±0.461 10.016±1.337 11.080±0.453 10.283±0.648 9.915±0.537 8.589±0.361* 8.683±0.581* 9.437±0.528 (cmH2O/mL)

Tissue Elastance# 45.516±2.766 42.270±5.500 45.186±2.607 37.516±2.306* 32.297±1.430* 29.710±2.145* 26.737±1.280* 31.893±2.973* (cmH2O/mL)

Total Lung Capacity 0.512±0.032 0.533±0.033 0.504±0.038 0.548±0.015 0.668±0.026* 0.701±0.025* 0.752±0.033* 0.635±0.032 in PV loop# (mL)

Inspiratory Capacity for 0.593±0.030 0.580±0.050 0.485±0.026 0.621±0.018 0.549±0.025 0.696±0.040 0.634±0.041 0.528±0.026 Zero Pressure# (mL)

Form of Deflating 0.160±0.014 0.154±0.007 0.133±0.005 0.144±0.004 0.128±0.005* 0.137±0.005 0.122±0.006* 0.142±0.030 PV Loop# (1/cmH2O)

Static Compliance 0.041±0.002 0.041±0.004 0.033±0.002 0.043±0.002 0.037±0.002 0.048±0.003 0.042±0.003 0.033±0.002*

Table 2. Pulmonary function following exposure to 85% O2.

Pulmonary function was assessed in mice exposed to 85% O2 for 14 days and RA recovery for 14 days. Data were analyzed by using one-way ANOVA with Newman- Keuls multiple comparison test to assess differences between groups. Data are expressed as mean ± SEM, p<0.05, n=8-13 per group. Significant effects of exposure are indicated by (#) and injection is indicated by (%). Significant differences are indicated with symbols: RA/vehicle (*), O2/vehicle ($).

Figure 25. Pressure-Volume loop following 85% O2.

Lung volumes were measured during increasing and decreasing pressure. Graph of pressure-volume loop from (A) aspirin and (B) celecoxib treatments. Data are expressed as mean, n=8-13 per group.

Chapter 4: COX-2 expression in mouse transformed Clara cells

Introduction

The airway epithelium is composed of multiple cell types and has many important functions that contribute to protecting the lung from invading pathogens and toxic particulates. It serves as a physical barrier, produces pro-inflammatory mediators, secretes mucus, and clears pathogens from the lung (238). Clara cells are identified as non-ciliated cells within the airway epithelium that express Clara cell secretory protein

(CCSP). Clara cells compose an estimated 22% of epithelial cells of the human distal airway (239). The murine lung has a larger Clara cell population than humans. Clara cells can be found from the tracheal epithelium to the distal airways in rodents (239)

(240).

CCSP is part of the secretoglobin protein family whose function remains poorly understood (241). Additional airway epithelial cells, such as goblet cells, express variable levels of CCSP and other secretoglobins (242, 243). The immune function of

Clara cells have been largely attributed to their high expression and secretion of CCSP.

CCSP is localized in granules within Clara cells (244). Studies in animal models have suggested that CCSP has an important anti-inflammatory role during lung injury. CCSP expression is reduced in mice exposed to hyperoxia (245), ozone (246), and LPS (247).

CCSP knockout and over expression models suggest CCSP has anti-inflammatory properties during lung injury (248-254). CCSP -/- mice have altered bronchoalveolar 81 lavage protein composition (255). Recent studies suggest that Clara cells may regulate macrophage phenotype though CCSP expression (256, 257). Studies have observed regulation of CCSP expression by pro-inflammatory cytokines and hyperoxia exposure

(258-262). The CCSP promoter includes AP-1 and HNF-3 binding sites (263). AP-1 activation during hyperoxia exposure results in reduction in CCSP expression (258), and

IFN-γ stimulates CCSP expression (259).

Patients who develop acute lung injury (ALI) have lower CCSP plasma and BAL levels suggesting it may be a useful biomarker for the development of ALI and acute respiratory distress syndrome (264, 265). CCSP expression was also shown to be decreased in preterm infants who developed mild and severe BPD (266). The use of

CCSP as a biomarker has recently been suggested for development of BPD (267, 268).

Clara cell number and CCSP expression are also reduced in smokers (269, 270). Further clinical studies are needed to provide more information about the reliability of CCSP as an indicator for the progression of lung disease in preterm infants and adults.

Recent studies in mice have suggested a pivotal role for Clara cells during initiation of pulmonary inflammation. Expression of a degradation resistant IκBα protein in transgenic mice under control of the CCSP promoter resulted in an inability to activate

NFκB and stimulate a pro-inflammatory response after LPS administration to the lung

(271). Compared to controls, these mice have diminished leukocyte infiltration and cytokine expression (271). Further studies using this model support a critical role for

Clara cell NFκB activation to stimulate an inflammatory response in the lung (272-277).

These studies suggest NFκB activation in Clara cells is critical for the initiation of lung inflammation.

Clara cells have also been shown to release inflammatory mediators.

Immortalized and primary Clara cells produce chemokines, KC and MCP-1, in response to LPS and TNF-α (278-280). Interestingly, in vitro and ex vivo studies suggest LPS does not induce secretion of TNF-α in Clara cells (278). During allergic airway inflammation in mice, Clara cells in the proximal airways produce mucus (281) and may modulate eosinophil infiltration (282). Furthermore, IL-13 increases Muc5A expression in mouse transformed Clara cells (281). Specific IL-13 expression in Clara cells increase mucus production and induces airway reactivity in mice (283). These data demonstrate that

Clara cells may modulate multiple pro-inflammatory processes during models of lung injury.

Clara cells are also important for the maintenance and restoration of the airway epithelium following lung injury. As a progenitor cell, Clara cells aid in maintaining epithelial renewal every 1-2 months (284, 285). Studies show that CCSP+/calcitonin gene related peptide (CGRP)+ progenitor cells proliferate to regenerate the bronchiolar epithelium following naphthalene injury (286). CCSP+ cells are able to differentiate into ciliated and goblet cells, but not alveolar cells, during naphthalene and hyperoxic injury

(287).

Mechanisms that regulate airway epithelial restoration remain poorly understood.

Extracellular matrix remodeling and signaling pathways within the airway epithelial microenvironment may influence the renewal process (288). Additional studies have

83 suggested that TGFβ signaling and the surrounding stroma regulate CCSP+ stem cells

(289).

Based on previous define roles of Clara cells during inflammatory processes, we hypothesized that Clara cells express additional pro-inflammatory mediators i.e. COX-2 and chemokines. Using immunohistochemical analysis, we found COX-2 expression within the airway epithelium in adult mice intratracheally challenged with Escherichia

Coli (E. Coli). We utilized mouse transformed Clara cells (MTCC) to model Clara cell, in vitro. We hypothesized that LPS treatment would increase COX-2 expression and activity. Additional studies investigated the effect of COX-2 on chemokine expression and activation of MAPK and NFκB pathways.

Materials and Methods

E. Coli Treatment and Immunohistochemistry. All mouse studies were performed using protocols that are approved by the IACUC at The Research Institute at Nationwide

Children’s Hospital. Eleven to twelve week old female C57B6 mice were anesthetized with isofluorane, and then inoculated intratracheally with vehicle (PBS) or 1 x 107

CFU/mouse E. coli (strain #12014, American Type Culture Collection, Manassas, VA).

Twenty four hours after infection, mice were sacrificed and lungs were fixed with formalin. Fixed lungs were paraffin embedded and microsections were mounted on microscope slides. Slides of lung sections were stained with antibodies specific for

COX-2 (rabbit polyclonal, 1:150, Cayman, Ann Arbor, MI) and CCSP (rabbit polyclonal,

1:1000, Seven Hill Bioreagents, Cincinnati, OH). Photomicrographs were taken of conducting or bronchiolar airways with a light microscope at 400x magnification.

Cell Culture and Treatments. Mouse transformed Clara cells (MTCC) and RAW 264.7 cells (a mouse macrophage cell line) were grown to 70-80% confluence for all studies.

MTCC or RAW 264.7 cells were cultured in 1X DMEM with 4.5 g/L glucose

(Mediatech, Inc., Manassas, VA) with 10% Fetal Bovine Serum (Mediatech, Inc.), and

1% Penicillin-Streptomycin (Mediatech, Inc.). MTCC or RAW 264.7 macrophages were treated with Dulbecco’s PBS (Mediatech, Inc.) or E. coli O111: B4 lipopolysaccaride

(cat# 437627, Calbiochem, Darmstadt, Germany) in serum-free media for the length of time indicated in each experiment. In additional studies, MTCC were treated with

DMSO, 20 µM SB203580 (Invivogen, San Diego, CA), 20 µM SP600125 (Invivogen), and 1 or 10 µM NS-398 (Cayman).

ELISA. MTCCs or RAW 264.7 macrophages were cultured on 24-well plates and allowed to adhere overnight. Cells were treated with DPBS, 1, 10, 100, or 1000 ng/mL

LPS and media was collected after 24 h. TNF-α, IL-6, and KC levels were measured in media using ELISA (Duoset ELISA kits, R&D Systems, Minneapolis, MN) according to the manufacturers’ protocols. Absorbance was determined spectrophometrically using a

Spectramax M2 Plate Reader (Molecular Devices, Sunnyvale, CA).

Western blots. Protein concentrations of cell lysates were determined by Bradford assay.

Samples (25 µg protein) were separated by SDS-PAGE, and transferred to nitrocellulose membranes. Following blocking for 1.5 h, blots were probed with primary antibodies for phospho-p38 (rabbit monoclonal, 1:1000, Cell Signaling, Danvers, MA), phospho-ERK

(rabbit monoclonal, 1:1000, Cell Signaling), phospho-JNK (rabbit monoclonal, 1:1000,

Cell Signaling), total p38 (rabbit monoclonal, 1:1000, Cell Signaling), total ERK (rabbit monoclonal, 1:1000, Cell Signaling), total JNK (rabbit monoclonal, 1:1000, Cell

Signaling), IκB-α (rabbit monoclonal, 1:1000, Cell Signaling), CCSP (1:1000), COX-1

(rabbit polyclonal, Cayman), or COX-2 (rabbit monoclonal, 1:200, Abcam, Cambridge,

MA). For loading controls, α-tubulin (rabbit polyclonal, 1:10000, Abcam) or β-actin

(rabbit monoclonal, 1:10000, Abcam) primary antibodies were used. Horseradish peroxidase conjugated goat anti-rabbit or goat anti-mouse secondary antibodies (1:12000,

BioRad Laboratories, Hercules, CA) were applied for 1 h. Immunoblots were developed using enhanced chemiluminescence western blotting detection (GE Healthcare,

Buckinghamshire, UK) and band densities were quantified using Image Quant TL software, version 5.0 (GE Healthcare). During band quantification, background was

86 subtracted.

Quantitative real-time PCR. RNA was isolated from cells using TRIzol reagent

(Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. cDNAs were synthesized using Oligo d(T) primers (Invitrogen), dNTP, Superscript III Reverse

Transcriptase kit (Invitrogen). cDNAs were loaded onto 96-well plates containing specific primers for KC, TNF-α, IL-6, CCSP, and β-actin (Table 1; Integrated DNA

Technologies, San Diego, CA), and SYBR green/ROX master mix (Qiagen, Valencia,

CA). Quantitative real time PCR (qRT-PCR) was performed using 7500 Applied

Biosystems Real time PCR System (Qiagen). CT values of specified proteins were normalized to the CT values of β-actin. Fold change was calculated by normalizing to vehicle (calculated using 2(-ΔΔCT)). Melt curves were utilized to ensure formation of a single product. Statistical analyses were performed on β-actin normalized values to assess differences between treatment groups.

Prostaglandin levels. Internal standard containing 0.5 ng/µL of deuterated PGF2α, TXB2,

PGD2, LTB4, and 5-HETE was added to each sample. To perform Bligh and Dyer lipid extraction, media was immediately added to 4X sample volume 2:1 chloroform/methanol and then centrifuged at 2000 rpm for 2 min. The organic phase was extracted and placed under a stream of N2. The chloroform/methanol extraction step was repeated and the extraxts combined and dried under N2. Following evaporation of the organics, lipids were reconstituted in 100 µL ethanol. Samples were loaded on Shimadzu high performance liquid chromatography coupled with Applied Biosystems 4000 Q trap

(HPLC/MS-MS). Analytes were separated at a flow rate of 0.3 mL/min using 8.3 mM

87 acetic acid, pH 5.7 (mobile phase A) and 1:1 acetonitrile/isopropanol (mobile B) on a

Zorbax SB-C18 column. Standard curves were used to quantify concentrations

(81).Statistics. Values from ELISA, density values, and prostaglandin levels were analyzed using one-way ANOVA with Newman-Keuls post-hoc. ΔCT values were analyzed by two-tailed student’s t test. Significant differences are indicated as p<0.05.

Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, La

Jolla, CA).

Results

COX-2 expression and activity

COX-2 expression in Clara cells during E. Coli infection was assessed by immunohistochemical staining of COX-2 and CCSP (Figure 26A). Intense COX-2 staining was observed in airway epithelial cells that also expressed CCSP.

Immunofluorescence co-locatization of COX-2 and CCSP confirm that CCSP expressing cells also express COX-2 (Figure 26B).

Compared to controls, COX-2 mRNA expression was increased in MTCC treated with 100 ng/mL LPS for 1 h (Figure 27). Treatment with LPS significantly increased

COX-2 protein expression at 2 h and was sustained to 8 h, while returning to baseline levels by 24 h (Figure 28A). COX-1 protein expression levels were not significantly altered by LPS treatment at the time points tested (Figure 28B).

To assess the effect of LPS on COX activity, MTCC were treated with vehicle or

100 ng/mL LPS for 2, 4, and 8 h (Figure 29). LPS treatment increased PGE2 and PGF2α levels after 4 h. TXB2 levels were not altered during the time points tested. MTCC were pretreated with vehicle or 10 µM NS-398, a COX-2 selective inhibitor, for 1 h and then

MTCC 100 ng/mL LPS for 4 h. NS-398 inhibited LPS-induced increases in PGE2 and

PGF2α. These data suggest that increases in PGE2 and PGF2α levels are due to increased

COX-2 activity.

Chemokine and cytokine expression

KC, MIP-2, and MCP-1 mRNA and protein levels were measured in MTCC treated with vehicle or 100 ng/mL LPS (Figure 30). Compared to vehicle treatment, LPS increased KC, MIP-2, and MCP-1 mRNA levels 8 h after LPS treatment. Chemokine secretion was measured by ELISA. KC, MIP-2, and MCP-1 was increased by LPS compared to vehicle controls (Figure 31).

Additional pro-inflammatory cytokines, IL-6 and TNFα levels were also assessed in MTCC following treatment with LPS. IL-6 and TNFα mRNA levels were significantly increased by LPS (Figure 32A). Surprisingly, IL-6 and TNFα protein concentrations were below limit of detection upon stimulation with 100 ng/mL LPS

(Figure 32B). In contrast, RAW 264.7 macrophages treated with 100 ng/mL LPS significantly increased TNFα and IL-6 in media compared to vehicle controls. However, there were detectable levels of TNFα in cell lysates from cells treated with 100 ng/mL

LPS (Figure 32C).

Effect of COX inhibition on Chemokine Expression

Production of PGE2 and PGF2α can induce autocrine signaling via binding GPCRs

(290, 291). We hypothesized that inhibiting the increases in PGE2 and PGF2α would affect chemokine expression. Pretreatment with 10 µM NS-398 did not significantly alter

KC expression following LPS treatment (Figure 33).

MAPK Phosphoylation and IκBα protein levels

LPS and Pam2CSK stimulated phosphorylation of p38, JNK, and ERK within 15 min treatment (Figure 34A). Decreases in IκBα protein levels, evidence of NFκB activation, were also observed following LPS treatment (Figure 34B).

MTCC were pretreated with SB203580 and SP600125 for 1 h and then vehicle or

LPS for 2 h. SB203580 and SP600125 did not alter LPS induced COX-2 expression after

2 h. MTCC were pretreated with vehicle, SB203580 (p38 inhibitor), and SP600125 (JNK inhibitor) for 1 h then vehicle or 1 ng/mL LPS. Media was collected after 4 h and 24 h.

SB203580 and SP600125 significantly reduced LPS induced KC expression after 4 h compared to LPS only treated cells (Figure 36A). After 24 h, SB203580 significantly increased KC secretion, while pretreatment with SB203580 and SP600125 significantly reduced KC secretion into media (Figure 36B). Unfortunately, the IκBα inhibitor, BAY-

117082, was cytotoxic to MTCC therefore the effect of NFκB activation was not assessed.

Discussion

Clara cells are emerging as an important cell population within the airway epithelium during lung injury (284). However, the specific mediators that are produced by Clara cells during inflammation are not well characterized. The role of COX-2 during inflammation is dynamic and recent studies demonstrate that COX-2 has a central role during the initiation as well as resolution of inflammation (292). A seminal report by

Gilroy et al. (128) indicated bi-phasic responses associated with COX-2 activation during a model of acute inflammation in the pleural cavity. The first phase was a classical pro- inflammatory response early in the course of injury, followed by a second anti- inflammatory phase involving the production of lipids with anti-inflammatory properties

(128). We observed changes in COX-2 expression in MTCC treated with LPS suggesting a potential role for Clara cells in response to LPS.

Immunohistochemical analysis show that COX-2 expression in small airways is present in non-ciliated airway epithelial cells in addition to other cell types (Figure 26A).

Positive staining in vehicle treated mice suggests that COX-2 may have a homeostatic role in the small airways. Immunofluorescence staining indicates that Clara cells express

COX-2 (Figure 26B). Recent studies in animal models have found that COX-2 is expressed during bacterial or viral challenge to the lung. COX-2 was detected in bronchial epithelial cells in nonhuman primates with acute severe pneumonia (205).

COX-2 expression was increased in the bronchiolar epithelium following parainfluenza virus and respiratory syncytial virus infection in neonatal lambs (206). While elevations in COX-2 and subsequent prostaglandin formation during inflammation are likely

92 transient and tightly regulated, we speculate that Clara cells exhibit increased COX-2 mRNA and protein, and production of prostaglandins during initial responses to E. coli infection.

The expression of COX-2 in the airway epithelium (Figure 26A) led us to investigate COX-2 expression and activity in an immortalized Clara cell line, using LPS treatment as a model of interaction with Gram negative bacteria. Our studies in MTCC indicated a rapid and substantial increase in COX-2 mRNA and protein expression in response to LPS (Figures 27-28). These data suggest that COX-2 expression may be regulated post-transcriptionally. Previous studies have shown that COX-2 mRNA is stabilized by pro-inflammatory stimuli (120). We speculate that LPS is increasing COX-

2 expression through similar mechanisms in MTCC.

Increases in COX-2 expression was preceded by an elevation in PGE2 and PGF2α levels in response to LPS (Figure 29). The role of PGE2 during inflammatory responses is complex and depends on the cell type, expression of receptors, and the microenvironment. During the initiation of inflammation in the lung, PGE2 regulates vascular and airway constriction increasing vascular and airway permeability (214, 293).

After the initial response, PGE2 is implicated in “class switching” to initiate pathways involved in reprogramming of inflammatory cells and activates inflammatory resolution pathways (139, 294, 295). Studies using COX-2 deficient mice have demonstrated an essential role for PGE2 expression in prevention of pulmonary fibrosis induced by vanadium pentoxide or bleomycin (131, 133) and bronchoconstriction in response to LPS

(130). Increases in PGE2 production by MTCC suggest an active role for Clara cells in

93 both stages of the inflammatory response.

The specific effects of elevated PGF2α remain less characterized than other prostaglandins, however PGF2α is associated with inflammatory responses (214, 296).

The primary physiological functions of PGF2α include vaso- and broncho-constriction by stimulation of smooth muscle cells that is observed early in the course of pulmonary infection (214). The lack of changes in the levels of TXB2 was surprising. One possible explanation is that the subsequent enzyme activity required for TXB2 formation, thromboxane A2 synthase, is not altered by LPS in Clara cells.

Cytokine and chemokine responses to LPS by MTCC were similar to previous reports investigating inflammatory responses of Clara cells (Figures 30-32). Elizur et al. reported increased chemokine mRNA and protein secretion in both immortalized and primary Clara cells treated with LPS (278). Similarly, we observed increases in KC,

MIP-2, and MCP-1 mRNA expression and secretion in MTCC treated with LPS (Figures

30-31). We evaluated the effects of COX-2 inhibition on LPS induced chemokine expression. In our studies, inhibition of COX-2 and thus prostaglandin production did not alter LPS-induced KC secretion in MTCC. These data suggest that COX-2 metabolic products do not influence KC expression in response to LPS in MTCC.

Interestingly, expression of TNF-α and IL-6 mRNA were increased in MTCC treated with LPS compared to vehicle-treated controls however protein levels in media were below the limit of detection (Figure 32). TNF-α, but not IL-6, protein was detected in cell lysates from MTCC but was not different between LPS and vehicle treated cells

(Figure 32C). We observed a similar finding upon treatment with Pam2CSK4, a TLR2

94 agonist, which stimulated KC secretion but did not stimulate TNF-α and IL-6 secretion in

MTCC (data not shown), suggesting these mechanisms are not specific to LPS treatment.

Similar observations have been previously reported in primary Clara cells treated with

LPS (278). Furthermore, CCSP-positive primary airway epithelial cells isolated from mice expressing constitutively active NFκB produce multiple chemokines but do not produce TNF-α or interleukin-1β (IL-1β) protein (272). The physiological significance or mechanisms responsible for the absence of significant cytokine production upon LPS stimulation are not clear. However, we speculate that Clara cells may contribute to chemotactic gradients for leukocyte recruitment and facilitate production of cytokines by other cell types such as macrophages.

Our data indicate that LPS treatment induces phosphorylation of p38, JNK, and

ERK and reduces IκBα protein levels in MTCC within 30 min (Figure 35). We demonstrated decreases in IκBα protein levels which is an indirect marker for NFκB nuclear translocation. Previous studies have suggested that rapid induction of COX-2 expression may be regulated by p38 (297). We found that COX-2 protein expression was not directly related to activation of p38 or JNK in MTCC, but may be dependent on

NFκB-mediated mechanisms (Figure 35). Additional studies using the IκBα inhibitor,

BAY-117082, resulted in cytotoxicity of MTCC; consequently, we were unable to test this hypothesis. We speculate that other cell signaling pathways may be involved in the regulation of LPS induced COX-2 expression in Clara cells.

Inhibition of p38 and JNK activation significantly reduced protein expression of

KC after 4 h LPS treatment but had little effect on KC protein levels at 24 h (Figure 36).

These data suggest that p38 and JNK may regulate early induction of KC expression, but their effects were not sustained, likely due to continued enhanced transcription of KC mRNA. Further investigation is needed to understand the transcriptional regulation of

COX-2 and KC expression in Clara cells.

In addition to the previously described roles for Clara cells in the pathogenesis of acute lung injury, our studies indicate that LPS modulates COX-2 expression in MTCC and the subsequent production of prostaglandins. Furthermore, these novel findings suggest a previously unidentified mechanism by which Clara cells may modulate pro- inflammatory responses within the small airways. Enhanced understanding of the role of

COX-2 in response to LPS in Clara cells could lead to directed therapies that would be clinically useful in the treatment of acute lung injury.

Figure 26. COX-2 expression in nonciliated airway epithelial cells.

Eleven to twelve week old mice were treated with vehicle or intratracheal E. coli and tissues fixed 24 h post treatment. (A) Lung serial sections were stained with CCSP or COX-2 antibodies. Photomicrographs were taken at 400X magnification. CCSP expressing cells are identified by solid arrow and non-CCSP expressing cells are identified by dashed arrow. (B) To assess localization, lung sections were stained with COX-2 and CCSP. Fluorescent images were taken at 100X magnification.

Figure 27. COX-2 mRNA expression.

MTCC were treated with vehicle or 100 ng/mL LPS for 1, 4, or 8 h. COX-2 mRNA expression levels were determined by qRT-PCR. Data are presented as fold change. Statistical analyses were performed on dCT values and analyzed using unpaired student t test. Data represent means ± SEM from 2 independent experiments (n=6, p<0.05). Significant differences are indicated by symbols: Vehicle (*).

Figure 28. COX-2 and COX-1 protein expression levels.

MTCC were treated with vehicle or 100 ng/mL LPS for 2, 4, 8, or 24 h. (A) COX-2 and (B) COX-1 protein levels were determined by western blot. Data were analyzed using one-way ANOVA with Newman Keuls post-hoc. Data represents means ± SEM (normalized to vehicle controls) from at least 3 independent experiments (n=9-12, p<0.05). Significant differences are indicated by symbols: Vehicle (*), 4 h (%).

Figure 29. LPS-induced prostanoid levels.

MTCC were treated with vehicle or 100 ng/mL LPS for 2, 4, or 8 h. Additionally, MTCC were pretreated with vehicle or 10 µM NS-398 for 30 min, then vehicle or 100 ng/mL LPS for 4 h. Data was analyzed using one-way ANOVA with Newman Keuls post-hoc. Data represents means ± SEM (normalized to vehicle controls) from at least 3 independent experiments (n=9-12, p<0.05). Significant differences are indicated by symbols: Vehicle (*), 2 h ($), 8 h (#), NS-398 (%).

100

Figure 30. Chemokine mRNA levels.

MTCC were treated with vehicle or 100 ng/mL LPS for 8 h. KC, MIP-2, and MCP-1 mRNA expression levels were determined by qRT-PCR. Data are presented as fold change from at least 3 independent experiments. Statistical analyses were performed on dCT values and analyzed using two tailed student’s t test (n=9-10, p<0.05). Significant differences are indicated by symbols: Vehicle (**).

101

Figure 31. Chemokine secretion.

MTCC were treated with vehicle or 100 ng/mL LPS for 24 h. Data was analyzed using one-way ANOVA with Newman Keuls post-hoc. Data represents means ± SEM (normalized to vehicle controls) from 5 independent experiments (n=15). Significant differences are indicated by symbols: Vehicle (*).

102

Figure 32. TNF-α and IL-6 expression levels.

(A) MTCC were treated with vehicle or 100 ng/mL LPS for 8 h. Transcription of TNF-α and IL-6 was evaluated by qRT-PCR. Data are presented as fold change. Statistical analyses were performed on dCT values and analyzed using two tailed student’s t test. Significant differences are indicated by symbols: Vehicle (*). Data represent means ± SEM from 3 independent experiments (n=10, p<0.05). (B) MTCC and RAW 264.7 macrophages were treated with vehicle or 100 ng/mL LPS for 24 h. Media was collected, TNF-α and IL-6 protein levels were measured in media by ELISA (n=6). (C) MTCC were treated with vehicle or 100 ng/mL LPS for 24 h. TNF-α protein levels were measured in cell lysates by ELISA. Data represent means ± SEM from 2 independent experiments (n=6). Significant differences are indicated by symbols: Vehicle (*).

103

Figure 33. Effect of NS-398 on LPS-induced KC expression.

MTCC were pre-treated with vehicle, 1, or 10 µM NS-398 for 30 min, and then treated with vehicle or 1 ng/mL LPS for 24 h. Media was collected and KC protein levels were measured by ELISA. Data were analyzed using one-way ANOVA with Newman Keuls post-hoc (n=9, p<0.05). Significant differences are indicated by symbols: Vehicle (*).

104

Figure 34. LPS induces MAP kinase phosphorylation and IκB-α degradation.

MTCC were treated with vehicle or 100 ng/mL LPS for 0, 15, 30, 45, 60, or 120 min. (A) p38, JNK, and ERK phosphorylation and total levels were assessed by Western blot. (B) IκB-α and α-tubulin protein levels were also evaluated by western blot. Blots are representative of at least 3 independent experiments.

105

Figure 35. Regulation of COX-2 by SB203580 and SP600125.

MTCC were pretreated with vehicle, 20 µM SB203580 (p38 inhibitor), or 20 µM SP600125 (JNK inhibitor) for 1 h. COX-2 protein levels were assessed after treatment with 100 ng/mL LPS for 2 h by western. Significant differences are indicated by symbols: Vehicle (*).

106

Figure 36. Regulation chemokines by SB203580 and SP600125.

MTCC were pretreated with vehicle, 20 µM SB203580 (p38 inhibitor), or 20 µM SP600125 (JNK inhibitor) for 1 h. Media was collected and KC secretion was analyzed by ELISA after treatment with 1 ng/mL LPS for (A) 4 h or (B) 24 h. Data were analyzed using one-way ANOVA with Newman Keuls post-hoc. Data represents means ± SEM (normalized to vehicle controls) from 2-3 independent experiments (n=6-9 p<0.05). Significant differences are indicated by symbols: Vehicle (*), LPS (#, $).

107

Chapter 5: Conclusions

After 25-30 years of research and significant improvements in neonatal care, chronic lung disease in preterm infants remains as significant problem in NICUs throughout the United States. The use of surfactant supplementation and lower oxygen concentrations for oxygen therapy has improved outcomes to some extent; however, more importantly the progression and phenotype of BPD has changed to a disease characterized primarily by decreased alveolarization. Inflammation remains a significant contributor to development of chronic lung disease through both maternal and neonatal sources which results in impaired postnatal alveolar and vascular development. In addition to hyperoxia exposure, preterm infants are subject to inflammatory responses due to nosocomial infections. Currently, there are no therapeutic strategies that target the pro-inflammatory responses in neonates at risk of developing BPD.

COX-1 and COX-2 are expressed in the developing lung of term and preterm infants (201). Preterm infants have increased levels of prostaglandins in tracheal aspirates compared to controls (16). The contributions of COX expression and activity in to development of BPD are unknown. Our goal was to investigate the role of COX-2 during hyperoxia exposure in the developing lung. Newborn mice and preterm infants

(<32 weeks gestation) are born in the saccular stage of lung development. Due to the similarity in postnatal lung development between humans and mice, we utilized a mouse

108 model of newborn born hyperoxic lung injury to study COX-2 expression. Our studies assessed the effect of pharmacologic nonselective and selective COX-2 inhibition on inflammation, alveolarization, and pulmonary function in two models of newborn hyperoxic lung injury. Acetylation of COX-2 by aspirin initiates the formation of lipoxins and resolvins (151). We evaluated the effect of 15-epi-LXA4 during exposure to

85% O2. Additionally, we have identified COX-2 expression in the airway epithelium particularly in Clara cells, and shown that LPS modulates COX-2 expression and activity in MTCC.

Resident alveolar macrophage populations regularly clear pathogens and airborne toxins from alveolar spaces. Following initiation of lung injury, monocytes infiltrated into the lung and differentiate into macrophages. Pro-inflammatory or M1 macrophages are responsible for production of pro-inflammatory cytokines, ROS, and proteases which contribute to lung injury (298). These macrophages also phagocytose pathogens and cellular debris from necrotic and apoptotic cells i.e. epithelial cells and neutrophils.

During the resolution phase of lung injury, macrophages help the lung return to homeostasis and acquire what is termed an alternative phenotype (M2) (299). This alternative phenotype is characterized by inflammatory resolution mechanisms (298).

Maintaining the balance between pro- and anti-inflammatory roles of macrophages is important for preventing the development of chronic inflammation.

COX inhibition, via aspirin and celecoxib, reduced the number of macrophages in the lung (Figures 7 and 20) and reduced TXB2, PGE2, and PGD2 metabolite levels

(Figures 6 and 21). Our studies suggest that COX-2 activity and subsequently its

109 products may be important for macrophage infiltration into alveolar walls and airspaces during exposure to hyperoxia. Macrophages begin to infiltrate the lung in newborn pups as early as 3 days in >95% O2 (81, 300). It is possible that COX inhibition prevented macrophage infiltration or stimulated clearance of macrophage from lung more rapidly than control pups exposed to hyperoxia. Additional studies are needed to determine the initiating events in macrophage infiltration and the effect of COX-2 on this process.

Modulation of COX-2 may be a useful strategy in maintaining the macrophage phenotype balance.

COX-2 activity gives rise to a diverse and complex array of metabolic products including lipoxins. Therefore we assessed the effects of 15-epi-LXA4, a product of

COX-2 acetylation by aspirin. Similar to aspirin and celecoxib treatment, 15-epi-LXA4 reduced macrophage infiltration into the lung during exposure to 85% O2 (Figure 20C).

Lipoxins have been implicated in the mechanisms that return the lung to homeostasis following injury (139, 301). In fact, there is accumulating evidence that lipoxins modulate macrophages and stimulate phenotypic changes (237). Additional studies have implicated resolvin D1, an ALXR agonist, in initiating the M2 macrophage phenotype

(184, 302), suggesting that acetylated COX-2 derived lipid mediators could influence macrophage function. Therefore further investigations are needed to determine effects of lipoxins and resolvins on the macrophage phenotype present during newborn hyperoxic lung injury.

The effect on macrophage infiltration by 15-epi LXA4 could be related to its activation of the lipoxin receptor, ALXR. ALXR is expressed by multiple cell types in

110 the lung (184, 301). While LXA4 was not significantly increased in lung tissues of pups injected with aspirin, we cannot exclude the possibility that 15-epi LXA4 is formed in the lung. Further characterization is needed to determine the effects of aspirin and/or 15-epi

LXA4 on macrophage function during newborn hyperoxia lung injury.

CCSP expression is reduced in preterm infants that develop severe BPD (266).

Previous studies reported COX-2 expression in the bronchial epithelium is increased in preterm with and without BPD compared to infants born at term (201). We identified

COX-2 expression in Clara cells and have shown that LPS transiently augments COX-2 expression and activity in MTCC (Figure 27). Our data suggest the Clara cells may be able to produce multiple prostanoids including PGE2, PGF2α and TXB2 (Figure 30) in addition to chemokines, KC, MIP-2, and MCP-1 (Figure 32). Clara cells are beginning to be considered as an important contributor to the pro-inflammatory processes in the lung

(257, 271). We speculate that COX-2 is part of the many pro-inflammatory mediators produced by Clara cells. Future studies are required to further characterize the role of

COX-2 in Clara cells during lung injury.

Chronic inflammation has been implicated in the pathogenesis of airway disease and asthma (303) and often present in survivors of BPD (33, 34, 103). Reducing chronic inflammation may be a useful strategy to prevent the development of these conditions.

Improvements in nutritional fatty acid supplementation in preterm infants in the NICU are currently being investigated as a potential therapeutic approach to reduce the risk of developing diseases such as BPD (304-306). Understanding the expression pattern of

111

COX-2, in addition to other fatty acid metabolizing enzymes, in preterm infants would help understand the therapeutic value of fatty acid supplementation.

Exogenous administration or enhancement of endogenous lipoxin and resolvin levels may be a novel therapeutic strategy to reduced inflammation in infants at risk of developing BPD. Lipoxin and resolvin levels have not been measured in preterm infants.

Careful supplementation of fatty acids, i.e. arachidonic acid and docosahexanoic acid may be required to optimize endogenous production of lipoxins and resolvins. Our studies are the first step towards understanding how hyperoxia exposure effects COX-2 expression, activity, and downstream metabolites.

Pulmonary inflammation is regarded as a contributing factor to deficits in alveolarization observed in preterm infants (307). Recent studies have suggested that inflammatory stimuli can impair alveolar development. Newborn pups exposed to LPS develop similar deficits in lung development to pups exposed to hyperoxia (308).

Another study showed that localized lung expression of IL-1β to airway epithelial cells in newborn pups causes development of a phenotype similar to pups exposed to hyperoxia

(60, 309). Administration of agents used to inhibit chemokine signaling has been shown to inhibit neutrophil infiltration and improve alveolarization in mice exposed to hyperoxia (84, 93, 94). These data suggest that pro-inflammatory pathways can directly alter pathways critical for alveolar development. However, these studies did not determine if assessed improvement in alveolar development was sustained to adulthood.

Deficits in alveolarization in survivors of BPD results in persistent impairments in lung function (103). Our data are similar to previous studies showing that hyperoxia

112 exposure impairs lung structure and function during RA recovery (100, 112) and lung function assessments in survivors of BPD support this concept (103). In the present studies alveolarization was not affected in newborn pups injected with celecoxib and aspirin while exposed to RA (Figures 23-24). Few studies have investigated the role of

COX-2 on alveolarization. Nagai et al. reported that indomethacin treatment impaired alveolarization in newborn rats (310). These adverse effects were attenuated by administration of PGE2, suggesting that prostaglandins are important for alveolarization.

Additional studies have suggested that PGE2 is also important for the stimulation of surfactant synthesis by alveolar type II cells (311). The potencies of aspirin, celecoxib, and indomethacin are different and may explain the normal levels of alveolarization observed in our studies. Pharmacologic inhibition has a transient effect of on COX activity, therefore studies in a COX-2 transgenic model would help better determine the role of COX-2 in newborn hyperoxic lung injury.

Although newborn mice offer a simple and easily manipulated model, they have limitations and do not exactly mimic the newborn infant. Since newborn mice are born at term, they have endogenous surfactant synthesis and induction of antioxidant responses to adapt to the new oxidative environment. Meanwhile most preterm infants are surfactant deficient at birth and require administration of exogenous surfactant. Preterm infants at risk of developing BPD are subjected to additional risk factors such as in utero infection, ventilation, and nosocomial infections. The influence of these factors on COX-

2 expression and activity merits further investigation.

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Our studies suggest that COX-2 may have role in modulating hyperoxia-induced macrophage infiltration. COX-2 has been extensively studied in many disease models however the role of COX-2 in lung injury, particularly in preterm infants, remains poorly defined. Further studies are needed to determine the role of COX-2 expression in Clara cells and assess COX-2 as a therapeutic strategy for preterm infants at risk for developing

BPD.

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References

1. Northway WH, Jr., Rosan RC, Porter DY. Pulmonary disease following respirator therapy of hyaline-membrane disease. Bronchopulmonary dysplasia. N Engl J Med 1967;276:357-368. 2. Northway WH, Jr. Bronchopulmonary dysplasia: Then and now. Arch Dis Child 1990;65:1076-1081. 3. Jobe AH, Ikegami M. Mechanisms initiating lung injury in the preterm. Early Hum Dev 1998;53:81-94. 4. Walsh MC, Wilson-Costello D, Zadell A, Newman N, Fanaroff A. Safety, reliability, and validity of a physiologic definition of bronchopulmonary dysplasia. J Perinatol 2003;23:451-456. 5. Ehrenkranz RA, Walsh MC, Vohr BR, Jobe AH, Wright LL, Fanaroff AA, Wrage LA, Poole K. Validation of the national institutes of health consensus definition of bronchopulmonary dysplasia. Pediatrics 2005;116:1353-1360. 6. Stoll BJ, Hansen NI, Bell EF, Shankaran S, Laptook AR, Walsh MC, Hale EC, Newman NS, Schibler K, Carlo WA, Kennedy KA, Poindexter BB, Finer NN, Ehrenkranz RA, Duara S, Sanchez PJ, O'Shea TM, Goldberg RN, Van Meurs KP, Faix RG, Phelps DL, Frantz ID, 3rd, Watterberg KL, Saha S, Das A, Higgins RD. Neonatal outcomes of extremely preterm infants from the nichd neonatal research network. Pediatrics 2010;126:443-456. 7. Ambalavanan N, Walsh M, Bobashev G, Das A, Levine B, Carlo WA, Higgins RD. Intercenter differences in bronchopulmonary dysplasia or death among very low birth weight infants. Pediatrics 2011;127:e106-116. 8. Ryan RM, Ahmed Q, Lakshminrusimha S. Inflammatory mediators in the immunobiology of bronchopulmonary dysplasia. Clin Rev Allergy Immunol 2008;34:174-190. 9. Speer CP. Inflammation and bronchopulmonary dysplasia: A continuing story. Semin Fetal Neonatal Med 2006;11:354-362. 10. Schmidt B, Cao L, Mackensen-Haen S, Kendziorra H, Klingel K, Speer CP. Chorioamnionitis and inflammation of the fetal lung. Am J Obstet Gynecol 2001;185:173-177. 11. Jobe AH. Antenatal factors and the development of bronchopulmonary dysplasia. Semin Neonatol 2003;8:9-17. 12. Garland SM, Ni Chuileannain F, Satzke C, Robins-Browne R. Mechanisms, organisms and markers of infection in pregnancy. J Reprod Immunol 2002;57:169-183. 13. Schmatz M, Madan J, Marino T, Davis J. Maternal obesity: The interplay between inflammation, mother and fetus. J Perinatol 2010;30:441-446. 115

14. Jobe AH. The new bronchopulmonary dysplasia. Curr Opin Pediatr 2011;23:167- 172. 15. Ambalavanan N, Carlo WA, D'Angio CT, McDonald SA, Das A, Schendel D, Thorsen P, Higgins RD. Cytokines associated with bronchopulmonary dysplasia or death in extremely low birth weight infants. Pediatrics 2009;123:1132-1141. 16. Watterberg KL, Demers LM, Scott SM, Murphy S. Chorioamnionitis and early lung inflammation in infants in whom bronchopulmonary dysplasia develops. Pediatrics 1996;97:210-215. 17. Cheah FC, Hampton MB, Darlow BA, Winterbourn CC, Vissers MC. Detection of apoptosis by caspase-3 activation in tracheal aspirate neutrophils from premature infants: Relationship with nf-kappab activation. J Leukoc Biol 2005;77:432-437. 18. Bourbia A, Cruz MA, Rozycki HJ. Nf-kappab in tracheal lavage fluid from intubated premature infants: Association with inflammation, oxygen, and outcome. Arch Dis Child Fetal Neonatal Ed 2006;91:F36-39. 19. Kotecha S, Silverman M, Shaw RJ, Klein N. Soluble l-selectin concentration in bronchoalveolar lavage fluid obtained from infants who develop chronic lung disease of prematurity. Arch Dis Child Fetal Neonatal Ed 1998;78:F143-147. 20. Ramsay PL, O'Brian Smith E, Hegemier S, Welty SE. Early clinical markers for the development of bronchopulmonary dysplasia: Soluble e-selectin and icam-1. Pediatrics 1998;102:927-932. 21. Groneck P, Gotze-Speer B, Oppermann M, Eiffert H, Speer CP. Association of pulmonary inflammation and increased microvascular permeability during the development of bronchopulmonary dysplasia: A sequential analysis of inflammatory mediators in respiratory fluids of high-risk preterm neonates. Pediatrics 1994;93:712-718. 22. Munshi UK, Niu JO, Siddiq MM, Parton LA. Elevation of interleukin-8 and interleukin-6 precedes the influx of neutrophils in tracheal aspirates from preterm infants who develop bronchopulmonary dysplasia. Pediatr Pulmonol 1997;24:331-336. 23. Martin TR, Nakamura M, Matute-Bello G. The role of apoptosis in acute lung injury. Crit Care Med 2003;31:S184-188. 24. Oei J, Lui K, Wang H, Henry R. Decreased neutrophil apoptosis in tracheal fluids of preterm infants at risk of chronic lung disease. Arch Dis Child Fetal Neonatal Ed 2003;88:F245-249. 25. Kotecha S, Mildner RJ, Prince LR, Vyas JR, Currie AE, Lawson RA, Whyte MK. The role of neutrophil apoptosis in the resolution of acute lung injury in newborn infants. Thorax 2003;58:961-967. 26. Jones CA, Cayabyab RG, Kwong KY, Stotts C, Wong B, Hamdan H, Minoo P, deLemos RA. Undetectable interleukin (il)-10 and persistent il-8 expression early in hyaline membrane disease: A possible developmental basis for the predisposition to chronic lung inflammation in preterm newborns. Pediatr Res 1996;39:966-975.

116

27. Baier RJ, Majid A, Parupia H, Loggins J, Kruger TE. Cc chemokine concentrations increase in respiratory distress syndrome and correlate with development of bronchopulmonary dysplasia. Pediatr Pulmonol 2004;37:137-148. 28. Colin AA, McEvoy C, Castile RG. Respiratory morbidity and lung function in preterm infants of 32 to 36 weeks' gestational age. Pediatrics 2010;126:115-128. 29. Filbrun AG, Popova AP, Linn MJ, McIntosh NA, Hershenson MB. Longitudinal measures of lung function in infants with bronchopulmonary dysplasia. Pediatr Pulmonol 2011;46:369-375. 30. Robin B, Kim YJ, Huth J, Klocksieben J, Torres M, Tepper RS, Castile RG, Solway J, Hershenson MB, Goldstein-Filbrun A. Pulmonary function in bronchopulmonary dysplasia. Pediatr Pulmonol 2004;37:236-242. 31. Smith VC, Zupancic JA, McCormick MC, Croen LA, Greene J, Escobar GJ, Richardson DK. Rehospitalization in the first year of life among infants with bronchopulmonary dysplasia. J Pediatr 2004;144:799-803. 32. Fawke J, Lum S, Kirkby J, Hennessy E, Marlow N, Rowell V, Thomas S, Stocks J. Lung function and respiratory symptoms at 11 years in children born extremely preterm: The epicure study. Am J Respir Crit Care Med 2010;182:237-245. 33. Halvorsen T, Skadberg BT, Eide GE, Roksund O, Aksnes L, Oymar K. Characteristics of asthma and airway hyper-responsiveness after premature birth. Pediatr Allergy Immunol 2005;16:487-494. 34. Kim do K, Choi SH, Yu J, Yoo Y, Kim B, Koh YY. Bronchial responsiveness to methacholine and adenosine 5'-monophosphate in preschool children with bronchopulmonary dysplasia. Pediatr Pulmonol 2006;41:538-543. 35. Greenough A, Dimitriou G, Bhat RY, Broughton S, Hannam S, Rafferty GF, Leipala JA. Lung volumes in infants who had mild to moderate bronchopulmonary dysplasia. Eur J Pediatr 2005;164:583-586. 36. Narang I. Review series: What goes around, comes around: Childhood influences on later lung health? Long-term follow-up of infants with lung disease of prematurity. Chron Respir Dis 2010;7:259-269. 37. Wong PM, Lees AN, Louw J, Lee FY, French N, Gain K, Murray CP, Wilson A, Chambers DC. Emphysema in young adult survivors of moderate-to-severe bronchopulmonary dysplasia. Eur Respir J 2008;32:321-328. 38. Shi W, Bellusci S, Warburton D. Lung development and adult lung diseases. Chest 2007;132:651-656. 39. Saugstad OD. Bronchopulmonary dysplasia and oxidative stress: Are we closer to an understanding of the pathogenesis of bpd? Acta Paediatr 1997;86:1277-1282. 40. Saugstad OD, Aune D. In search of the optimal oxygen saturation for extremely low birth weight infants: A systematic review and meta-analysis. Neonatology 2011;100:1-8. 41. Vento M, Moro M, Escrig R, Arruza L, Villar G, Izquierdo I, Roberts LJ, 2nd, Arduini A, Escobar JJ, Sastre J, Asensi MA. Preterm resuscitation with low oxygen causes less oxidative stress, inflammation, and chronic lung disease. Pediatrics 2009;124:e439-449.

117

42. Smith CV, Hansen TN, Martin NE, McMicken HW, Elliott SJ. Oxidant stress responses in premature infants during exposure to hyperoxia. Pediatr Res 1993;34:360-365. 43. Boda D, Nemeth I, Pinter S. Surface tension, glutathione content and redox ratio of the tracheal aspirate fluid of premature infants with irds. Biol Neonate 1998;74:281-288. 44. Varsila E, Pesonen E, Andersson S. Early protein oxidation in the neonatal lung is related to development of chronic lung disease. Acta Paediatr 1995;84:1296-1299. 45. Moison RM, Palinckx JJ, Roest M, Houdkamp E, Berger HM. Induction of lipid peroxidation of pulmonary surfactant by plasma of preterm babies. Lancet 1993;341:79-82. 46. Gerber CE, Bruchelt G, Stegmann H, Schweinsberg F, Speer CP. Presence of bleomycin-detectable free iron in the alveolar system of preterm infants. Biochem Biophys Res Commun 1999;257:218-222. 47. Ahola T, Fellman V, Kjellmer I, Raivio KO, Lapatto R. Plasma 8-isoprostane is increased in preterm infants who develop bronchopulmonary dysplasia or periventricular leukomalacia. Pediatr Res 2004;56:88-93. 48. Nycyk JA, Drury JA, Cooke RW. Breath pentane as a marker for lipid peroxidation and adverse outcome in preterm infants. Arch Dis Child Fetal Neonatal Ed 1998;79:F67-69. 49. Carlsson LM, Jonsson J, Edlund T, Marklund SL. Mice lacking extracellular superoxide dismutase are more sensitive to hyperoxia. Proc Natl Acad Sci U S A 1995;92:6264-6268. 50. Folz RJ, Abushamaa AM, Suliman HB. Extracellular superoxide dismutase in the airways of transgenic mice reduces inflammation and attenuates lung toxicity following hyperoxia. J Clin Invest 1999;103:1055-1066. 51. Ahmed MN, Suliman HB, Folz RJ, Nozik-Grayck E, Golson ML, Mason SN, Auten RL. Extracellular superoxide dismutase protects lung development in hyperoxia-exposed newborn mice. Am J Respir Crit Care Med 2003;167:400-405. 52. Cho HY, Jedlicka AE, Reddy SP, Kensler TW, Yamamoto M, Zhang LY, Kleeberger SR. Role of nrf2 in protection against hyperoxic lung injury in mice. Am J Respir Cell Mol Biol 2002;26:175-182. 53. Frank L. Hyperoxic inhibition of newborn rat lung development: Protection by deferoxamine. Free Radic Biol Med 1991;11:341-348. 54. Frank L, McLaughlin GE. Protection against acute and chronic hyperoxic inhibition of neonatal rat lung development with the 21-aminosteroid drug u74389f. Pediatr Res 1993;33:632-638. 55. Rosenfeld W, Evans H, Concepcion L, Jhaveri R, Schaeffer H, Friedman A. Prevention of bronchopulmonary dysplasia by administration of bovine superoxide dismutase in preterm infants with respiratory distress syndrome. J Pediatr 1984;105:781-785. 56. Davis JM, Parad RB, Michele T, Allred E, Price A, Rosenfeld W. Pulmonary outcome at 1 year corrected age in premature infants treated at birth with recombinant human cuzn superoxide dismutase. Pediatrics 2003;111:469-476. 118

57. Saugstad OD. Bronchopulmonary dysplasia-oxidative stress and antioxidants. Semin Neonatol 2003;8:39-49. 58. Massaro GD, Massaro D, Chambon P. Retinoic acid receptor-alpha regulates pulmonary alveolus formation in mice after, but not during, perinatal period. Am J Physiol Lung Cell Mol Physiol 2003;284:L431-433. 59. McGowan S, Jackson SK, Jenkins-Moore M, Dai HH, Chambon P, Snyder JM. Mice bearing deletions of retinoic acid receptors demonstrate reduced lung elastin and alveolar numbers. Am J Respir Cell Mol Biol 2000;23:162-167. 60. Bry K, Lappalainen U. Pathogenesis of bronchopulmonary dysplasia: The role of interleukin 1beta in the regulation of inflammation-mediated pulmonary retinoic acid pathways in transgenic mice. Semin Perinatol 2006;30:121-128. 61. Veness-Meehan KA, Pierce RA, Moats-Staats BM, Stiles AD. Retinoic acid attenuates o2-induced inhibition of lung septation. Am J Physiol Lung Cell Mol Physiol 2002;283:L971-980. 62. Couroucli XI, Liang YW, Jiang W, Barrios R, Moorthy B. Attenuation of oxygen- induced abnormal lung maturation in rats by retinoic acid: Possible role of cytochrome p4501a enzymes. J Pharmacol Exp Ther 2006;317:946-954. 63. Zimova-Herknerova M, Myslivecek J, Potmesil P. Retinoic acid attenuates the mild hyperoxic lung injury in newborn mice. Physiol Res 2008;57:33-40. 64. Dirami G, Massaro GD, Clerch LB, Ryan US, Reczek PR, Massaro D. Lung retinol storing cells synthesize and secrete retinoic acid, an inducer of alveolus formation. Am J Physiol Lung Cell Mol Physiol 2004;286:L249-256. 65. Hind M, Gilthorpe A, Stinchcombe S, Maden M. Retinoid induction of alveolar regeneration: From mice to man? Thorax 2009;64:451-457. 66. Crapo JD. Morphologic changes in pulmonary oxygen toxicity. Annu Rev Physiol 1986;48:721-731. 67. Wispe JR, Roberts RJ. Molecular basis of pulmonary oxygen toxicity. Clin Perinatol 1987;14:651-666. 68. Gan Z, Roerig DL, Clough AV, Audi SH. Differential responses of targeted lung redox enzymes to rat exposure to 60 or 85% oxygen. J Appl Physiol 2011;111:95- 107. 69. Coalson JJ, Winter VT, Siler-Khodr T, Yoder BA. Neonatal chronic lung disease in extremely immature baboons. Am J Respir Crit Care Med 1999;160:1333- 1346. 70. D'Angio CT, LoMonaco MB, Chaudhry SA, Paxhia A, Ryan RM. Discordant pulmonary proinflammatory cytokine expression during acute hyperoxia in the newborn rabbit. Exp Lung Res 1999;25:443-465. 71. D'Angio CT, Sinkin RA, LoMonaco MB, Finkelstein JN. Interleukin-8 and monocyte chemoattractant protein-1 mrnas in oxygen-injured rabbit lung. Am J Physiol 1995;268:L826-831. 72. Piedboeuf B, Horowitz S, Johnston CJ, Gamache M, Belanger S, Poubelle PE, Welty SE, Watkins RH. Interleukin-1 expression during hyperoxic lung injury in the mouse. Free Radic Biol Med 1998;24:1446-1454.

119

73. Shea LM, Beehler C, Schwartz M, Shenkar R, Tuder R, Abraham E. Hyperoxia activates nf-kappab and increases tnf-alpha and ifn-gamma gene expression in mouse pulmonary lymphocytes. J Immunol 1996;157:3902-3908. 74. Wagenaar GT, ter Horst SA, van Gastelen MA, Leijser LM, Mauad T, van der Velden PA, de Heer E, Hiemstra PS, Poorthuis BJ, Walther FJ. Gene expression profile and histopathology of experimental bronchopulmonary dysplasia induced by prolonged oxidative stress. Free Radic Biol Med 2004;36:782-801. 75. Johnston CJ, Wright TW, Reed CK, Finkelstein JN. Comparison of adult and newborn pulmonary cytokine mrna expression after hyperoxia. Exp Lung Res 1997;23:537-552. 76. Alejandre-Alcazar MA, Kwapiszewska G, Reiss I, Amarie OV, Marsh LM, Sevilla-Perez J, Wygrecka M, Eul B, Kobrich S, Hesse M, Schermuly RT, Seeger W, Eickelberg O, Morty RE. Hyperoxia modulates tgf-beta/bmp signaling in a mouse model of bronchopulmonary dysplasia. Am J Physiol Lung Cell Mol Physiol 2007;292:L537-549. 77. Alejandre-Alcazar MA, Michiels-Corsten M, Vicencio AG, Reiss I, Ryu J, de Krijger RR, Haddad GG, Tibboel D, Seeger W, Eickelberg O, Morty RE. Tgf- beta signaling is dynamically regulated during the alveolarization of rodent and human lungs. Dev Dyn 2008;237:259-269. 78. Dasgupta C, Sakurai R, Wang Y, Guo P, Ambalavanan N, Torday JS, Rehan VK. Hyperoxia-induced neonatal rat lung injury involves activation of tgf-{beta} and wnt signaling and is protected by rosiglitazone. Am J Physiol Lung Cell Mol Physiol 2009;296:L1031-1041. 79. Phillips GJ, Mohammed W, Kelly FJ. Oxygen-induced lung injury in the pre-term guinea pig: The role of leukotriene b4. Respir Med 1995;89:607-613. 80. Hosford GE, Koyanagi KS, Leung WI, Olson DM. Hyperoxia increases protein mass of 5-lipoxygenase and its activating protein, flap, and leukotriene b(4) output in newborn rat lungs. Exp Lung Res 2002;28:671-684. 81. Rogers LK, Tipple TE, Nelin LD, Welty SE. Differential responses in the lungs of newborn mouse pups exposed to 85% or >95% oxygen. Pediatr Res 2009;65:33- 38. 82. Deng H, Mason SN, Auten RL, Jr. Lung inflammation in hyperoxia can be prevented by antichemokine treatment in newborn rats. Am J Respir Crit Care Med 2000;162:2316-2323. 83. Vozzelli MA, Mason SN, Whorton MH, Auten RL, Jr. Antimacrophage chemokine treatment prevents neutrophil and macrophage influx in hyperoxia- exposed newborn rat lung. Am J Physiol Lung Cell Mol Physiol 2004;286:L488- 493. 84. Auten RL, Jr., Mason SN, Tanaka DT, Welty-Wolf K, Whorton MH. Anti- neutrophil chemokine preserves alveolar development in hyperoxia-exposed newborn rats. Am J Physiol Lung Cell Mol Physiol 2001;281:L336-344. 85. Auten RL, Whorton MH, Nicholas Mason S. Blocking neutrophil influx reduces DNA damage in hyperoxia-exposed newborn rat lung. Am J Respir Cell Mol Biol 2002;26:391-397. 120

86. Park MS, Sohn MH, Kim KE, Namgung R, Lee C. 5-lipoxygenase-activating protein (flap) inhibitor mk-0591 prevents aberrant alveolarization in newborn mice exposed to 85% oxygen in a dose- and time-dependent manner. Lung 2011;189:43-50. 87. Manji JS, O'Kelly CJ, Leung WI, Olson DM. Timing of hyperoxic exposure during alveolarization influences damage mediated by leukotrienes. Am J Physiol Lung Cell Mol Physiol 2001;281:L799-806. 88. Boros V, Burghardt JS, Morgan CJ, Olson DM. Leukotrienes are indicated as mediators of hyperoxia-inhibited alveolarization in newborn rats. Am J Physiol 1997;272:L433-441. 89. Burghardt JS, Boros V, Biggs DF, Olson DM. Lipid mediators in oxygen-induced airway remodeling and hyperresponsiveness in newborn rats. Am J Respir Crit Care Med 1996;154:837-842. 90. Funk AJ, Mandrell TD, Lokey SJ, Kosanke SD, Li CS, Potters CF. Effects of leukotriene inhibition on pulmonary morphology in rat pup lungs exposed to hyperoxia. Comp Med 2007;57:186-192. 91. Sue RD, Belperio JA, Burdick MD, Murray LA, Xue YY, Dy MC, Kwon JJ, Keane MP, Strieter RM. Cxcr2 is critical to hyperoxia-induced lung injury. J Immunol 2004;172:3860-3868. 92. Okuma T, Terasaki Y, Sakashita N, Kaikita K, Kobayashi H, Hayasaki T, Kuziel WA, Baba H, Takeya M. Mcp-1/ccr2 signalling pathway regulates hyperoxia- induced acute lung injury via nitric oxide production. Int J Exp Pathol 2006;87:475-483. 93. Auten RL, Richardson RM, White JR, Mason SN, Vozzelli MA, Whorton MH. Nonpeptide cxcr2 antagonist prevents neutrophil accumulation in hyperoxia- exposed newborn rats. J Pharmacol Exp Ther 2001;299:90-95. 94. Yi M, Jankov RP, Belcastro R, Humes D, Copland I, Shek S, Sweezey NB, Post M, Albertine KH, Auten RL, Tanswell AK. Opposing effects of 60% oxygen and neutrophil influx on alveologenesis in the neonatal rat. Am J Respir Crit Care Med 2004;170:1188-1196. 95. Jankov RP, Luo X, Belcastro R, Copland I, Frndova H, Lye SJ, Hoidal JR, Post M, Tanswell AK. Gadolinium chloride inhibits pulmonary macrophage influx and prevents o(2)-induced pulmonary hypertension in the neonatal rat. Pediatr Res 2001;50:172-183. 96. Jankov RP, Johnstone L, Luo X, Robinson BH, Tanswell AK. Macrophages as a major source of oxygen radicals in the hyperoxic newborn rat lung. Free Radic Biol Med 2003;35:200-209. 97. Massaro D, Massaro GD. Invited review: Pulmonary alveoli: Formation, the "call for oxygen," and other regulators. Am J Physiol Lung Cell Mol Physiol 2002;282:L345-358. 98. Stenmark KR, Abman SH. Lung vascular development: Implications for the pathogenesis of bronchopulmonary dysplasia. Annu Rev Physiol 2005;67:623- 661.

121

99. Burri PH. Structural aspects of postnatal lung development - alveolar formation and growth. Biol Neonate 2006;89:313-322. 100. Velten M, Heyob KM, Rogers LK, Welty SE. Deficits in lung alveolarization and function after systemic maternal inflammation and neonatal hyperoxia exposure. J Appl Physiol 2010;108:1347-1356. 101. Warner BB, Stuart LA, Papes RA, Wispe JR. Functional and pathological effects of prolonged hyperoxia in neonatal mice. Am J Physiol 1998;275:L110-117. 102. Albertine KH, Jones GP, Starcher BC, Bohnsack JF, Davis PL, Cho SC, Carlton DP, Bland RD. Chronic lung injury in preterm lambs. Disordered respiratory tract development. Am J Respir Crit Care Med 1999;159:945-958. 103. Greenough A. Late respiratory outcomes after preterm birth. Early Hum Dev 2007;83:785-788. 104. Hershenson MB, Aghili S, Punjabi N, Hernandez C, Ray DW, Garland A, Glagov S, Solway J. Hyperoxia-induced airway hyperresponsiveness and remodeling in immature rats. Am J Physiol 1992;262:L263-269. 105. Hershenson MB, Garland A, Kelleher MD, Zimmermann A, Hernandez C, Solway J. Hyperoxia-induced airway remodeling in immature rats. Correlation with airway responsiveness. Am Rev Respir Dis 1992;146:1294-1300. 106. Hershenson MB, Wylam ME, Punjabi N, Umans JG, Schumacker PT, Mitchell RW, Solway J. Exposure of immature rats to hyperoxia increases tracheal smooth muscle stress generation in vitro. J Appl Physiol 1994;76:743-749. 107. Hershenson MB, Abe MK, Kelleher MD, Naureckas ET, Garland A, Zimmermann A, Rubinstein VJ, Solway J. Recovery of airway structure and function after hyperoxic exposure in immature rats. Am J Respir Crit Care Med 1994;149:1663-1669. 108. Schulman SR, Canada AT, Fryer AD, Winsett DW, Costa DL. Airway hyperreactivity produced by short-term exposure to hyperoxia in neonatal guinea pigs. Am J Physiol 1997;272:L1211-1216. 109. Schultz ED, Potts EN, Mason SN, Foster WM, Auten RL. Mast cells mediate hyperoxia-induced airway hyper-reactivity in newborn rats. Pediatr Res 2010;68:70-74. 110. Yao Q, Zaidi SI, Haxhiu MA, Martin RJ. Neonatal lung and airway injury: A role for neurotrophins. Semin Perinatol 2006;30:156-162. 111. Dauger S, Ferkdadji L, Saumon G, Vardon G, Peuchmaur M, Gaultier C, Gallego J. Neonatal exposure to 65% oxygen durably impairs lung architecture and breathing pattern in adult mice. Chest 2003;123:530-538. 112. Yee M, Chess PR, McGrath-Morrow SA, Wang Z, Gelein R, Zhou R, Dean DA, Notter RH, O'Reilly MA. Neonatal oxygen adversely affects lung function in adult mice without altering surfactant composition or activity. Am J Physiol Lung Cell Mol Physiol 2009;297:L641-649. 113. Yee M, White RJ, Awad HA, Bates WA, McGrath-Morrow SA, O'Reilly MA. Neonatal hyperoxia causes pulmonary vascular disease and shortens life span in aging mice. Am J Pathol 2011;178:2601-2610.

122

114. O'Reilly MA, Marr SH, Yee M, McGrath-Morrow SA, Lawrence BP. Neonatal hyperoxia enhances the inflammatory response in adult mice infected with influenza a virus. Am J Respir Crit Care Med 2008;177:1103-1110. 115. O'Reilly MA, Yee M, Buczynski BW, Vitiello PF, Keng PC, Welle SL, Finkelstein JN, Dean DA, Lawrence BP. Neonatal oxygen increases sensitivity to influenza a virus infection in adult mice by suppressing epithelial expression of ear1. Am J Pathol 2012. 116. Buczynski BW, Yee M, Paige Lawrence B, O'Reilly MA. Lung development and the host response to influenza a virus are altered by different doses of neonatal oxygen in mice. Am J Physiol Lung Cell Mol Physiol 2012;302:L1078-1087. 117. McGrath-Morrow SA, Lauer T, Collaco JM, Yee M, O'Reilly M, Mitzner W, Neptune E, Wise R, Biswal S. Neonatal hyperoxia contributes additively to cigarette smoke-induced chronic obstructive pulmonary disease changes in adult mice. Am J Respir Cell Mol Biol 2011;45:610-616. 118. D'Acquisto F, Iuvone T, Rombola L, Sautebin L, Di Rosa M, Carnuccio R. Involvement of nf-kappab in the regulation of cyclooxygenase-2 protein expression in lps-stimulated j774 macrophages. FEBS Lett 1997;418:175-178. 119. Wu KK. Control of cyclooxygenase-2 transcriptional activation by pro- inflammatory mediators. Prostaglandins Leukot Essent Fatty Acids 2005;72:89- 93. 120. Mbonye UR, Song I. Posttranscriptional and posttranslational determinants of cyclooxygenase expression. BMB Rep 2009;42:552-560. 121. Xu F, Xu Z, Zhang R, Wu Z, Lim JH, Koga T, Li JD, Shen H. Nontypeable haemophilus influenzae induces cox-2 and pge2 expression in lung epithelial cells via activation of p38 mapk and nf-kappa b. Respir Res 2008;9:16. 122. N'Guessan PD, Etouem MO, Schmeck B, Hocke AC, Scharf S, Vardarova K, Opitz B, Flieger A, Suttorp N, Hippenstiel S. Legionella pneumophila-induced pkcalpha-, mapk-, and nf-kappab-dependent cox-2 expression in human lung epithelium. Am J Physiol Lung Cell Mol Physiol 2007;292:L267-277. 123. N'Guessan PD, Hippenstiel S, Etouem MO, Zahlten J, Beermann W, Lindner D, Opitz B, Witzenrath M, Rosseau S, Suttorp N, Schmeck B. Streptococcus pneumoniae induced p38 mapk- and nf-kappab-dependent cox-2 expression in human lung epithelium. Am J Physiol Lung Cell Mol Physiol 2006;290:L1131- 1138. 124. Smith WL, Urade Y, Jakobsson PJ. Enzymes of the cyclooxygenase pathways of prostanoid biosynthesis. Chem Rev 2011;111:5821-5865. 125. Harizi H, Corcuff JB, Gualde N. Arachidonic-acid-derived eicosanoids: Roles in biology and immunopathology. Trends Mol Med 2008;14:461-469. 126. Gilroy DW, Tomlinson A, Willoughby DA. Differential effects of inhibition of isoforms of cyclooxygenase (cox-1, cox-2) in chronic inflammation. Inflamm Res 1998;47:79-85. 127. Gilroy DW, Tomlinson A, Willoughby DA. Differential effects of inhibitors of cyclooxygenase (cyclooxygenase 1 and cyclooxygenase 2) in acute inflammation. Eur J Pharmacol 1998;355:211-217. 123

128. Gilroy DW, Colville-Nash PR, Willis D, Chivers J, Paul-Clark MJ, Willoughby DA. Inducible cyclooxygenase may have anti-inflammatory properties. Nat Med 1999;5:698-701. 129. Scher JU, Pillinger MH. The anti-inflammatory effects of prostaglandins. J Investig Med 2009;57:703-708. 130. Zeldin DC, Wohlford-Lenane C, Chulada P, Bradbury JA, Scarborough PE, Roggli V, Langenbach R, Schwartz DA. Airway inflammation and responsiveness in prostaglandin h synthase-deficient mice exposed to bacterial lipopolysaccharide. Am J Respir Cell Mol Biol 2001;25:457-465. 131. Bonner JC, Rice AB, Ingram JL, Moomaw CR, Nyska A, Bradbury A, Sessoms AR, Chulada PC, Morgan DL, Zeldin DC, Langenbach R. Susceptibility of cyclooxygenase-2-deficient mice to pulmonary fibrogenesis. Am J Pathol 2002;161:459-470. 132. Card JW, Voltz JW, Carey MA, Bradbury JA, Degraff LM, Lih FB, Bonner JC, Morgan DL, Flake GP, Zeldin DC. Cyclooxygenase-2 deficiency exacerbates bleomycin-induced lung dysfunction but not fibrosis. Am J Respir Cell Mol Biol 2007;37:300-308. 133. Hodges RJ, Jenkins RG, Wheeler-Jones CP, Copeman DM, Bottoms SE, Bellingan GJ, Nanthakumar CB, Laurent GJ, Hart SL, Foster ML, McAnulty RJ. Severity of lung injury in cyclooxygenase-2-deficient mice is dependent on reduced prostaglandin e(2) production. Am J Pathol 2004;165:1663-1676. 134. Dackor RT, Cheng J, Voltz JW, Card JW, Ferguson CD, Garrett RC, Bradbury JA, DeGraff LM, Lih FB, Tomer KB, Flake GP, Travlos GS, Ramsey RW, Jr., Edin ML, Morgan DL, Zeldin DC. Prostaglandin e(2) protects murine lungs from bleomycin-induced pulmonary fibrosis and lung dysfunction. Am J Physiol Lung Cell Mol Physiol 2011;301:L645-655. 135. Maher TM, Evans IC, Bottoms SE, Mercer PF, Thorley AJ, Nicholson AG, Laurent GJ, Tetley TD, Chambers RC, McAnulty RJ. Diminished prostaglandin e2 contributes to the apoptosis paradox in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 2010;182:73-82. 136. Oguma T, Asano K, Ishizaka A. Role of prostaglandin d(2) and its receptors in the pathophysiology of asthma. Allergol Int 2008;57:307-312. 137. Carey MA, Germolec DR, Bradbury JA, Gooch RA, Moorman MP, Flake GP, Langenbach R, Zeldin DC. Accentuated t helper type 2 airway response after allergen challenge in cyclooxygenase-1-/- but not cyclooxygenase-2-/- mice. Am J Respir Crit Care Med 2003;167:1509-1515. 138. Li H, Bradbury JA, Dackor RT, Edin ML, Graves JP, DeGraff LM, Wang PM, Bortner CD, Maruoka S, Lih FB, Cook DN, Tomer KB, Jetten AM, Zeldin DC. Cyclooxygenase-2 regulates th17 cell differentiation during allergic lung inflammation. Am J Respir Crit Care Med 2011;184:37-49. 139. Fukunaga K, Kohli P, Bonnans C, Fredenburgh LE, Levy BD. Cyclooxygenase 2 plays a pivotal role in the resolution of acute lung injury. J Immunol 2005;174:5033-5039.

124

140. Mochizuki M, Ishii Y, Itoh K, Iizuka T, Morishima Y, Kimura T, Kiwamoto T, Matsuno Y, Hegab AE, Nomura A, Sakamoto T, Uchida K, Yamamoto M, Sekizawa K. Role of 15-deoxy delta(12,14) prostaglandin j2 and nrf2 pathways in protection against acute lung injury. Am J Respir Crit Care Med 2005;171:1260- 1266. 141. Morris T, Stables M, Gilroy DW. New perspectives on aspirin and the endogenous control of acute inflammatory resolution. ScientificWorldJournal 2006;6:1048-1065. 142. Vane JR. Adventures and excursions in bioassay: The stepping stones to prostacyclin. Nobel lecture, 8 december 1982. Biosci Rep 1983;3:683-711. 143. Vane JR, Botting RM. The mechanism of action of aspirin. Thromb Res 2003;110:255-258. 144. Roth GJ, Stanford N, Majerus PW. Acetylation of prostaglandin synthase by aspirin. Proc Natl Acad Sci U S A 1975;72:3073-3076. 145. Loll PJ, Picot D, Garavito RM. The structural basis of aspirin activity inferred from the crystal structure of inactivated prostaglandin h2 synthase. Nat Struct Biol 1995;2:637-643. 146. Xiao G, Tsai AL, Palmer G, Boyar WC, Marshall PJ, Kulmacz RJ. Analysis of hydroperoxide-induced tyrosyl radicals and lipoxygenase activity in aspirin- treated human prostaglandin h synthase-2. Biochemistry 1997;36:1836-1845. 147. Holtzman MJ, Turk J, Shornick LP. Identification of a pharmacologically distinct prostaglandin h synthase in cultured epithelial cells. J Biol Chem 1992;267:21438-21445. 148. Lecomte M, Laneuville O, Ji C, DeWitt DL, Smith WL. Acetylation of human prostaglandin endoperoxide synthase-2 (cyclooxygenase-2) by aspirin. J Biol Chem 1994;269:13207-13215. 149. Meade EA, Smith WL, DeWitt DL. Differential inhibition of prostaglandin endoperoxide synthase (cyclooxygenase) isozymes by aspirin and other nonsteroidal anti-inflammatory drugs. J Biol Chem 1993;268:6610-6614. 150. Mancini JA, O'Neill GP, Bayly C, Vickers PJ. Mutation of serine-516 in human prostaglandin g/h synthase-2 to methionine or aspirin acetylation of this residue stimulates 15-r-hete synthesis. FEBS Lett 1994;342:33-37. 151. Claria J, Lee MH, Serhan CN. Aspirin-triggered lipoxins (15-epi-lx) are generated by the human lung adenocarcinoma cell line (a549)-neutrophil interactions and are potent inhibitors of cell proliferation. Mol Med 1996;2:583-596. 152. Groeger AL, Cipollina C, Cole MP, Woodcock SR, Bonacci G, Rudolph TK, Rudolph V, Freeman BA, Schopfer FJ. Cyclooxygenase-2 generates anti- inflammatory mediators from omega-3 fatty acids. Nat Chem Biol 2010;6:433- 441. 153. Serhan CN, Hamberg M, Samuelsson B. Lipoxins: Novel series of biologically active compounds formed from arachidonic acid in human leukocytes. Proc Natl Acad Sci U S A 1984;81:5335-5339.

125

154. Papayianni A, Serhan CN, Brady HR. Lipoxin a4 and b4 inhibit leukotriene- stimulated interactions of human neutrophils and endothelial cells. J Immunol 1996;156:2264-2272. 155. Serhan CN, Maddox JF, Petasis NA, Akritopoulou-Zanze I, Papayianni A, Brady HR, Colgan SP, Madara JL. Design of lipoxin a4 stable analogs that block transmigration and adhesion of human neutrophils. Biochemistry 1995;34:14609- 14615. 156. Colgan SP, Serhan CN, Parkos CA, Delp-Archer C, Madara JL. Lipoxin a4 modulates transmigration of human neutrophils across intestinal epithelial monolayers. J Clin Invest 1993;92:75-82. 157. Takano T, Clish CB, Gronert K, Petasis N, Serhan CN. Neutrophil-mediated changes in vascular permeability are inhibited by topical application of aspirin- triggered 15-epi-lipoxin a4 and novel lipoxin b4 stable analogues. J Clin Invest 1998;101:819-826. 158. Fiore S, Ryeom SW, Weller PF, Serhan CN. Lipoxin recognition sites. Specific binding of labeled lipoxin a4 with human neutrophils. J Biol Chem 1992;267:16168-16176. 159. Fiore S, Maddox JF, Perez HD, Serhan CN. Identification of a human cdna encoding a functional high affinity lipoxin a4 receptor. J Exp Med 1994;180:253- 260. 160. Takano T, Fiore S, Maddox JF, Brady HR, Petasis NA, Serhan CN. Aspirin- triggered 15-epi-lipoxin a4 (lxa4) and lxa4 stable analogues are potent inhibitors of acute inflammation: Evidence for anti-inflammatory receptors. J Exp Med 1997;185:1693-1704. 161. Chiang N, Takano T, Arita M, Watanabe S, Serhan CN. A novel rat lipoxin a4 receptor that is conserved in structure and function. Br J Pharmacol 2003;139:89- 98. 162. Dufton N, Hannon R, Brancaleone V, Dalli J, Patel HB, Gray M, D'Acquisto F, Buckingham JC, Perretti M, Flower RJ. Anti-inflammatory role of the murine formyl-peptide receptor 2: Ligand-specific effects on leukocyte responses and experimental inflammation. J Immunol 2010;184:2611-2619. 163. Levy BD, Fokin VV, Clark JM, Wakelam MJ, Petasis NA, Serhan CN. Polyisoprenyl phosphate (pipp) signaling regulates phospholipase d activity: A 'stop' signaling switch for aspirin-triggered lipoxin a4. FASEB J 1999;13:903- 911. 164. Wu SH, Wu XH, Lu C, Dong L, Zhou GP, Chen ZQ. Lipoxin a4 inhibits connective tissue growth factor-induced production of chemokines in rat mesangial cells. Kidney Int 2006;69:248-256. 165. Wu SH, Lu C, Dong L, Zhou GP, He ZG, Chen ZQ. Lipoxin a4 inhibits tnf-alpha- induced production of interleukins and proliferation of rat mesangial cells. Kidney Int 2005;68:35-46. 166. El Kebir D, Jozsef L, Khreiss T, Pan W, Petasis NA, Serhan CN, Filep JG. Aspirin-triggered lipoxins override the apoptosis-delaying action of serum

126

amyloid a in human neutrophils: A novel mechanism for resolution of inflammation. J Immunol 2007;179:616-622. 167. Maddox JF, Serhan CN. Lipoxin a4 and b4 are potent stimuli for human monocyte migration and adhesion: Selective inactivation by dehydrogenation and reduction. J Exp Med 1996;183:137-146. 168. Maddox JF, Hachicha M, Takano T, Petasis NA, Fokin VV, Serhan CN. Lipoxin a4 stable analogs are potent mimetics that stimulate human monocytes and thp-1 cells via a g-protein-linked lipoxin a4 receptor. J Biol Chem 1997;272:6972-6978. 169. Godson C, Mitchell S, Harvey K, Petasis NA, Hogg N, Brady HR. Cutting edge: Lipoxins rapidly stimulate nonphlogistic phagocytosis of apoptotic neutrophils by monocyte-derived macrophages. J Immunol 2000;164:1663-1667. 170. Mitchell S, Thomas G, Harvey K, Cottell D, Reville K, Berlasconi G, Petasis NA, Erwig L, Rees AJ, Savill J, Brady HR, Godson C. Lipoxins, aspirin-triggered epi- lipoxins, lipoxin stable analogues, and the resolution of inflammation: Stimulation of macrophage phagocytosis of apoptotic neutrophils in vivo. J Am Soc Nephrol 2002;13:2497-2507. 171. El Kebir D, Jozsef L, Pan W, Wang L, Petasis NA, Serhan CN, Filep JG. 15-epi- lipoxin a4 inhibits myeloperoxidase signaling and enhances resolution of acute lung injury. Am J Respir Crit Care Med 2009;180:311-319. 172. Jin SW, Zhang L, Lian QQ, Liu D, Wu P, Yao SL, Ye DY. Posttreatment with aspirin-triggered lipoxin a4 analog attenuates lipopolysaccharide-induced acute lung injury in mice: The role of heme oxygenase-1. Anesth Analg 2007;104:369- 377. 173. Planaguma A, Pfeffer MA, Rubin G, Croze R, Uddin M, Serhan CN, Levy BD. Lovastatin decreases acute mucosal inflammation via 15-epi-lipoxin a4. Mucosal Immunol 2010;3:270-279. 174. Martins V, Valenca SS, Farias-Filho FA, Molinaro R, Simoes RL, Ferreira TP, e Silva PM, Hogaboam CM, Kunkel SL, Fierro IM, Canetti C, Benjamim CF. Atla, an aspirin-triggered lipoxin a4 synthetic analog, prevents the inflammatory and fibrotic effects of bleomycin-induced pulmonary fibrosis. J Immunol 2009;182:5374-5381. 175. Karp CL, Flick LM, Park KW, Softic S, Greer TM, Keledjian R, Yang R, Uddin J, Guggino WB, Atabani SF, Belkaid Y, Xu Y, Whitsett JA, Accurso FJ, Wills- Karp M, Petasis NA. Defective lipoxin-mediated anti-inflammatory activity in the cystic fibrosis airway. Nat Immunol 2004;5:388-392. 176. Levy BD, De Sanctis GT, Devchand PR, Kim E, Ackerman K, Schmidt BA, Szczeklik W, Drazen JM, Serhan CN. Multi-pronged inhibition of airway hyperresponsiveness and inflammation by lipoxin a(4). Nat Med 2002;8:1018-1023. 177. Levy BD, Lukacs NW, Berlin AA, Schmidt B, Guilford WJ, Serhan CN, Parkinson JF. Lipoxin a4 stable analogs reduce allergic airway responses via mechanisms distinct from cyslt1 receptor antagonism. FASEB J 2007;21:3877- 3884.

127

178. Levy BD, Kohli P, Gotlinger K, Haworth O, Hong S, Kazani S, Israel E, Haley KJ, Serhan CN. Protectin d1 is generated in asthma and dampens airway inflammation and hyperresponsiveness. J Immunol 2007;178:496-502. 179. Levy BD, Bonnans C, Silverman ES, Palmer LJ, Marigowda G, Israel E. Diminished lipoxin biosynthesis in severe asthma. Am J Respir Crit Care Med 2005;172:824-830. 180. Planaguma A, Kazani S, Marigowda G, Haworth O, Mariani TJ, Israel E, Bleecker ER, Curran-Everett D, Erzurum SC, Calhoun WJ, Castro M, Chung KF, Gaston B, Jarjour NN, Busse WW, Wenzel SE, Levy BD. Airway lipoxin a4 generation and lipoxin a4 receptor expression are decreased in severe asthma. Am J Respir Crit Care Med 2008;178:574-582. 181. Seki H, Fukunaga K, Arita M, Arai H, Nakanishi H, Taguchi R, Miyasho T, Takamiya R, Asano K, Ishizaka A, Takeda J, Levy BD. The anti-inflammatory and proresolving mediator resolvin e1 protects mice from bacterial pneumonia and acute lung injury. J Immunol 2010;184:836-843. 182. Haworth O, Cernadas M, Yang R, Serhan CN, Levy BD. Resolvin e1 regulates interleukin 23, interferon-gamma and lipoxin a4 to promote the resolution of allergic airway inflammation. Nat Immunol 2008;9:873-879. 183. Haworth O, Cernadas M, Levy BD. Nk cells are effectors for resolvin e1 in the timely resolution of allergic airway inflammation. J Immunol 2011;186:6129- 6135. 184. Rogerio AP, Haworth O, Croze R, Oh SF, Uddin M, Carlo T, Pfeffer MA, Priluck R, Serhan CN, Levy BD. Resolvin d1 and aspirin-triggered resolvin d1 promote resolution of allergic airways responses. J Immunol 2012. 185. Eickmeier O, Seki H, Haworth O, Hilberath JN, Gao F, Uddin M, Croze RH, Carlo T, Pfeffer MA, Levy BD. Aspirin-triggered resolvin d1 reduces mucosal inflammation and promotes resolution in a murine model of acute lung injury. Mucosal Immunol 2012. 186. Tegeder I, Pfeilschifter J, Geisslinger G. Cyclooxygenase-independent actions of cyclooxygenase inhibitors. FASEB J 2001;15:2057-2072. 187. Wu KK. Aspirin and other cyclooxygenase inhibitors: New therapeutic insights. Semin Vasc Med 2003;3:107-112. 188. Chiabrando C, Castelli MG, Cozzi E, Fanelli R, Campoleoni A, Balotta C, Latini R, Garattini S. Antiinflammatory action of salicylates: Aspirin is not a prodrug for salicylate against rat carrageenin pleurisy. Eur J Pharmacol 1989;159:257-264. 189. Frantz B, O'Neill EA. The effect of sodium salicylate and aspirin on nf-kappa b. Science 1995;270:2017-2019. 190. Kopp E, Ghosh S. Inhibition of nf-kappa b by sodium salicylate and aspirin. Science 1994;265:956-959. 191. Grilli M, Pizzi M, Memo M, Spano P. Neuroprotection by aspirin and sodium salicylate through blockade of nf-kappab activation. Science 1996;274:1383- 1385. 192. Bayon Y, Alonso A, Sanchez Crespo M. 4-trifluoromethyl derivatives of salicylate, triflusal and its main metabolite 2-hydroxy-4-trifluoromethylbenzoic 128

acid, are potent inhibitors of nuclear factor kappab activation. Br J Pharmacol 1999;126:1359-1366. 193. Yin MJ, Yamamoto Y, Gaynor RB. The anti-inflammatory agents aspirin and salicylate inhibit the activity of i(kappa)b kinase-beta. Nature 1998;396:77-80. 194. Cianferoni A, Schroeder JT, Kim J, Schmidt JW, Lichtenstein LM, Georas SN, Casolaro V. Selective inhibition of interleukin-4 gene expression in human t cells by aspirin. Blood 2001;97:1742-1749. 195. Huang C, Ma WY, Hanenberger D, Cleary MP, Bowden GT, Dong Z. Inhibition of ultraviolet b-induced activator protein-1 (ap-1) activity by aspirin in ap-1- luciferase transgenic mice. J Biol Chem 1997;272:26325-26331. 196. Dong Z, Huang C, Brown RE, Ma WY. Inhibition of activator protein 1 activity and neoplastic transformation by aspirin. J Biol Chem 1997;272:9962-9970. 197. Cronstein BN, Van de Stouwe M, Druska L, Levin RI, Weissmann G. Nonsteroidal antiinflammatory agents inhibit stimulated neutrophil adhesion to endothelium: Adenosine dependent and independent mechanisms. Inflammation 1994;18:323-335. 198. Cronstein BN, Montesinos MC, Weissmann G. Salicylates and sulfasalazine, but not glucocorticoids, inhibit leukocyte accumulation by an adenosine-dependent mechanism that is independent of inhibition of prostaglandin synthesis and p105 of nfkappab. Proc Natl Acad Sci U S A 1999;96:6377-6381. 199. Hiller KO, Hodd PL, Willson RL. Antiinflammatory drugs: Protection of a bacterial virus as an in vitro biological measure of free radical activity. Chem Biol Interact 1983;47:293-305. 200. Ghiselli A, Laurenti O, De Mattia G, Maiani G, Ferro-Luzzi A. Salicylate hydroxylation as an early marker of in vivo oxidative stress in diabetic patients. Free Radic Biol Med 1992;13:621-626. 201. Lassus P, Wolff H, Andersson S. Cyclooxygenase-2 in human perinatal lung. Pediatr Res 2000;47:602-605. 202. Brannon TS, MacRitchie AN, Jaramillo MA, Sherman TS, Yuhanna IS, Margraf LR, Shaul PW. Ontogeny of cyclooxygenase-1 and cyclooxygenase-2 gene expression in ovine lung. Am J Physiol 1998;274:L66-71. 203. Ermert L, Ermert M, Goppelt-Struebe M, Walmrath D, Grimminger F, Steudel W, Ghofrani HA, Homberger C, Duncker H, Seeger W. Cyclooxygenase isoenzyme localization and mrna expression in rat lungs. Am J Respir Cell Mol Biol 1998;18:479-488. 204. Cho WS, Chae C. Immunohistochemical detection of cyclooxygenase-2 in lungs of pigs naturally infected with actinobacillus pleuropneumoniae. J Comp Pathol 2002;127:274-279. 205. Khan KN, Stanfield K, Trajkovic D, Harris RK. Cyclooxygenase-2 expression in inflammatory lung lesions of nonhuman primates. Vet Pathol 2000;37:512-516. 206. Radi ZA, Meyerholz DK, Ackermann MR. Pulmonary cyclooxygenase-1 (cox-1) and cox-2 cellular expression and distribution after respiratory syncytial virus and parainfluenza virus infection. Viral Immunol 2010;23:43-48.

129

207. Adawi A, Zhang Y, Baggs R, Finkelstein J, Phipps RP. Disruption of the cd40- cd40 ligand system prevents an oxygen-induced respiratory distress syndrome. Am J Pathol 1998;152:651-657. 208. Hageman JR, Babler S, Lee SC, Cobb M, Pachman LM, Smith LJ, Hunt CE. The early involvement of pulmonary prostaglandins in hyperoxic lung injury. Prostaglandins Leukot Med 1986;25:105-122. 209. Amann R, Peskar BA. Anti-inflammatory effects of aspirin and sodium salicylate. Eur J Pharmacol 2002;447:1-9. 210. Rogers LK, Tipple TE, Britt RD, Welty SE. Hyperoxia exposure alters hepatic eicosanoid metabolism in newborn mice. Pediatr Res 2010;67:144-149. 211. Kamata H, Hirata H. Redox regulation of cellular signalling. Cell Signal 1999;11:1-14. 212. Chung SW, Chung HY, Toriba A, Kameda T, Tang N, Kizu R, Hayakawa K. An environmental quinoid polycyclic aromatic hydrocarbon, acenaphthenequinone, modulates cyclooxygenase-2 expression through reactive oxygen species generation and nuclear factor kappa b activation in a549 cells. Toxicol Sci 2007;95:348-355. 213. Nakahata N. Thromboxane a2: Physiology/pathophysiology, cellular signal transduction and pharmacology. Pharmacol Ther 2008;118:18-35. 214. Park GY, Christman JW. Involvement of cyclooxygenase-2 and prostaglandins in the molecular pathogenesis of inflammatory lung diseases. Am J Physiol Lung Cell Mol Physiol 2006;290:L797-805. 215. Ishitsuka Y, Moriuchi H, Isohama Y, Tokunaga H, Hatamoto K, Kurita S, Irikura M, Iyama K, Irie T. A selective thromboxane a2 (txa2) synthase inhibitor, ozagrel, attenuates lung injury and decreases monocyte chemoattractant protein-1 and interleukin-8 mrna expression in oleic acid-induced lung injury in guinea pigs. J Pharmacol Sci 2009;111:211-215. 216. Ishitsuka Y, Moriuchi H, Hatamoto K, Yang C, Takase J, Golbidi S, Irikura M, Irie T. Involvement of thromboxane a2 (txa2) in the early stages of oleic acid- induced lung injury and the preventive effect of ozagrel, a txa2 synthase inhibitor, in guinea-pigs. J Pharm Pharmacol 2004;56:513-520. 217. Thies SD, Corbin RS, Goff CD, Binns OA, Buchanan SA, Shockey KS, Frierson HF, Jr., Young JS, Tribble CG, Kron IL. Thromboxane receptor blockade improves oxygenation in an experimental model of acute lung injury. Ann Thorac Surg 1996;61:1453-1457. 218. Sinuff T, Cook DJ, Peterson JC, Fuller HD. Development, implementation, and evaluation of a ketoconazole practice guideline for ards prophylaxis. J Crit Care 1999;14:1-6. 219. Ketoconazole for early treatment of acute lung injury and acute respiratory distress syndrome: A randomized controlled trial. The ards network. JAMA 2000;283:1995-2002. 220. Kanaoka Y, Urade Y. Hematopoietic prostaglandin d synthase. Prostaglandins Leukot Essent Fatty Acids 2003;69:163-167.

130

221. Inoue K, Takano H, Yanagisawa R, Morita M, Ichinose T, Sadakane K, Yoshino S, Yamaki K, Kumagai Y, Uchiyama K, Yoshikawa T. Effect of 15-deoxy-delta 12,14-prostaglandin j2 on acute lung injury induced by lipopolysaccharide in mice. Eur J Pharmacol 2003;481:261-269. 222. Cho HY, Gladwell W, Wang X, Chorley B, Bell D, Reddy SP, Kleeberger SR. Nrf2-regulated ppar{gamma} expression is critical to protection against acute lung injury in mice. Am J Respir Crit Care Med 2010;182:170-182. 223. Kliewer SA, Lenhard JM, Willson TM, Patel I, Morris DC, Lehmann JM. A prostaglandin j2 metabolite binds peroxisome proliferator-activated receptor gamma and promotes adipocyte differentiation. Cell 1995;83:813-819. 224. Rossi A, Kapahi P, Natoli G, Takahashi T, Chen Y, Karin M, Santoro MG. Anti- inflammatory cyclopentenone prostaglandins are direct inhibitors of ikappab kinase. Nature 2000;403:103-108. 225. Cernuda-Morollon E, Pineda-Molina E, Canada FJ, Perez-Sala D. 15-deoxy-delta 12,14-prostaglandin j2 inhibition of nf-kappab-DNA binding through covalent modification of the p50 subunit. J Biol Chem 2001;276:35530-35536. 226. Straus DS, Pascual G, Li M, Welch JS, Ricote M, Hsiang CH, Sengchanthalangsy LL, Ghosh G, Glass CK. 15-deoxy-delta 12,14-prostaglandin j2 inhibits multiple steps in the nf-kappa b signaling pathway. Proc Natl Acad Sci U S A 2000;97:4844-4849. 227. Bell-Parikh LC, Ide T, Lawson JA, McNamara P, Reilly M, FitzGerald GA. Biosynthesis of 15-deoxy-delta12,14-pgj2 and the ligation of ppargamma. J Clin Invest 2003;112:945-955. 228. Ishizuka T, Sawada S, Sugama K, Kurita A. Thromboxane a2 (txa2) receptor blockade suppresses monocyte chemoattractant protein-1 (mcp-1) expression by stimulated vascular endothelial cells. Clin Exp Immunol 2000;120:71-78. 229. Amiri KI, Richmond A. Fine tuning the transcriptional regulation of the cxcl1 chemokine. Prog Nucleic Acid Res Mol Biol 2003;74:1-36. 230. Rangachari PK, Betti PA. Biological activity of metabolites of pgd2 on canine proximal colon. Am J Physiol 1993;264:G886-894. 231. Kostenis E, Ulven T. Emerging roles of dp and crth2 in allergic inflammation. Trends Mol Med 2006;12:148-158. 232. Hirai H, Tanaka K, Yoshie O, Ogawa K, Kenmotsu K, Takamori Y, Ichimasa M, Sugamura K, Nakamura M, Takano S, Nagata K. Prostaglandin d2 selectively induces chemotaxis in t helper type 2 cells, eosinophils, and basophils via seven- transmembrane receptor crth2. J Exp Med 2001;193:255-261. 233. Spik I, Brenuchon C, Angeli V, Staumont D, Fleury S, Capron M, Trottein F, Dombrowicz D. Activation of the prostaglandin d2 receptor dp2/crth2 increases allergic inflammation in mouse. J Immunol 2005;174:3703-3708. 234. Tajima T, Murata T, Aritake K, Urade Y, Hirai H, Nakamura M, Ozaki H, Hori M. Lipopolysaccharide induces macrophage migration via prostaglandin d(2) and prostaglandin e(2). J Pharmacol Exp Ther 2008;326:493-501. 235. Honda K, Arima M, Cheng G, Taki S, Hirata H, Eda F, Fukushima F, Yamaguchi B, Hatano M, Tokuhisa T, Fukuda T. Prostaglandin d2 reinforces th2 type 131

inflammatory responses of airways to low-dose antigen through bronchial expression of macrophage-derived chemokine. J Exp Med 2003;198:533-543. 236. Ohara M, Sawa T, Kurahashi K, Wiener-Kronish JP, Doshi V, Kudoh I, Gropper MA. Induction of cyclooxygenase-2 in alveolar macrophages after acid aspiration: Selective cyclooxygenase-2 blockade reduces interleukin-6 production. Anesthesiology 1998;88:1014-1022. 237. Maderna P, Godson C. Taking insult from injury: Lipoxins and lipoxin receptor agonists and phagocytosis of apoptotic cells. Prostaglandins Leukot Essent Fatty Acids 2005;73:179-187. 238. Kato A, Schleimer RP. Beyond inflammation: Airway epithelial cells are at the interface of innate and adaptive immunity. Curr Opin Immunol 2007;19:711-720. 239. Boers JE, Ambergen AW, Thunnissen FB. Number and proliferation of basal and parabasal cells in normal human airway epithelium. Am J Respir Crit Care Med 1998;157:2000-2006. 240. Pack RJ, Al-Ugaily LH, Morris G, Widdicombe JG. The distribution and structure of cells in the tracheal epithelium of the mouse. Cell Tissue Res 1980;208:65-84. 241. Mukherjee AB, Zhang Z, Chilton BS. Uteroglobin: A steroid-inducible immunomodulatory protein that founded the secretoglobin superfamily. Endocr Rev 2007;28:707-725. 242. Kim S, Shim JJ, Burgel PR, Ueki IF, Dao-Pick T, Tam DC, Nadel JA. Il-13- induced clara cell secretory protein expression in airway epithelium: Role of egfr signaling pathway. Am J Physiol Lung Cell Mol Physiol 2002;283:L67-75. 243. Reynolds SD, Reynolds PR, Pryhuber GS, Finder JD, Stripp BR. Secretoglobins scgb3a1 and scgb3a2 define secretory cell subsets in mouse and human airways. Am J Respir Crit Care Med 2002;166:1498-1509. 244. Dodge DE, Plopper CG, Rucker RB. Regulation of clara cell 10 kd protein secretion by pilocarpine: Quantitative comparison of nonciliated cells in rat bronchi and bronchioles based on laser scanning confocal microscopy. Am J Respir Cell Mol Biol 1994;10:259-270. 245. Johnston CJ, Mango GW, Finkelstein JN, Stripp BR. Altered pulmonary response to hyperoxia in clara cell secretory protein deficient mice. Am J Respir Cell Mol Biol 1997;17:147-155. 246. Mango GW, Johnston CJ, Reynolds SD, Finkelstein JN, Plopper CG, Stripp BR. Clara cell secretory protein deficiency increases oxidant stress response in conducting airways. Am J Physiol 1998;275:L348-356. 247. Arsalane K, Broeckaert F, Knoops B, Wiedig M, Toubeau G, Bernard A. Clara cell specific protein (cc16) expression after acute lung inflammation induced by intratracheal lipopolysaccharide administration. Am J Respir Crit Care Med 2000;161:1624-1630. 248. Wang SZ, Rosenberger CL, Bao YX, Stark JM, Harrod KS. Clara cell secretory protein modulates lung inflammatory and immune responses to respiratory syncytial virus infection. J Immunol 2003;171:1051-1060. 249. Wang SZ, Rosenberger CL, Espindola TM, Barrett EG, Tesfaigzi Y, Bice DE, Harrod KS. Ccsp modulates airway dysfunction and host responses in an ova- 132

challenged mouse model. Am J Physiol Lung Cell Mol Physiol 2001;281:L1303- 1311. 250. Harrod KS, Mounday AD, Stripp BR, Whitsett JA. Clara cell secretory protein decreases lung inflammation after acute virus infection. Am J Physiol 1998;275:L924-930. 251. Watson TM, Reynolds SD, Mango GW, Boe IM, Lund J, Stripp BR. Altered lung gene expression in ccsp-null mice suggests immunoregulatory roles for clara cells. Am J Physiol Lung Cell Mol Physiol 2001;281:L1523-1530. 252. Hayashida S, Harrod KS, Whitsett JA. Regulation and function of ccsp during pulmonary pseudomonas aeruginosa infection in vivo. Am J Physiol Lung Cell Mol Physiol 2000;279:L452-459. 253. Mandal AK, Zhang Z, Ray R, Choi MS, Chowdhury B, Pattabiraman N, Mukherjee AB. Uteroglobin represses allergen-induced inflammatory response by blocking pgd2 receptor-mediated functions. J Exp Med 2004;199:1317-1330. 254. Chen LC, Zhang Z, Myers AC, Huang SK. Cutting edge: Altered pulmonary eosinophilic inflammation in mice deficient for clara cell secretory 10-kda protein. J Immunol 2001;167:3025-3028. 255. Stripp BR, Reynolds SD, Boe IM, Lund J, Power JH, Coppens JT, Wong V, Reynolds PR, Plopper CG. Clara cell secretory protein deficiency alters clara cell secretory apparatus and the protein composition of airway lining fluid. Am J Respir Cell Mol Biol 2002;27:170-178. 256. Reynolds SD, Reynolds PR, Snyder JC, Whyte F, Paavola KJ, Stripp BR. Ccsp regulates cross talk between secretory cells and both ciliated cells and macrophages of the conducting airway. Am J Physiol Lung Cell Mol Physiol 2007;293:L114-123. 257. Snyder JC, Reynolds SD, Hollingsworth JW, Li Z, Kaminski N, Stripp BR. Clara cells attenuate the inflammatory response through regulation of macrophage behavior. Am J Respir Cell Mol Biol 2010;42:161-171. 258. Ramsay PL, Luo Z, Major A, Park MS, Finegold M, Welty SE, Kwak I, Darlington G, Demayo FJ. Multiple mechanisms for oxygen-induced regulation of the clara cell secretory protein gene. FASEB J 2003;17:2142-2144. 259. Yao XL, Ikezono T, Cowan M, Logun C, Angus CW, Shelhamer JH. Interferon- gamma stimulates human clara cell secretory protein production by human airway epithelial cells. Am J Physiol 1998;274:L864-869. 260. Yao XL, Levine SJ, Cowan MJ, Logun C, Shelhamer JH. Tumor necrosis factor- alpha stimulates human clara cell secretory protein production by human airway epithelial cells. Am J Respir Cell Mol Biol 1998;19:629-635. 261. Cassel TN, Nordlund-Moller L, Andersson O, Gustafsson JA, Nord M. C/ebpalpha and c/ebpdelta activate the clara cell secretory protein gene through interaction with two adjacent c/ebp-binding sites. Am J Respir Cell Mol Biol 2000;22:469-480. 262. Harrod KS, Jaramillo RJ. Pseudomonas aeruginosa and tumor necrosis factor- alpha attenuate clara cell secretory protein promoter function. Am J Respir Cell Mol Biol 2002;26:216-223. 133

263. Sawaya PL, Stripp BR, Whitsett JA, Luse DS. The lung-specific cc10 gene is regulated by transcription factors from the ap-1, octamer, and hepatocyte nuclear factor 3 families. Mol Cell Biol 1993;13:3860-3871. 264. Kropski JA, Fremont RD, Calfee CS, Ware LB. Clara cell protein (cc16), a marker of lung epithelial injury, is decreased in plasma and pulmonary edema fluid from patients with acute lung injury. Chest 2009;135:1440-1447. 265. McAuley DF, Matthay MA. Clara cell protein cc16. A new lung epithelial biomarker for acute lung injury. Chest 2009;135:1408-1410. 266. Ramsay PL, DeMayo FJ, Hegemier SE, Wearden ME, Smith CV, Welty SE. Clara cell secretory protein oxidation and expression in premature infants who develop bronchopulmonary dysplasia. Am J Respir Crit Care Med 2001;164:155- 161. 267. Sarafidis K, Stathopoulou T, Diamanti E, Soubasi V, Agakidis C, Balaska A, Drossou V. Clara cell secretory protein (cc16) as a peripheral blood biomarker of lung injury in ventilated preterm neonates. Eur J Pediatr 2008;167:1297-1303. 268. Schrama AJ, Bernard A, Poorthuis BJ, Zwinderman AH, Berger HM, Walther FJ. Cord blood clara cell protein cc16 predicts the development of bronchopulmonary dysplasia. Eur J Pediatr 2008;167:1305-1312. 269. Lumsden AB, McLean A, Lamb D. Goblet and clara cells of human distal airways: Evidence for smoking induced changes in their numbers. Thorax 1984;39:844-849. 270. Shijubo N, Itoh Y, Yamaguchi T, Shibuya Y, Morita Y, Hirasawa M, Okutani R, Kawai T, Abe S. Serum and bal clara cell 10 kda protein (cc10) levels and cc10- positive bronchiolar cells are decreased in smokers. Eur Respir J 1997;10:1108- 1114. 271. Poynter ME, Irvin CG, Janssen-Heininger YM. A prominent role for airway epithelial nf-kappa b activation in lipopolysaccharide-induced airway inflammation. J Immunol 2003;170:6257-6265. 272. Cheng DS, Han W, Chen SM, Sherrill TP, Chont M, Park GY, Sheller JR, Polosukhin VV, Christman JW, Yull FE, Blackwell TS. Airway epithelium controls lung inflammation and injury through the nf-kappa b pathway. J Immunol 2007;178:6504-6513. 273. Ather JL, Hodgkins SR, Janssen-Heininger YM, Poynter ME. Airway epithelial nf-kappab activation promotes allergic sensitization to an innocuous inhaled antigen. Am J Respir Cell Mol Biol 2011;44:631-638. 274. Pantano C, Ather JL, Alcorn JF, Poynter ME, Brown AL, Guala AS, Beuschel SL, Allen GB, Whittaker LA, Bevelander M, Irvin CG, Janssen-Heininger YM. Nuclear factor-kappab activation in airway epithelium induces inflammation and hyperresponsiveness. Am J Respir Crit Care Med 2008;177:959-969. 275. Poynter ME, Cloots R, van Woerkom T, Butnor KJ, Vacek P, Taatjes DJ, Irvin CG, Janssen-Heininger YM. Nf-kappa b activation in airways modulates allergic inflammation but not hyperresponsiveness. J Immunol 2004;173:7003-7009. 276. Sadikot RT, Han W, Everhart MB, Zoia O, Peebles RS, Jansen ED, Yull FE, Christman JW, Blackwell TS. Selective i kappa b kinase expression in airway 134

epithelium generates neutrophilic lung inflammation. J Immunol 2003;170:1091- 1098. 277. Skerrett SJ, Liggitt HD, Hajjar AM, Ernst RK, Miller SI, Wilson CB. Respiratory epithelial cells regulate lung inflammation in response to inhaled endotoxin. Am J Physiol Lung Cell Mol Physiol 2004;287:L143-152. 278. Elizur A, Adair-Kirk TL, Kelley DG, Griffin GL, deMello DE, Senior RM. Clara cells impact the pulmonary innate immune response to lps. Am J Physiol Lung Cell Mol Physiol 2007;293:L383-392. 279. Elizur A, Adair-Kirk TL, Kelley DG, Griffin GL, Demello DE, Senior RM. Tumor necrosis factor-alpha from macrophages enhances lps-induced clara cell expression of keratinocyte-derived chemokine. Am J Respir Cell Mol Biol 2008;38:8-15. 280. Park MS, Zhao B, Ramsay PL, Chang AS, Reardon MJ, DeMayo FJ. Expression of inflammatory cytokines in a mouse transformed clara cell line by tumor necrosis factor-alpha. Ann N Y Acad Sci 2000;923:336-337. 281. Evans CM, Williams OW, Tuvim MJ, Nigam R, Mixides GP, Blackburn MR, DeMayo FJ, Burns AR, Smith C, Reynolds SD, Stripp BR, Dickey BF. Mucin is produced by clara cells in the proximal airways of antigen-challenged mice. Am J Respir Cell Mol Biol 2004;31:382-394. 282. Sonar SS, Ehmke M, Marsh LM, Dietze J, Dudda JC, Conrad ML, Renz H, Nockher WA. Clara cells drive eosinophil accumulation in allergic asthma. Eur Respir J 2012;39:429-438. 283. Kuperman DA, Huang X, Koth LL, Chang GH, Dolganov GM, Zhu Z, Elias JA, Sheppard D, Erle DJ. Direct effects of interleukin-13 on epithelial cells cause airway hyperreactivity and mucus overproduction in asthma. Nat Med 2002;8:885-889. 284. Stripp BR, Reynolds SD. Maintenance and repair of the bronchiolar epithelium. Proc Am Thorac Soc 2008;5:328-333. 285. Reynolds SD, Malkinson AM. Clara cell: Progenitor for the bronchiolar epithelium. Int J Biochem Cell Biol 2010;42:1-4. 286. Giangreco A, Reynolds SD, Stripp BR. Terminal bronchioles harbor a unique airway stem cell population that localizes to the bronchoalveolar duct junction. Am J Pathol 2002;161:173-182. 287. Rawlins EL, Okubo T, Xue Y, Brass DM, Auten RL, Hasegawa H, Wang F, Hogan BL. The role of scgb1a1+ clara cells in the long-term maintenance and repair of lung airway, but not alveolar, epithelium. Cell Stem Cell 2009;4:525- 534. 288. Snyder JC, Zemke AC, Stripp BR. Reparative capacity of airway epithelium impacts deposition and remodeling of extracellular matrix. Am J Respir Cell Mol Biol 2009;40:633-642. 289. Teisanu RM, Chen H, Matsumoto K, McQualter JL, Potts E, Foster WM, Bertoncello I, Stripp BR. Functional analysis of two distinct bronchiolar progenitors during lung injury and repair. Am J Respir Cell Mol Biol 2011;44:794-803. 135

290. Wang D, Wang H, Brown J, Daikoku T, Ning W, Shi Q, Richmond A, Strieter R, Dey SK, DuBois RN. Cxcl1 induced by prostaglandin e2 promotes angiogenesis in colorectal cancer. J Exp Med 2006;203:941-951. 291. Sales KJ, Maldonado-Perez D, Grant V, Catalano RD, Wilson MR, Brown P, Williams AR, Anderson RA, Thompson EA, Jabbour HN. Prostaglandin f(2alpha)-f-prostanoid receptor regulates cxcl8 expression in endometrial adenocarcinoma cells via the calcium-calcineurin-nfat pathway. Biochim Biophys Acta 2009;1793:1917-1928. 292. Serhan CN, Brain SD, Buckley CD, Gilroy DW, Haslett C, O'Neill LA, Perretti M, Rossi AG, Wallace JL. Resolution of inflammation: State of the art, definitions and terms. Faseb J 2007;21:325-332. 293. Goggel R, Hoffman S, Nusing R, Narumiya S, Uhlig S. Platelet-activating factor- induced pulmonary edema is partly mediated by prostaglandin e(2), e-prostanoid 3-receptors, and potassium channels. Am J Respir Crit Care Med 2002;166:657- 662. 294. Bonnans C, Levy BD. Lipid mediators as agonists for the resolution of acute lung inflammation and injury. American journal of respiratory cell and molecular biology 2006. 295. Levy BD, Clish CB, Schmidt B, Gronert K, Serhan CN. Lipid mediator class switching during acute inflammation: Signals in resolution. Nat Immunol 2001;2:612-619. 296. Basu S. Novel cyclooxygenase-catalyzed bioactive prostaglandin f2alpha from physiology to new principles in inflammation. Med Res Rev 2007;27:435-468. 297. Lasa M, Mahtani KR, Finch A, Brewer G, Saklatvala J, Clark AR. Regulation of cyclooxygenase 2 mrna stability by the mitogen-activated protein kinase p38 signaling cascade. Mol Cell Biol 2000;20:4265-4274. 298. Murray PJ, Wynn TA. Protective and pathogenic functions of macrophage subsets. Nat Rev Immunol 2011;11:723-737. 299. Byers DE, Holtzman MJ. Alternatively activated macrophages and airway disease. Chest 2011;140:768-774. 300. Rogers LK, Valentine CJ, Pennell M, Velten M, Britt RD, Dingess K, Zhao X, Welty SE, Tipple TE. Maternal docosahexaenoic acid supplementation decreases lung inflammation in hyperoxia-exposed newborn mice. J Nutr 2011;141:214- 222. 301. Haworth O, Levy BD. Endogenous lipid mediators in the resolution of airway inflammation. Eur Respir J 2007;30:980-992. 302. Titos E, Rius B, Gonzalez-Periz A, Lopez-Vicario C, Moran-Salvador E, Martinez-Clemente M, Arroyo V, Claria J. Resolvin d1 and its precursor docosahexaenoic acid promote resolution of adipose tissue inflammation by eliciting macrophage polarization toward an m2-like phenotype. J Immunol 2011;187:5408-5418. 303. Hamid Q, Tulic M. Immunobiology of asthma. Annu Rev Physiol 2009;71:489- 507.

136

304. Valentine CJ, Morrow G, Fernandez S, Gulati P, Bartholomew D, Long D, Welty SE, Morrow AL, Rogers LK. Docosahexaenoic acid and amino acid contents in pasteurized donor milk are low for preterm infants. J Pediatr 2010;157:906-910. 305. Valentine CJ, Fernandez S, Rogers LK, Gulati P, Hayes J, Lore P, Puthoff T, Dumm M, Jones A, Collins K, Curtiss J, Hutson K, Clark K, Welty SE. Early amino-acid administration improves preterm infant weight. J Perinatol 2009;29:428-432. 306. Valentine CJ. Maternal dietary dha supplementation to improve inflammatory outcomes in the preterm infant. Adv Nutr 2012;3:370-376. 307. Speer CP. Pulmonary inflammation and bronchopulmonary dysplasia. J Perinatol 2006;26 Suppl 1:S57-62; discussion S63-54. 308. Ueda K, Cho K, Matsuda T, Okajima S, Uchida M, Kobayashi Y, Minakami H, Kobayashi K. A rat model for arrest of alveolarization induced by antenatal endotoxin administration. Pediatric research 2006;59:396-400. 309. Bry K, Whitsett JA, Lappalainen U. Il-1beta disrupts postnatal lung morphogenesis in the mouse. American journal of respiratory cell and molecular biology 2007;36:32-42. 310. Nagai A, Katayama M, Thurlbeck WM, Matsui R, Yasui S, Konno K. Effect of indomethacin on lung development in postnatal rats: Possible role of prostaglandin in alveolar formation. Am J Physiol 1995;268:L56-62. 311. Torday JS, Rehan VK. Developmental cell/molecular biologic approach to the etiology and treatment of bronchopulmonary dysplasia. Pediatr Res 2007;62:2-7.

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