Timothy S. Blackwell - US grants

Acute lung injury is an event that occurs as a consequence of a variety of disease states which share a common pathobiological process, neutrophilic lung inflammation (NLI). NLI likely results from increased production of inflammatory cytokines and endothelial-leukocyte adhesion molecules, many of which are regulated by the ubiquitous transcription factor complex, NF-kappaB. Although NF-kappaB is necessary for directing high level transcription of many cytokines, adhesion molecules, and other pro-inflammatory genes in tissue culture, the extent to which NF-kappaB controls specific biological processes in vivo remains unanswered. Systemic inflammation induced by injection of bacterial lipopolysaccharide (LPS) results in a reproducible pattern of NF-kappaB activation in lung tissue, gene expression of several NF- kappaB-dependent cytokines, and NLI. At present, the proximate stimulus for NF-kappaB activation in the lung following LPS injection is unknown. NF-kappaB activation could occur as a result of direct stimulation of lung cells by LPS. Conversely, other mediators, such as TNFalpha or IL-1, could be primarily responsible for lung NF-kappaB activation in this setting. Another uncertainty is whether NF-kappaB is activated diffusely and simultaneously in all lung cells, or whether the timing and intensity of NF-kappaB activation differs among subpopulations of lung cells. The larger question concerns the extent to which NF-kappaB activation regulates production of cytokines and other pro-inflammatory molecules in vivo. It is uncertain whether NF-kappaB activation is merely a marker of significant acute inflammation. The following hypotheses are proposed to address these issues: 1) systemic LPS induces NF-kappaB activation in the lung through both direct stimulation of lung cells and action of the LPS-induced cytokines TNFalpha and IL-1,2) specific subsets of lung cells are differentially activated following LPS injection, and 3) targeting NF-kappaB in lung cells for molecular intervention will result in attenuation of LPS-induced neutrophilic lung inflammation. We have four specific aims to address these hypotheses using a murine model system of NLI following intraperitoneal injection of LPS. The first is to define the characteristic pattern of NF-kappaB activation in lung (compared to other organs) following intraperitoneal injection of LPS and relate NF-kappaB activation to expression of NF- kappaB dependent cytokine genes and NLI. The second specific aim is to investigate the specific cell types in the lung which respond to intraperitoneal LPS injection by activating NF-kappaB. The third specific aim is to modulate LPS-induced activation of NF-kappaB in the lungs by inhibiting the cytokine cascade or blocking IKappaB degradation. The last specific aim is to specifically target the NF- kappaB complex in the lung for molecular intervention using a trans- dominant inhibitor of the NF-kappaB complex. These studies should lead to a better understanding of the role of NF-kappaB in the molecular regulation of NLI, and may ultimately lead to better treatment strategies for the adult respiratory distress syndrome and other inflammatory lung diseases.

Activated fibroblasts determine the extent of pulmonary fibrosis by their production of collagen and other matrix components. Fibroblast specific protein 1 (FSP1) is a member of the S100 protein superfamily and appears to play an early role in establishing the fibroblast phenotype. Although the origin of activated fibroblasts in pulmonary fibrosis is uncertain, recent studies in the kidney have shown that fibroblasts may arise from epithelium through a phenomenon called epithelial- mesenchymal transformation. Transforming growth factor-beta and other phenotypic modulators appear to regulate this process through up- regulation of FSP1 and other proteins that control fibroblast phenotype. In this project, we propose to investigate the following hypothesis. In lung fibrosis, activated lung fibroblasts are derived from airway epithelial cells as well as from resident interstitial fibroblasts. FSP1 is both a marker and important determinant of this cellular phenotype. The appearance and persistence of FSP1 expressing cells are crucial for determining the extent of lung fibrosis. We propose three specific aims: 1) to identify the role of FSP1+ cells in experimental lung fibrosis, 2) to determine whether epithelial-mesenchymal transformation occurs in the lungs and contributes to lung fibrosis in the mouse, 3) to modulate FSP1 expression and determine the effects on epithelial-mesenchymal transformation and induction of lung fibrosis. Identification of a more useful fibroblast marker in the lungs would allow identification and monitoring of this cell population after fibrogenic stimuli and could foster targeted treatments that alter the accumulation or function of cells exhibiting the fibroblast phenotype. In addition, we plan to explore the origins of lung fibroblasts in experimental lung fibrosis with the hope that determining the presence and extent of epithelial-mesenchymal transformation in the lungs in these models will lead to innovative interventions to inhibit or reverse this transformation, thus limiting fibrosis and lung dysfunction.

The mouse husbandry and genotyping core will support the use of mice in this program by: 1) performing routine husbandry of transgenic, knockout, and other mice for all proposed, 2) determining genotypes of transgenic and knockout mice, 3) cross-breeding genetically defined mouse strains, 2) distributing and knockout mice to investigators in the individual projects. This core will work with the Vanderbilt University Animal Care Facility the use of genetically defined mice for use in the study of inflammation.

DESCRIPTION (provided by applicant): Recent evidence points to a critical role for lung epithelial cells in determining the extent and progression of lung fibrosis. The importance of epithelial cells in fibrogenesis is highlighted by the identification of mutations in the epithelia-restricted genes, surfactant protein C (SFTPC) and SFTPA2, that are associated with familial IPF. Mutant forms of SFTPC impact protein folding in the endoplasmic reticulum (ER), resulting in ER stress. During this funding period, we found that ER stress is common in patients with sporadic and familial IPF and that markers of ER stress localize to epithelial cells in areas of lung parenchymal remodeling. We generated a transgenic mouse model in which expression of L188Q SFTPC is localized to type II alveolar epithelial cells (AECs). Transgene induction in this model leads to ER stress, but is not sufficient to cause fibrosis. However, treating these mice with low dose bleomycin results in a marked exacerbation of lung fibrosis as well as increased apoptosis of AECs and increased accumulation of fibroblasts, including fibroblasts derived via epithelial-mesenchymal transition (EMT). Our studies support the concept that ER stress in AECs makes these cells vulnerable to fibrotic stimuli, but a 'second hit' is necessary for development of lung fibrosis. In order to begin to identify relevant stimuli (i.e. - second hits) tat lead to progressive fibrosis in the setting of 'vulnerable' epithelial cells, we infected L188Q SFTPC expressing mice with the murine gamma herpesvirus (MHV68) and found that MVH68 infection in L188Q SFTPC expressing mice (but not wild type mice) results in progressive fibrosis. In addition, we recently showed that induction of ER stress in AECs in vitro results in EMT via endogenous activation of Src and Smad2/3 pathways. Based on preliminary data, we propose the hypothesis that ER stress in lung epithelial cells contributes to fibrotic remodeling by altering susceptibility to environmental stimuli (including herpesviruses and cigarette smoke). Depending on the severity and timing of the insult, ER stress predisposes epithelial cells to apoptosis, EMT, or a pro-fibrotic epithelial phenotype through alterations in intracellular signaling pathways, including Smad2/3 and Src. Interventions to reduce or modify the ER stress response will be effective in limiting progressive lung fibrosis. Specific aims will: 1) define the mechanisms by which herpesvirus infection causes lung fibrosis in mice with inducible expression of mutant surfactant protein C in type II AECs, 2) investigate ER stress dependent pathways in type II AECs that mediate profibrotic phenotypes, and 3) test the impact of cigarette smoke on development of lung fibrosis in the setting of underlying ER stress and investigate whether ER stress reducing agents can block fibrotic remodeling. Together, these studies will advance the study of lung fibrosis by developing more relevant mouse models to study lung fibrosis, defining the vulnerable state of AECs that facilitates lung fibrosis, and identifying novl therapeutic approaches to ameliorate lung fibrosis based on improved ER function.

[unreadable] DESCRIPTION (provided by applicant): Although the etiology of idiopathic pulmonary fibrosis (IPF) remains poorly defined, several lines of evidence suggest that alveolar epithelial cells (AECs) play key roles in disease pathogenesis. We have identified mutations in the gene encoding surfactant protein C (SP-C) that affect AEC function in some patients with the familial form of IPF, familial interstitial pneumonia (FIP). Data presented in this application indicate that abnormal processing of pro-SP-C by AECs leads to endoplasmic reticulum (ER) stress, activation of the unfolded protein response, and cell death. In addition, our data show that ER stress occurs frequently in AECs in late stage IPF (and FIP), suggesting that this pathway commonly contributes to disease progression. Other preliminary data show that herpesviruses, which are commonly identified in IPF, are localized to AECs and could contribute to ER stress and AEC injury. Further, we have identified loss-of-function mutations in genes for essential components of telomerase that segregate with disease in some FIP families. In the lungs, it is possible that defective telomerase leads to telomere shortening followed by cell cycle arrest and apoptosis of type II AECs. Since most patients have advanced disease at the time of diagnosis, it has not been possible to define early events in IPF pathogenesis and controversy continues about whether mediators and pathways implicated in fibrotic remodeling represent primary disease mechanisms or occur as a result of an ongoing injury-repair process. These issues are particularly relevant to the study of AECs, which become hyperplastic in areas of fibrotic remodeling. Identification of patients with early FIP provides a valuable resource for investigations aimed at defining primary disease mechanisms. We have currently identified 61 asymptomatic individuals with radiographic changes consistent with early FIP by screening 417 at risk individuals. This group and additional subjects with early FIP identified by screening with high resolution computed tomography scanning will undergo bronchoscopy for sample collection to test the following hypothesis. Genetic or acquired factors that increase the susceptibility of lung epithelial cells to apoptosis underlie the pathogenesis of familial and sporadic forms of IPF. Exposure of vulnerable epithelial cells to common injurious/toxic environmental stimuli results in extensive injury with limited capacity for alveolar repair, leading to fibrotic remodeling. Identification of common phenotypic manifestations of vulnerable epithelial cells, such as ER stress or telomere shortening, will better define disease pathogenesis and identify novel targets for future therapeutic interventions. The following specific aims will investigate the role of alveolar epithelium in early FIP: 1) to evaluate epithelial cell injury/apoptosis, markers of ER stress, and surfactant protein production in the lungs of patients with early FIP, 2) to investigate whether herpesvirus infection occurs in early FIP, contributes to ER stress, and is associated with alveolar epithelial cell injury and, 3) to determine whether differential telomere length occurs in epithelial cells from patients with early FIP and correlates with epithelial cell injury. PROJECT NARRATIVE: Diseases that result in progressive fibrosis of the lung, including idiopathic pulmonary fibrosis (IPF), are a substantial cause of morbidity and mortality for which there are no effective treatments. We have identified a unique cohort of individuals with the earliest form of pulmonary fibrosis that will be studied in order to determine factors that are essential for disease initiation and early progression. By elucidating the critical components of early stage disease, our study will define novel targets for future therapeutic interventions. [unreadable] [unreadable] [unreadable]

Description (provided by applicant): Acute and chronic lung diseases are major causes of morbidity and mortality throughout the world. For many of these diseases, the fundamental pathobiology is not well understood and effective, disease-modifying treatments are not available. Therefore, there is a pressing need to train researchers focused on elucidating disease mechanisms. The Division of Allergy, Pulmonary, and Critical Care Medicine at Vanderbilt University and the Center for Lung Research have a long, successful history in developing well trained researchers who have the vision and the skills with which to embark on successful careers in academic research. This new program will train young investigators to study basic mechanisms of lung inflammation, repair, and remodeling. The Vanderbilt Center for Lung Research (VCLR), which was developed to coordinate and enhance collaborative interdisciplinary research and training related to the lungs, will house this program, and Dr. Timothy Blackwell (director of the VCLR) will serve as program director. This training program will support postdoctoral trainees who have completed M.D. and/or Ph.D. programs and show exceptional aptitude for successfully pursuing an academic research career. Trainees will concentrate on one of several disease focused areas of existing expertise in the VCLR: acute lung inflammation/injury, asthma, pulmonary fibrosis, pulmonary hypertension, or lung carcinogenesis. A customized mentoring team will be formed for each trainee, consisting of a mentor with nationally recognized expertise in the area and a research advisory committee to provide additional guidance, mentoring, and feedback. The trainee's experience will be enhanced by interactions with other investigators and trainees in existing lung disease-focused research programs in the VCLR, an extensive program of seminars and conferences, and coursework tailored to meet the needs of each trainee. The unique environment of the VCLR - its rich collaborative interactions between basic scientists and clinical researchers, wide range of supporting Cores and Centers, and exposure to state-of-the-art clinical care - provides an outstanding opportunity to train successful scientists whose discoveries regarding fundamental aspects of lung diseases can be translated into improved patient care.

DESCRIPTION (provided by applicant): Bronchopulmonary Dysplasia (BPD) is a frequent complication of preterm birth that results from arrested lung development in the saccular and alveolar stages of lung morphogenesis. While innate immune signaling has been implicated in the disruption of epithelial-mesenchymal interactions that regulate normal branching morphogenesis, the mechanisms linking innate immunity and abnormal lung development in BPD remain unclear. This proposal will specifically test the role of macrophages in BPD pathogenesis through regulation of innate immune signaling in the fetal lung. Our preliminary data indicate that exposing the fetal lung to inflammatory stimuli induces expression of NF-kB dependent inflammatory cytokine by fetal lung macrophages. Depletion of macrophages or targeted disruption of the NF-kB signaling pathway in macrophages prevents alterations in lung morphogenesis induced by Gram negative bacterial lipopolysaccharide (LPS). While prenatal exposure to inflammatory stimuli increases the risk of BPD in preterm infants, a second episode of significant inflammation may be key for progression to clinical disease, suggesting that exposure to prenatal inflammatory stimuli primes the innate immunity for a heightened response to subsequent noxious stimuli. Consistent with this idea, we found that in utero exposure to LPS results in a marked increase in neutrophilic lung inflammation following subsequent intratracheal LPS treatment of pups after weaning. Therefore, structural abnormalities in the lungs resulting from prenatal inflammation may define a vulnerable host in which additional insults, such as oxygen toxicity, mechanical ventilation, and recurrent infection, result in excessive lung inflammation, injury, and remodeling that is clinically manifest as BPD. In this proposal, we will test the hypothesis that infections and inflammatory stimuli lead to BPD through activation of innate immune signaling in fetal lung macrophages. Activation of NF-kB signaling in macrophages results in production of cytokines, including TNF1 and IL-12, that inhibit epithelial-mesenchymal cell interactions required for branching morphogenesis in the lungs. Activation of NF-kB in fetal lung macrophages also primes the lung for excessive immune responses later in life by altering the lung macrophage phenotype. Three specific aims are proposed to investigate this hypothesis: 1) to determine whether fetal lung macrophages are required for inhibition of lung development by Toll-Like Receptor (TLR) agonists, 2) to identify the role of the NF-kB pathway in inhibition of lung development following innate immune activation, 3) to determine whether activation of fetal lung macrophages alters the macrophage phenotype as lungs mature. Determining the cell types and pathways that lead to arrested lung development and altered innate immunity in the setting of perinatal inflammation could lead to targeted therapies that prevent BPD or improve outcomes. PUBLIC HEALTH RELEVANCE: This proposal will test novel mechanisms of disease explaining how infection and inflammation disrupt normal lung development and immune function in preterm infants. These processes contribute to the pathogenesis of bronchopulmonary dysplasia, a chronic lung disease that affects up to 10,000 former preterm children each year in the United States. (End of Abstract)

PROJECT 1: The pathogenesis of idiopathic pulmonary fibrosis (IPF) remains poorly defined;however, identificafion of mutafions in the gene encoding surfactant protein C (SP-C) in the familial form of IPF (FIP), along with several other lines of evidence, suggests that alveolar epithelial cells (AECs) play a key role in disease progression. Our data indicate that abnormal processing of pro-SP-C by AECs leads to endoplasmic reficulum (ER) stress, acfivafion ofthe unfolded protein response, and cell death. We also show that ER stress occurs frequently in AECs in IPF, suggesfing that this pathway contributes to disease. In addifion, we show that herpesviruses are commonly localized to AECs in IPF and could contribute to ER stress and AEC injury. Finally, we have identified loss-of-funcfion mutations in telomerase genes that segregate with disease in some FIP families, suggesting that defective telomerase leads to telomere shortening and apoptosis of type II AECs. Identificafion of individuals in FIP families with early fibrotic changes will provide a valuable resource for invesfigafions aimed at defining primary disease mechanisms. In this study, we will ufilize CT scanning to identify asymptomafic individuals at risk for FIP who have radiographic changes consistent with early fibrosis. Subjects with eariy FIP and controls will undergo bronchoscopy for sample collecfion to test the following hypothesis. Genefic or acquired factors that increase the suscepfibility of lung epithelial cells to injury and/or apoptosis underiie the pathogenesis of IPF. Exposure of vulnerable epithelial cells to common injurious/toxic environmental sfimuli results in extensive injury with limited capacity for alveolar repair, leading to fibrofic remodeling. The following specific aims will invesfigate the role of AECs in early FIP: 1) to evaluate epithelial cell injury/apoptosis, markers of ER stress, and surfactant protein producfion in the lungs of pafients with eariy FIP, 2) to invesfigate whether herpesvirus infection occurs in eariy FIP, contributes to ER stress, and is associated with alveolar epithelial cell injury and, 3) to determine whether differenfial telomere length occurs in epithelial cells from patients with eariy FIP and correlates with epithelial cell injury. By elucidafing crifical components of eariy stage disease, our study will define novel therapeufic targets.

DESCRIPTION (provided by applicant): Bronchopulmonary Dysplasia (BPD) is a frequent complication of preterm birth that results from arrested lung development in the saccular and alveolar stages of lung morphogenesis. While innate immune signaling has been implicated in the disruption of epithelial-mesenchymal interactions that regulate normal branching morphogenesis, the mechanisms linking innate immunity and abnormal lung development in BPD remain unclear. This proposal will specifically test the role of macrophages in BPD pathogenesis through regulation of innate immune signaling in the fetal lung. Our preliminary data indicate that exposing the fetal lung to inflammatory stimuli induces expression of NF-kB dependent inflammatory cytokine by fetal lung macrophages. Depletion of macrophages or targeted disruption of the NF-kB signaling pathway in macrophages prevents alterations in lung morphogenesis induced by Gram negative bacterial lipopolysaccharide (LPS). While prenatal exposure to inflammatory stimuli increases the risk of BPD in preterm infants, a second episode of significant inflammation may be key for progression to clinical disease, suggesting that exposure to prenatal inflammatory stimuli primes the innate immunity for a heightened response to subsequent noxious stimuli. Consistent with this idea, we found that in utero exposure to LPS results in a marked increase in neutrophilic lung inflammation following subsequent intratracheal LPS treatment of pups after weaning. Therefore, structural abnormalities in the lungs resulting from prenatal inflammation may define a vulnerable host in which additional insults, such as oxygen toxicity, mechanical ventilation, and recurrent infection, result in excessive lung inflammation, injury, and remodeling that is clinically manifest as BPD. In this proposal, we will test the hypothesis that infections and inflammatory stimuli lead to BPD through activation of innate immune signaling in fetal lung macrophages. Activation of NF-kB signaling in macrophages results in production of cytokines, including TNF1 and IL-12, that inhibit epithelial-mesenchymal cell interactions required for branching morphogenesis in the lungs. Activation of NF-kB in fetal lung macrophages also primes the lung for excessive immune responses later in life by altering the lung macrophage phenotype. Three specific aims are proposed to investigate this hypothesis: 1) to determine whether fetal lung macrophages are required for inhibition of lung development by Toll-Like Receptor (TLR) agonists, 2) to identify the role of the NF-kB pathway in inhibition of lung development following innate immune activation, 3) to determine whether activation of fetal lung macrophages alters the macrophage phenotype as lungs mature. Determining the cell types and pathways that lead to arrested lung development and altered innate immunity in the setting of perinatal inflammation could lead to targeted therapies that prevent BPD or improve outcomes. PUBLIC HEALTH RELEVANCE: This proposal will test novel mechanisms of disease explaining how infection and inflammation disrupt normal lung development and immune function in preterm infants. These processes contribute to the pathogenesis of bronchopulmonary dysplasia, a chronic lung disease that affects up to 10,000 former preterm children each year in the United States. (End of Abstract)

CORE A: Given the size, complexity, duration, and scope involved, Core A will be fundamental In enabling the successful establishment and operation ofthe entire program. All costs for administrative support for the Cores and Projects are included here. Dr. Timothy Blackwell will assume ultimate administrative responsibility for the management of this Core. Administrative and clerical support for the entire program will be shared by all Project Leaders, Core Leaders and investigators. This Core will provide and maintain all of the administrative space and functions for the entire program, including but not limited to, offices for all investigators, an administrative area with secretarial and administrative support, a copy/work room, and a conference room with up to date audiovisual amenities. Administrative functions will include ordering of supplies and equipment, maintenance of all records, keeping and monitoring of budgets, maintenance ofthe personnel database for grant effort, interactions with University administrative offices and the NIH regarding budgetary and other administrative matters, and scheduling and organizing meetings and presentations. This Core will support all of the computer hardware and software resources for the administrative and clerical functions ofthe program. This Core will coordinate and support the activities ofthe Internal Advisory Board and the External Scientific Advisory Board. The basic functions and objectives ofthe Core include: quality management of program resources, integration ofthe program, oversight of deliverables within the timeframe, assistance with data management, and leading the vision. To achieve these objectives, detailed plans are presented for administrative structure and leadership, project management, external Scientific Advisory Board, and communication/meetings.

DESCRIPTION (provided by applicant): This program has arisen from our long-standing interest in familial lung diseases, including familial interstitial pneumonia (FIP). We intend to use this unique cohort of families with FIP to investigate the underlying mechanisms that lead to progressive lung fibrosis. This information will be highly relevant to individuals with sporadic idiopathic pulmonary fibrosis (IPF) and other forms of idiopathic interstitial pneumonia (IIP). There are important advantages to investigating lung fibrosis in families. First, it is possible to study genetic causes of disease in this group. Three genes linked to disease, surfactant protein C and components of the telomerase complex, have already been identified using these FIP families, and this identification has led to new insights into disease pathogenesis. Second, this population allows us the unprecedented opportunity to identify pre-symptomatic patients that can be studied at the earliest stages of disease. This gives us the ability to study the primary manifestations of disease as opposed to pathobiological changes that occur as a result of progressive lung remodeling. Project 1 will identify pre-symptomatic individuals at risk for FIP families by high resolution CT scanning that have radiographic abnormalities in the lungs. At risk individuals (with and without radiographic abnormalities) will undergo bronchoscopy to identify phenotypic characteristics of alveolar epithelial cells, such as ER stress, herpesvirus infection, or telomere shortening that contribute to disease progression in FIP. Project 2 will utilize a candidate gene approach to identify and characterize rare genetic vanants in the surfactant and telomerase pathways, as well as genes known to be associated with secondary forms of pulmonary fibrosis, that impact development of FIP. Project 3 will perform a linkage study to discover new genetic loci associated with disease in FIP and sporadic MP patients. Additional studies will identify viruses present in lung tissue of patients with IIP and investigate gene-gene and gene-environment interactions that influence disease manifestation. The integrated approach in this Program will lead to new concepts in pathogenesis of FIP and sporadic IPF (as well as other forms of IIP), and suggest opportunities for novel treatment or prevention strategies.

DESCRIPTION (provided by applicant): Lung macrophages are critical for initiating the innate immune response to microbial and environmental stimuli, resolving acute inflammation, and promoting repair following injury. Persistent or dysregulated macrophage activation plays an important role in the pathogenesis of multiple lung diseases; however, studying the phenotype of the pleiotropic macrophage population in human lung disease is complicated by the invasive methods currently required to obtain sufficient quantities of cells. Development of new methodologies to non- invasively identify activated macrophages could facilitate improved understanding and treatment options for inflammatory lung diseases. A subset of macrophages at sites of inflammation expresses high levels of the folate receptor ? (FR?). Our preliminary data in mice demonstrate that FR? is not expressed in normal, quiescent lungs but is up-regulated specifically in a subset of macrophages after intratracheal or systemic treatment with E. coli lipopolysaccharide (LPS). We also identified FR? expression in lung macrophages in mouse models of bronchopulmonary dysplasia (BPD), lung fibrosis, and chronic pulmonary obstructive disease (COPD). Further, we found that lung macrophages from humans with COPD, idiopathic pulmonary fibrosis (IPF), and BPD express FR?, whereas expression in healthy human macrophages is minimal. Based on expression of this receptor, we have found that we can image activated macrophages in mouse lungs using a folate derivative conjugated with a fluorescent probe. In this proposal, we hypothesize that developing molecular imaging techniques to identify functional subsets of activated macrophages will advance understanding of inflammatory lung diseases and could lead to novel, macrophage-targeted therapies. Although our preliminary studies have employed optical imaging techniques, for applicability to humans we plan to develop positron emission tomography (PET) techniques to image activated lung macrophages. Specific aims are to: 1) identify and characterize specific cell-surface markers present on activated lung macrophages, 2) validate pre-clinical imaging approaches to visualize macrophage activation in vivo, and 3) develop PET-based strategies for imaging activated lung macrophages in vivo. Together, these studies will optimize imaging probes based on FR? expression and explore new imaging targets present on the surface of activated macrophages. These new strategies can then be applied to the study of inflammatory lung diseases in humans.

PROJECT SUMMARY Ascertainment of kindreds with familial interstitial pneumonia (FIP) has proven to be a valuable resource for studying genetic susceptibility and underlying mechanisms that promote interstitial lung disease. Studies of FIP kindreds during the current funding period have led to several important findings, including the observation that rare variants (RVs) in a variety of genes can predispose to FIP. Based on whole exome sequencing (WES) from FIP patients during the current funding cycle, we have identified novel RVs in several genes that we believe are causative in FIP, including the telomerase pathway genes DKC1 and RTEL1, a G protein- coupled receptor GPR87, a centromere gene CENPN, and a gene of unknown function SYDE1. Preliminary studies to examine the functional importance of RVs in these genes have linked RTEL1 and GPR87 to the p53 pathway, suggesting that this pathway may integrate at least a portion of our genetic findings. To identify additional gene candidates, we used an unbiased approach to uncover pathways that were over-represented in the set of candidate RV-containing genes implicated by WES. Using this approach, two interrelated pathways containing microtubule/cilia genes and centrosome genes were found to be markedly over- represented. Together, our preliminary data point to a defined set of interacting pathways that show promise for containing RVs that predispose to FIP. The primary goal of this project during the current funding period was to identify, enroll, and phenotype members of families at risk for developing FIP. To date, we have enrolled 372 asymptomatic first-degree relatives of FIP patients in this ongoing cohort study and obtained blood samples and high resolution CT (HRCT) scans, as well as 100 bronchoscopies. In addition, through our ongoing collaboration with Dr. Schwartz (PI of Project 3), we have recently enrolled another 503 individuals at risk for FIP in this ongoing cohort study. Together, this cohort of 875 asymptomatic individuals provides a powerful resource to identify the earliest manifestations of FIP, better define the natural history of FIP, and determine endophenotypes based on genetic predisposition. In this competitive renewal, we hypothesize that damaging rare genetic variants in several inter-related pathways (including telomere genes, microtubule/cilia/centrosome genes, and p53-related genes) predispose to FIP through a common mechanism related to altered cell survival/proliferation. Specific aims are designed to: 1) evaluate genes with candidate rare genetic variants identified in FIP families and prioritize them for functional studies, 2) perform functional analysis of candidate rare genetic variants in targeted pathways, and 3) characterize genotype-phenotype relationships in individuals at risk for FIP. By validating and testing the function of new disease-associated alleles and using this information to determine genotype-phenotype relationships in individuals enrolled in our ongoing cohort study, these proposed studies provide an unprecedented opportunity to define the impact of genetic predisposition on key aspects of interstitial lung disease pathogenesis.

ABSTRACT Although prostaglandins and their receptors have been studied extensively in pulmonary fibrosis, there is a paucity of data regarding thromboxane A2 (TXA2) and the thromboxane-prostanoid receptor (TPr) in the lungs. We found that TPr is expressed in lung fibroblasts and that expression of this receptor is upregulated in fibroblasts from patients with idiopathic pulmonary fibrosis (IPF), as well as lung fibroblasts from mice treated with bleomycin. Genetic deletion of TPr in mice or treatment with a TPr antagonist (Ifetroban) markedly attenuated bleomycin-induced lung fibrosis. In addition, TPr deficiency or Ifetroben treatment reduced Smad2/3 phosphorylation, ?-smooth muscle actin (?-SMA) expression, and collagen 1 production in lung tissue and isolated lung fibroblasts following bleomycin treatment, without effects on inflammation or epithelial apoptosis. In contrast, treatment with a thromboxane synthesis inhibitor (Ozagrel) was minimally effective at inhibiting lung fibrosis. These findings, along with data showing that thromboxane expression was only transiently upregulated following bleomycin treatment, suggested that TPr activation in fibrosis is mediated through an alternative ligand. F2-isoprostanes (F2-isoPs) are a non-enzymatic product of reactive oxygen species (ROS)-induced peroxidation of arachidonic acid that have structural similarities to TXA2 and can activate TPr signaling. Following treatment with bleomycin, F2-isoPs in mouse lungs were persistently upregulated, suggesting that these ROS products could mediate lung fibrosis via TPr activation. To further investigate mechanisms by which TPr regulates fibrosis, we exposed mouse lung fibroblasts to F2-isoPs (or the specific TPr agonist U-46619) and observed myofibroblast differentiation, increased proliferation, and Smad2/3 phosphorylation, and collagen production, all of which were blocked by deletion of TPr or Ifetroban treatment. Further, in primary lung fibroblasts from IPF patients, we found that TPr antagonism reduced cell proliferation and expression of ?-smooth muscle actin and collagen 1. Together, these data support the hypothesis that reactive oxygen species produced in the lungs of IPF patients generate F2-isoprostanes which activate TPr signaling in lung fibroblasts, leading to myofibroblast differentiation and persistent collagen and matrix production through downstream activation of the Smad/TGF-? pathway. Interventions that block TPr signaling could provide novel therapeutic options to limit progressive pulmonary fibrosis. Specific Aims will: 1) determine the role of TPr signaling in lung fibroblasts in relevant pre-clinical models of lung fibrosis, 2) identify mechanisms by which TPr signaling regulates myofibroblast differentiation and activation, and 3) examine the anti-fibrotic effects of TPr inhibition in human lung fibroblasts and 3-D pulmosphere cultures. Since TPr antagonists, including Ifetroban, are currently available for human use, these studies are likely to set the stage for future clinical studies targeting this pathway (alone or in combination with current drugs) to improve outcomes in IPF and related diseases characterized by progressive pulmonary fibrosis.