Clay B. Marsh - US grants

DESCRIPTION (adapted from the application): This revised proposal will focus on a central hypothesis: monocyte and macrophage accumulation and activation in the lung induce inflammation and initiate fibrosis. The goal of this proposal is to dissect the mechanisms underlying the lung injury and fibrosis seen in idiopathic pulmonary fibrosis (IPF). IPF is a progressive, fatal lung disease of unknown cause and precious few therapeutic options to effectively treat patients. There is firm agreement in academic pulmonary medicine that studies are needed to define the etiology of the disease and to dissect the mechanisms underlying the fibrosis and lung injury. Mice deficient in either the tyrosine phosphatase SHP-1 or the inositol 5-phosphatase SHIP suffer unregulated accumulations of macrophages in their lungs and die of a pulmonary fibrosis-like state. In comparison, bleomycin-treated animals also suffer pulmonary fibrosis that appears to be dependent on macrophage accumulation in the lung. Since macrophage colony-stimulating factor appears to be the primary survival factor for monocytes, including monocytes that are precursors to tissue macrophages, the applicant will investigate the hypothesis that these phosphatases suppress M-CSF-induced cellular activation and survival, and that the lack of these phosphatases leads to macrophage accumulation and possibly fibrosis. The specific aims of the proposal are: 1) to determine the molecular targets of the phosphatases SHP-1 and SHIP in M-CSF activated human monocytes; 2) to determine the functional outcome of SHP-1 and SHIP deficiency in M-CSF-induced monocyte survival and determine how M-CSF suppresses phosphatase activity; and 3) to define the role of monocytes and M-CSF in lung injury and fibrosis induced by bleomycin or found in SHP-1-/- and SHIP-/- mice. They will perform both in vitro and in vivo studies to dissect these aims, using human monocytes, murine macrophages and animals that develop pulmonary fibrosis.

DESCRIPTION (Applicant's abstract): This proposal will seek to further define the fundamental immunological and biochemical events that lead to pulmonary fibrosis. In animal models and humans with pulmonary fibrosis, monocyte recruitment and survival in the lung and activation of TGF-beta appear to be important components of the remodeling and fibrosis, however, the specific mechanisms involved have not been well elucidated. To address this issue, we found that in addition to promoting monocyte survival, macrophage colony-stimulating factor (M-CSF) also induces monocytes to release soluble factor(s) capable of activating latent TGF-beta. Since activation of TGF-beta appears to be casually associated with lung fibrosis, we will seek to identify the specific factor(s) released by M-CSF-stimulated monocytes that cleave latent TGF-beta and define the biochemical pathways responsible for stimulating the production of these factor(s). We will then focus on defining if active TGF-beta can facilitate monocyte survival directly or through inducing M-CSF release by fibroblasts. Finally, we will focus on "proof-of-concept" studies to define if monocyte proteases are critical in the activation of latent TGF-beta to assess the potential therapeutic application for agents that block these factors. The short-term goals of this proposal are to define the mechanisms by which monocyte recruitment and survival facilitate tissue remodeling and fibrosis. The long-term goal of this project is to better define the pathological events regulating the genesis of pulmonary fibrosis and to define novel targets to direct innovative therapies to suppress lung destruction and fibrosis associated with this devastating lung disease.

This project will explore the role of macrophages in the pathogenesis of chronic in organ transplantation. We hypothesize that HLA class I and/or II antigens are expressed by cells in the graft serve as the ligand for anti- donor MHC antibodies to be deposited. We speculate that recipient macrophages are stimulated by these antibodies to produce and secreted TGF-beta that appears to be important in organ acceptance. Consistent with this hypothesis, we found that deposited IgG stimulates human monocytes to produce TGF-beta, although whether this TGF-beta is activated is not clear. Thus, we will seek to define how monocyte production of TGF-beta induced by deposited IgG is regulated, determine the biochemical pathways modulating this production, determine if these in vitro data apply to endomyocardial biopsies from patients with heart transplants and test "proof of concept" studies in animal models. This project will interact with the other two projects in the proposal in a manner that will provide additional opportunities for discovery. Through these studies we hope to define the molecular mechanisms by which monocytes, macrophages and IgG may facilitate the generation of chronic rejection in organ transplantation and define new molecular targets to direct new strategies.

DESCRIPTION (Applicant's Abstract): This proposal will focus on the central hypothesis that survival and accumulation of monocytes and macrophages in the lung promotes injury and fibrosis. Pathological data from mice with genetically- or drug-induced pulmonary fibrosis supports this hypothesis, as monocyte and macrophage accumulation in the lungs temporally associates with pulmonary fibrosis. Similarly, human patients with pulmonary fibrosis have increased numbers of young, activated macrophages in their alveoli that produce heightened amounts of reactive oxygen species (ROS). In small studies, antioxidants have been successfully used to treat patients with pulmonary fibrosis. We found evidence that activation of phosphatidylinositol 3-kinase, Akt and the production of ROS in response to M-CSF stimulation were important to activate NF-kB and promote cell survival in human monocytes. Inhibitors of PI 3-kinase, proteosome activity or ROS suppressed NF-kB activation and monocyte survival in M-CSF-treated cells, suggesting that these processes may be linked. Since NF-kB is a known downstream effector of activated Akt, we hypothesize that activation of NF-kB in M-CSF-treated cells may be an essential biochemical mediator of monocyte survival, and thus may regulate macrophage-mediated inflammation in lung diseases like IPF. The goal of these studies is to define molecular targets that will help patients with this devastating lung disease. To investigate the role of NF-kB in monocyte survival we will test the following Specific Aims. 1) Does M-CSF activate Akt to promote NF-kB translocation and induce the breakdown of IkB? 2) Determine how NF-kB activation is regulated and mediates survival of M-CSF-stimulated cells. 3) Determine the mechanism by which NF-kB translocation facilitates cellular survival. We will use studies in human rnonocytes, murine macrophages from animals that are genetically programmed to suffer lung fibrosis and cell lines to directly assess the contribution of NF-kB in the survival of monocytes and macrophages. These studies will initially be largely in vitro or ex vivo, which are designed to define the role and mechanisms by which NF-kB may lead to monocyte survival with particular attention focused at the serine/threonine kinase Akt and ROS. Our long-term goal is to apply information gained from these studies to reduce monocyte- and macrophage-mediated lung inflammation and offer new treatments to patients with pulmonary fibrosis.

DESCRIPTION (provided by applicant): Mononuclear phagocytes mediate physiological and pathological processes in the lung. In host defense and in the regulation of the pulmonary environment, there is evolving evidence that these cells participate in the response to lung injury. Our data suggest that mononuclear phagocytes are intimately involved in lung fibrosis. Others agree, as the number of mononuclear phagocytes in the lungs of IFF patients predicts pulmonary function and symptoms. SHIP1 and SHIP2 negatively regulate M-CSF-induced Akt and NF-?B activation and cellular activation and survival. The serine threonine kinase Akt1 is important in cellular survival and differentiation. In this revised proposal we will focus on understanding how SHIP 1/2 regulates monocyte survival and differentiation and Akt1 in M-CSF-stimulated cells. Using macrophages that express constitutively active myristoylated Akt1 (myr-Akt1), we will investigate the role of Akt1 in survival and differentiation of these cells. We will also investigate the molecular mechanism by which bleomycin worsens fibrosis in myr-Akt1 mice. We will also define the role of the src family kinase Lyn in SHIP1 function. We will investigate the role of Akt1 expression in mononuclear phagocytes in vitro and in vivo in this revised proposal and determine the regulation of Akt1 by SHIP1 and SHIP2 in M-CSF-stimulated cells as important regulators of this biochemical pathway and seek to understand the relevance of these events in lung fibrosis and remodeling. To accomplish these goals, we will pursue the following Specific Aims. Specific Aim 1) To understand the role of Akt1 in the survival and differentiation of mononuclear phagocytes. Specific Aim 2) To understand the role of SHIP-1 and SHIP-2 in mononuclear phagocyte differentiation and survival. Specific Aim 3) To understand the molecular mechanisms of bleomycin-induced lung fibrosis and inflammation in mice with macrophage selective myr-Akt1 expression compared to wild type animals.

DESCRIPTION (provided by applicant): Pulmonary fibrosis is a progressive interstitial lung disease with a mean survival of 3-5 yrs. The underlying cause or genetic factors involved in the pathogenesis of idiopathic pulmonary fibrosis (IPF) are unknown. Thus, little practical improvement has been made in treatment. While genetic changes are reported in two genes that regulate telomerase activity, these occur in only a small subset of individuals and clearly do not represent the majority of the patients with this disease. MicroRNAs (miRNA, miR) are regulatory RNAs that regulate gene expression by targeting mRNAs for degradation or preventing protein translation. It has been estimated that a single miRNA can regulate the expression of 20-30 proteins. The miR-17~92 miRNA cluster targets genes, such as metalloproteinases, collagen, and transforming growth factor that are highly expressed in IPF. Our preliminary data indicate that there are decreases in expression of the miR-17~92 miRNA cluster in the lungs from patients with IPF. We hypothesize that decrease expression of miRNAs contained within this cluster are critical for the pathogenesis of IPF. In this proposal, we will investigate the specific role of this miRNA cluster. We will also explore which members of this cluster are critical in the development and progression of IPF. Lastly, we determine if intervention in murine models of pulmonary fibrosis to replete the cluster can be effective treatment of this disease. For these goals we plan to investigate the following specific aims: Specific Aim 1: Determine the mechanisms and cell types involved in miR-17~92 suppression in IPF. Specific Aim 2: To define the impact of manipulating miR-17~92 cluster on cellular phenotype and gene expression in cells from patients with IPF. Specific Aim 3: To define the effect of manipulating miR-17~92 expression in a murine model of pulmonary fibrosis. PUBLIC HEALTH RELEVANCE: Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease with unknown etiology and a mean survival of 3-5 years survival. Despite ongoing studies to define the etiology of the disease, little practical improvement has been made in treatment and expected or observed outcomes. Complexity of IPF suggests that there are likely numerous interactions between a person's genes and environment. We speculate that searching for gene networks altered in patients with IPF would be a better approach to understand the disease. Since microRNAs can regulate multiple genes, we will focus on gene regulation through microRNAs in this proposal. We will use the integrative field of systems biology to classify and define molecular networks activated in these patients using miRNA and mRNA profiling of lung tissue. These data will be used to construct and predict gene networks that are activated in these patients to determine if the alterations can be used to classify and predict disease progression and severity as well as identify targets for therapeutic purposes.

DESCRIPTION (provided by applicant): Idiopathic Pulmonary Fibrosis (IPF) is a chronic progressive lung disease. This application will examine the hypothesis that epigenetic dysregulation underpins pulmonary fibrosis. We will examine miRs-19b and -20a, two members of the miR-17~92, which target DNA methyltransferase-1, DNMT1. We found that miRs-19b and -20a expression are reduced in the lung tissue of patients with IPF, accompanied by increased expression of DNMT1. Thus, the grant application investigates the role of DNMT1 in IPF and the relationship of miRs-19b and - 20a in this regulation. Moreover, we believe that the cluster itself is also regulated by DNA methylation of CpG islands in its promoter, forming a negative feedback loop between DNMT1 and miRs-19b and - 20a expression. In this proposal, we will use in vitro and in vivo studies to assess this hypothesis. To address our hypothesis in vivo, we have generated cell-specific miR-17~92 and DNMT1 knockdowns in transgenic murine models. We will also examine whether miRs-19b and -20a regulates components involved in epigenetic regulation. We anticipate that understanding the relationship between miRs-19b and -20a and DNMT1 expression in lung fibrosis will identify novel therapeutic strategies and underlying mechanisms of fibrosis. We predict that our findings will lead to the development of human clinical trials. To accomplish these goals, we propose the following two specific aims: Specific Aim 1) Understand the role of DNMT1 in expression and methylation of the miR-17~92 cluster in vitro and the reciprocal role of miRs-19b and -20a on DNMT1 expression. Specific Aim 2) Determine the in vivo role of DNMT1 in pulmonary fibrosis. PUBLIC HEALTH RELEVANCE: There are significant gaps in understanding the pathogenesis of Idiopathic Pulmonary Fibrosis (IPF) at the cellular and genetic levels. This disease is associated with high mortality due to the lack of therapies. We have found changes in regulatory proteins that are critical for gene expression and repair of lung fibrosis. Interestingly, current FDA approved treatments for hematological malignancies target some of these proteins. Thus, in this application, we propose in vitro and in vivo pre-clinical testing to determine the efficacyof these potential treatments. We anticipate that our findings will lead to the development of therapies to individuals with IPF.