Richard P. Phipps - US grants

The objective of this research is to use neoplastic and normal accessory cells in a novel model system to study the regulation of B cell triggering and tolerance. We have shown that haptenated-immunoglobulin molecules can be presented directly to B cells by normal peritoneal macrophages and certain macrophage-like tumor lines to induce specific unresponsiveness. The role of the B cell FcR and the mechanisms of macrophage elicited tolerance will be investigated. Variant clones derived from the J774 macrophage-like tumor line have been isolated which possess a differential ability to induce B cell unresponsiveness. These clones will be studied to determine characteristics which confer the ability to tolerize B cells. In contrast to the action of macrophages, a lymphoid dendritic cell-like tumor line can convert a tolerogenic signal into an immunogenic one and elicit augmented antibody responses. The mechanism and role of T cells in this conversion will be studied using these tumor cells, as well as the normal splenic lymphoid dendritic cells. The ability of macrophages and lymphoid dendritic cells to present immunoglobulin tolerogen in vivo will be determined. In addition, the ability of these accessory cells to present immune complexes in a tolerogenic or immunogenic fashion will be assessed. Since relatively little is known about how accessory cells modulate B cell responsiveness, this research proposal would help fill that gap. Furthermore, an understanding of macrophage and lymphoid dendritic cell modulation of B cell signalling could lead to more rational approaches when attempting immunological modulation of cancer and autoimmunity.

The long-term objective of this research is to delineate the mechanisms hereby macrophage-secreted prostanoids (i.e. prostaglandins and thromboxanes) inhibit the activation of normal B cells and B lymphomas. First, selected prostanoids will be examined for their ability to promote B cell unresponsiveness, in conjunction with antigen-specific signals. This will be accomplished using an in vitro assay and purified hapten-specific B cells. Second, the role of B cell receptors for antigen and for the Fc portion of immunoglobulin in prostaglandin(PG)-enhanced negative signalling will be determined. This is important because in the presence of PG, cells may be negatively signalled or growth- inhibited via sIgM or Fc receptors. Third, since PGE2 inhibits macrophage activation, it may similarly block B cell activation. The effects of PGE2 on the activation of normal B cells and B lymphomas, stimulated by IL-4 or anti-IgM reagents, will be assessed. Activation will be monitored by increases in size, induction of membrane IL-I and class II expression. Inhibition of these events by PG may block proliferation, differentiation and ability to present antigen. Fourth, the presence of high or low affinity receptors for PGE2 will be examined using B lymphomas. Some lymphoma clones are growth-inhibited by PGE2, whereas others are resistant. The presence of PGE2 receptors on B lymphomas may confer susceptibility to this prostanoid. A binding assay will be used to detect this receptor and to determine whether its expression is altered by cell activation. Since relatively little is known about how arachidonic acid metabolites like PGE2 affect normal and neoplastic B cells, this research proposal would help fill that gap. Furthermore, understanding how prostanoids regulate B lymphomas may provide an avenue for new strategies to inhibit their growth in vivo.

The purpose of this research is to determine the effects of ionizing radiation on cytokine production by subsets of lung fibroblasts and ascertain the key cytokines produced in lung during fibrosis induced by radiation. Lung fibrosis is characterized by extensive accumulation of extracellular matrix and increased cellularity of the fibroblast compartment. The causes of lung fibrosis are many and include treatment for malignancy with radiation. Research from this laboratory has shown that murine lung fibroblasts are divisible into two major subsets based on whether or not they express the Thy 1 antigen. Lines of Thy 1+ and Thy 1- fibroblasts differ in morphology, display of class II MHC antigens, ability to stimulate T lymphocytes, production of IL-1 and IL-1 receptors and amounts of extracellular matrix synthesized; including fibronectin and types I and III collagen. These fibroblast subsets may play differing roles in fibrotic development. The ability of a fibroblast subset to respond to or produce certain cytokines may lead to a reparative process or to a potentially fatal fibrosis. Thy 1+ and Thy 1- fibroblasts will be characterized for the cytokines they synthesize, both constitutively and after radiation treatment, using the polymerase chain reaction to detect cytokine mRNA. The products of these mRNAs will be detected and quantitated using specific bioassays or by ELISA. Determination of a specific cytokine profile or fingerprint may permit further subsetting of fibroblasts and elucidation of their role in radiation-induced lung fibrosis. Studies will also be performed with selected cytokines (e.g. IL- 1, TNFalpha, TGFbeta1, gamma-IFN) to assess their role in regulating proliferation and collagen production by fibroblast subsets. The important cytokines/growth factors involved in promoting radiation-induced fibrosis in a murine model will be determined. Characterization of the cytokines crucial for fibrotic development induced by radiation will allow new interventions to be developed. Novel treatments include using cytokines to inhibit lung fibroblast proliferation and collagen synthesis, or alternatively, using neutralizing antibodies or other cytokine inhibitors to block the stimulatory activity of pro-fibrotic cytokines. In this way it will be possible to block or arrest potentially fatal radiation-induced pulmonary fibrosis.

pulmonary fibrosis /granuloma; protein biosynthesis; cytokine; fibroblasts; interferon gamma; tumor necrosis factor beta; interleukin 6; interleukin 4; cell population study; collagen; human tissue; flow cytometry; laboratory mouse;

This project will determine whether CD40, a member of the TNF receptor superfamily, is a critical element in mediating pulmonary inflammation, dysplasia and fibrosis. The lung is susceptible to damage by treatments for respiratory distress in infants and adults (i.e., oxygen), by therapies for cancer (e.g. chemotherapy) and by exposure to environmental agents (e.g. ozone). Lung damage may induce chronic inflammation, which stimulates fibroblast proliferation and fibrosis. Pulmonary derangement, including bronchopulmonary dysplasia (BPD) in premature infants, is a potentially fetal consequence of severe lung damage. Treatments for pulmonary fibrotic conditions are essentially nonexistent. The fibroblast is the key effector cell responsible for lung fibrosis. T lymphocytes and mast cells are closely associated with lung inflammation and fibrosis and may directly stimulate the fibroblast. The mechanism of interaction between these cells is largely unknown. This laboratory discovered that pulmonary fibroblasts display CD40, a receptor previously thought to be associated with bone marrow derived cells (e.g. B cells). The importance of CD40 is that its ligand (L) is found on T lymphocytes and mast cells and the CD40-CD40L interaction is critical for the stimulation of the CD40 displaying cell. The hypothesis to be tested in this proposal is that CD40 is also a critical regulatory molecule for fibroblasts, important for their proliferation and synthesis of proinflammatory cytokines and collagen. Disruption of the fibroblast CD40 signaling mechanism may inhibit lung inflammation and fibrosis. The significance of fibroblast CD40 for lung inflammation and fibrosis will be determined by answering the following questions as our specific aims. (1) Does CD40 act as a cytokine/growth factor receptor and induce protein tyrosine phosphorylation and fibroblast proliferation? (2) Does crosslinkage of CD40 induce lung fibroblasts to synthesize proinflamatory/profibrotic cytokines and is their production of collagen increased? (3) Does disruption of the CD40-DC40L system in vivo prevent the onset of a BPD-like syndrome in an animal model? Results of this project may lead to the development of new therapeutic strategies to arrest the progression of lung dysplasia in premature infants, and to prevent lung fibrosis in patients' lungs damaged by cancer treatments or environmental agents.

The objectives of the research are to understand how natural small lipid molecules called prostaglandins (PGs) regulate normal and malignant B and T lymphocytes. The hypothesis developed by the investigator's laboratory is that prostaglandin E2 (PGE2), the major PG found in the lymphocyte milieu, is not solely immunoinhibitory, but also exhibits positive regulatory influences on B lymphocytes, shapes the class of immunoglobulin secreted and controls the types of cytokines produced by T lymphocytes. In support of this concept, this lab discovered that PGE2, which mainly signals via cAMP, is a powerful promoter of B lymphocyte immunoglobulin class switching to IgE. In contrast, 15-deoxy PGJ2, is strongly inhibitory for normal and cancerous B lymphocytes. A second key concept is that PGE2 tips the 'immunologic balance' away from a TH1 cytokine response and towards a TH2 response. The focus of this project is to study the mechanisms by which key PGs control normal and malignant B lymphocyte activation and differentiation and to ascertain how they regulate T lymphocyte development into TH1 and TH2 cells. The investigator will answer the following four questions to accomplish these goals. (1) What are the functional consequences of exposure to 15-deoxy-PGJ2 and by what mechanisms does it so profoundly inhibit normal and cancerous B lineage cell activation and growth? (2) Why do Balb/c mice generate exuberant PGE2 responses in contrast to C57BL/6 mice and is this the reason that these strains are predisposed to generate TH2 and TH1 cytokine responses, respectively? (3) Can naive T lymphocytes be preferentially driven in vitro to generate a TH1 or TH2 cytokine response by manipulation of the prostaglandin environment? (4) Can a TH1 or TH2 lymphocyte response be elicited in vivo by regulating exposure to prostaglandins? These studies will provide a basis for understanding the mechanisms whereby prostaglandins regulate the growth and differentiation of normal and malignant B- and T-lineage cells. Potential applications of this project include: targeting PG receptors on malignant B lymphoma cells to kill them, understanding how the abundance of PGs in periodontal disease contributes to disease progression, and using selected PGs or inhibitors thereof to generate polarized TH1 or TH2 responses. These applications are important in developing vaccines for cancer and infectious diseases where specific TH1 or TH2 responses are most efficacious. Immunodeficient patients such as the very young and elderly and those afflicted with cancer and AIDS might benefit from approaches to shape the immune response to vaccination at the outset.

DESCRIPTION (provided by applicant): As part of our long-term goal to develop new approaches to treat thrombotic diseases, we will investigate a novel mechanism that links platelets, inflammation and thrombosis. Platelets are crucial for the process of thrombosis, but are not widely considered as key elements in inflammation. An emerging view of platelets is that they contain and produce many immunomodulatory and proinflammatory mediators including CD40 ligand (CD40L), prostaglandins and cytokines. Platelets are not thought to possess transcription factors. However, our compelling preliminary data demonstrate that the transcription factor peroxisome proliferators activated receptor gamma (PPARgamma), believed only present in nucleated cells to regulate lipid metabolism, is highly expressed by platelets. Further, our experiments show that small molecule PPARgamma agonists, such as rosiglitazone and prostaglandin J2, inhibit platelet aggregation and ATP release. Remarkably, PPARgamma agonists inhibit the thrombin-induced production and release of key platelet mediators of inflammation including CD40L and thromboxanes. These findings are important because (1) thrombin is an extremely potent platelet activator, and (2) rosiglitazone and similar agents are already widely clinically employed in patients with diabetes. In addition to CD40L's known critical role in inflammation and atherosclerosis, it is now recognized as a primary platelet agonist important in thrombosis. CD40L is also a marker for disease activity and prognosis in cardiac and vascular diseases. Therefore, platelet PPARgamma is a new and previously unsuspected potential target to attenuate thrombosis, vascular injury and inflammation. We have developed the overall hypothesis that the transcription factor PPARgamma is expressed by platelets and modulates their ability to become activated and to produce mediators of inflammation that contribute to vascular injury. To provide a scientific framework to evaluate PPARgamma agonists as a possible therapy for thrombosis and inflammation, we will answer the following questions posed as our specific aims. Aim 1: What are the subcellular locations of platelet PPARgamma and what are the effects of PPARgamma agonists on platelet release of key proinflammatory mediators? Aim 2: How do PPARgamma agonists inhibit platelet activation and function? Aim 3: What is the significance of the PPARgamma protein that is expelled from platelets after activation? Completion of these studies will provide a scientific justification for clinical trials that employ existing PPARgamma agonists to attenuate atherothrombotic disease through modulation of platelet thrombotic and inflammatory functions.

DESCRIPTION (provided by applicant): Thyroid associated ophthalmopathy (TAO, also called Graves'ophthalmopathy) is a serious autoimmune disease involving orbital tissue inflammation. Orbital tissue is infiltrated mainly by T lymphocytes and contains proinflammatory cytokines and prostaglandins. A hallmark of TAO is an increase in orbital fat that pushes the eye out of the orbit (proptosis). TAO is disfiguring and may lead to blindness. Pre-adipocyte orbital fibroblasts are key effector cells in this process. Orbital inflammation is postulated to drive their differentiation to fat cells called adipocytes. The inflammatory signals that induce this differentiation remain an important, yet poorly studied aspect of TAO. Adipocytic differentiation is believed to be mainly controlled by a transcription factor called peroxisome proliferator activated receptor gamma (PPARgamma). Our research shows that human orbital fibroblasts highly express PPARgamma and that both natural (15d-PGJ2) and synthetic (e.g. the insulin-sensitizing drugs rosiglitazone) PPARgamma ligands induce a subset of Graves'orbital fibroblasts to differentiate to adipocytes. Our compelling new data show that circulating T lymphocytes from Graves'patients'highly express the prostaglandin-generating enzyme cycloxygenase-2 (Cox-2) and produce the PPARgamma ligand 15d-PGJ2. Moreover, Graves'T cells drive a subset of PPARgamma expressing TAO fibroblasts to adipocytes. These exciting data are the first to demonstrate that human T cells produce a functional PPARgamma ligand and supports that the process of orbital inflammation drives adipogenesis. The overall hypothesis we will test is that Graves'T lymphocytes produce a PPARgamma ligand that induces a subset of PPARgamma expressing orbital fibroblasts to differentiate to adipocytes. The following three questions posed as specific aims will be answered to test the overall hypothesis. Aim 1: Are Graves'disease human T lymphocytes unique in their ability to highly express Cox-2 and produce prostaglandins (e.g. 15d-PGJ2) that act as PPARgamma ligands? Aim 2: Is the ability of Graves'orbital fibroblasts to differentiate to adipocytes dependent on the pro-adipogenic transcription factor PPARgamma? Aim 3: What are the differences between Thy1+ and Thy1- Graves'orbital fibroblasts that determine why only ThyT fibroblasts differentiate to adipocytes? These studies will help delineate the inflammatory cells and pathways that incite orbital fibroblast differentiation to adipocytes. This new information will permit the development of new approaches to control inflammation and the pathologic differentiation of orbital fibroblasts important for TAO and other diseases that involve fat deposition.

DESCRIPTION (provided by applicant): Platelets play a key role in hemostasis and function as essential cellular elements for blood to clot. They also play a fundamental role in myocardial infarction, stroke and deep vein thrombosis. Platelets from type-2 diabetics circulate in a partially activated condition, which likely contributes to the diabetic's underlying cardiovascular disease and proinflammatory state. Interestingly, platelets are now also recognized as key regulators of inflammation, due to their release of potent proinflammatory and prothrombotic mediators such as CD40 ligand and thromboxane A2. Our recent work also shows that human platelets, which lack a nucleus, express the transcription factor peroxisome proliferator activated receptor gamma (PPARy), which when exposed to a PPARy ligand, dampens platelet activation. Further interest in platelets arises from the fact that millions of doses of platelets are transfused into patients with cancer, blood diseases, etc. Platelets can be stored for up to five days prior to transfusion. However, this causes a "platelet storage lesion" that includes partial platelet activation and release of bioactive protein mediators that are also transfused. These "proteomic changes" have been poorly characterized and likely mediate/contribute to post-transfusion adverse effects such as fever, heart rate increases and multi-organ failure. Our multidisciplinary team will test the hypothesis that the platelet proteome differs between normal and type-2 diabetics. We will also begin to characterize platelet proteomic changes that occur during the storage period prior to transfusion. Storage of platelets and subsequent proteomic changes may also reflect ongoing partial platelet activation in vivo, such as occurs in type-2 diabetics. Our studies will identify proteins that could be new biomarkers of platelet function abnormalities and/or new therapeutic targets to dampen unwanted platelet activation, both in healthy platelet donors and those platelet donors with asymptomatic platelet-activating diseases, such as type-2 diabetes. Our specific aims are as follows: Aim 1: Characterize the platelet proteome of fresh normal platelets versus tvpe-2 diabetic platelets. Differences in the proteome between normal and type-2 diabetic platelets will be determined using advanced chromatographic separation coupled with high sensitivity mass spectrometry and verified using a battery of complementary approaches. Whole human platelets and purified platelet membranes will be investigated. Aim 2: Determine changes in the normal and type-2 diabetic platelet proteome during storage for transfusion. Changes in the platelet proteome during the storage (up to 5 days) of platelets from normal donors and from newly diagnosed type-2 diabetics will be determined. We will also determine whether potentially deleterious changes in stored platelets can be blocked by PPARy ligands. Overall, our findings will form the foundation for clinical studies to evaluate new biomarkers of platelet function and improve the platelet deterioration that occurs during the storage period prior to transfusion. (End of Abstract)

[unreadable] DESCRIPTION (provided by applicant): Optimal humoral (i.e. antibody) responses to vaccination are essential if humans are to be protected against potentially devastating infectious agents. These include viruses such as variola that cause smallpox. Variola and other infectious agents have received significant attention due to their potential use as weapons for bioterrorism against both the civilian and military populations. B lymphocytes are crucial elements of the immune system responsible for the synthesis of antibodies that play key roles in host defense against infectious microorganisms including viruses. This laboratory has shown that human and mouse B lymphocytes highly express the prostaglandin-generating enzyme cyclooxygenase-2 (Cox-2) when activated. Importantly, commonly used non-steroidal anti-inflammatory drugs (NSAIDs) such as dual Cox-1/Cox-2 inhibitors (e.g. Indomethacin) and newer Cox-2 selective drugs (e.g. Celebrex) significantly blunt the ability of B cells to produce antibody in response to both polyclonal and antigen-specific stimulation. Mice genetically deficient in Cox-2 also respond with reduced antibody titers to virus-like particles and to infection with vaccinia virus (used in vaccination to smallpox). These observations led to the hypothesis that Cox-2 is required for B lymphocytes to optimally respond to stimulation and produce antibody. Thus NSAIDs, especially those that target Cox-2, may blunt humoral immunity. If proven, antibody responses to vaccination/infection would be reduced, especially where the immunizing agent weakly stimulates B cell immunity. Further ramifications include effects on humans with weakened immune systems such as the elderly and immuno-compromised patients (HIV, cancer, etc), who frequently use NSAIDs. Herein, two specific aims are proposed to study the role of Cox-2 in the antibody response to vaccinia. Aim 1 will determine the role of Cox-2 in the antibody response of mice to infection with vaccinia virus. Two complementary approaches will be used: a genetic strategy employing Cox-2 knock out mice and a pharmacologic one using normal mice treated with small molecule inhibitors of either Cox-1/Cox-2 or Cox-2. Humoral responses to vaccinia infection, including immune phenotyping will be evaluated and the critical time frame when Cox-2 activity is required for antibody responses will be determined. Aim 2 will determine the role of Cox-2 in antibody production to vaccinia for human B cells. Blood donors will consist of those vaccinated against smallpox prior to 1972 and those who participated in a recent clinical trial evaluating the smallpox vaccine. Polyclonal B cell activation in concert with NSAID inhibitors of either Cox-1/Cox-2 or Cox-2 will determine if these drugs dampen a recall antibody response to vaccinia. The overall significance of this research is that the use of NSAIDs or other drugs that inhibit Cox-2 activity or expression may be contraindicated during critical time periods after infection or vaccination. Such a finding will improve the ability of humans to respond to routine vaccination, as well as any bioterror threats. Impact on public health Optimal responses to vaccination (immunization) are essential to protect against devastating infectious microorganisms. The proposed research will study the potentially negative impact of the widespread use of non-steroidal anti-inflammatory drugs (NSAIDs) (e.g. Celebrex, Ibuprofen, etc.) on antibody responses to immunization. This research is especially important where vaccines are in short supply, are poor at inducing protective responses or the recipients have weakened immune systems. The overall significance of this research is that the use of NSAIDs may be contraindicated during critical time periods after infection or vaccination. Such a finding would improve our ability to respond to routine vaccination, as well as any bioterror threats. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Platelets play a key role in hemostasis and function as essential cellular elements for blood to clot. They also play a fundamental role in myocardial infarction, stroke and deep vein thrombosis. Platelets from type-2 diabetics circulate in a partially activated condition, which likely contributes to the diabetic's underlying cardiovascular disease and proinflammatory state. Interestingly, platelets are now also recognized as key regulators of inflammation, due to their release of potent proinflammatory and prothrombotic mediators such as CD40 ligand and thromboxane A2. Our recent work also shows that human platelets, which lack a nucleus, express the transcription factor peroxisome proliferator activated receptor gamma (PPARy), which when exposed to a PPARy ligand, dampens platelet activation. Further interest in platelets arises from the fact that millions of doses of platelets are transfused into patients with cancer, blood diseases, etc. Platelets can be stored for up to five days prior to transfusion. However, this causes a "platelet storage lesion" that includes partial platelet activation and release of bioactive protein mediators that are also transfused. These "proteomic changes" have been poorly characterized and likely mediate/contribute to post-transfusion adverse effects such as fever, heart rate increases and multi-organ failure. Our multidisciplinary team will test the hypothesis that the platelet proteome differs between normal and type-2 diabetics. We will also begin to characterize platelet proteomic changes that occur during the storage period prior to transfusion. Storage of platelets and subsequent proteomic changes may also reflect ongoing partial platelet activation in vivo, such as occurs in type-2 diabetics. Our studies will identify proteins that could be new biomarkers of platelet function abnormalities and/or new therapeutic targets to dampen unwanted platelet activation, both in healthy platelet donors and those platelet donors with asymptomatic platelet-activating diseases, such as type-2 diabetes. Our specific aims are as follows: Aim 1: Characterize the platelet proteome of fresh normal platelets versus tvpe-2 diabetic platelets. Differences in the proteome between normal and type-2 diabetic platelets will be determined using advanced chromatographic separation coupled with high sensitivity mass spectrometry and verified using a battery of complementary approaches. Whole human platelets and purified platelet membranes will be investigated. Aim 2: Determine changes in the normal and type-2 diabetic platelet proteome during storage for transfusion. Changes in the platelet proteome during the storage (up to 5 days) of platelets from normal donors and from newly diagnosed type-2 diabetics will be determined. We will also determine whether potentially deleterious changes in stored platelets can be blocked by PPARy ligands. Overall, our findings will form the foundation for clinical studies to evaluate new biomarkers of platelet function and improve the platelet deterioration that occurs during the storage period prior to transfusion. (End of Abstract)

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] Abstract Pulmonary fibrosis affects 200,000 patients per year in the USA. Idiopathic pulmonary fibrosis (IPF) is a severe form of pulmonary fibrosis for which there are no effective therapies except lung transplantation. The pathogenesis of IPF is poorly understood, but the cytokine TGF2 is upregulated and likely important. TGF2 drives both myofibroblast and extracellular matrix (ECM) accumulation. Peroxisome proliferator activated receptor gamma (PPAR3) is a receptor and transcription factor that regulates adipogenesis and insulin sensitization. It is now appreciated that it is also a down regulator of inflammation. Our new data, as well as that of others, has identified PPAR3 as an attenuator of fibrogenesis. Specifically, PPAR3 activation by PPAR3 ligands (endogenous or synthetic) inhibits key TGF2-mediated profibrogenic activities (myofibroblast and ECM accumulation) in vitro and pulmonary fibrosis in vivo. Tissue transglutaminase 2 (TG2) is an enzyme that cross-links and stabilizes matrix molecules such as collagen and fibronectin. Excess TG2 activity could excessively cross-link matrix proteins. Our new preliminary data demonstrate that TG2 expression is increased in fibrotic lung disease and that TGF2 up-regulates TG2 in fibroblasts, while PPAR3 activation suppresses TG2 expression. The overall hypothesis to be tested is that deficiency or dysregulation of PPAR3 in IPF leads to uncontrolled TGF2-mediated downstream bioactivities that include myofibroblast differentiation, excess extracellular matrix production and TG2 upregulation. Two specific aims are proposed to test this hypothesis: Aim 1. Determine whether there is cell type specific deficiency or dysregulation of PPAR3 protein expression, and/or alterations in the ratio of PPAR3 1 and 2 isoforms in tissues from patients with mild, moderate or severe IPF compared to controls. Aim 2. Evaluate if there is up-regulation of TG2 activity in lung tissues from patients with IPF resulting in excessive cross-linking of matrix, and whether this is related to PPAR3 deficiency (determined in Aim 1). These findings will reveal new insights into the pathogenesis of IPF and may identify new targets for therapy. The translational potential is high as several PPAR3 ligands are now FDA approved for human use in diabetes and are being evaluated for therapy of cancer. (End of Abstract) [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: [unreadable] [unreadable] Relevance Pulmonary fibrosis (scarring) is a severe disease with few effective therapies. In this proposal we will identify new pathways which are important in controlling the disease and which can be targeted for future therapy. We propose that a protective protein called the peroxisome proliferator activated receptor gamma is deficient in subjects with lung fibrosis, leading to increased deposition of stiff scar tissue. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Red cell transfusions are associated with inflammation and thrombosis, both arterial and venous, the mechanisms of which are not understood. Platelets are critically involved in hemostasis, thrombosis and inflammation. Our hypothesis is that storage-induced changes in red cells including the release of free hemoglobin and the accumulation of biologically active mediators in the supernatant have a direct deleterious effect on the recipient's platelets. We propose that unwanted effects on platelets contribute to the inflammatory and pro-thrombotic properties of red cell transfusions. We will study changes in stored leukoreduced Adsol (AS)-1 red cells and their supernatants over time, and ascertain the effects of supernatant mediators and stored red cells on recipient platelets. In Aim 1, red cell-free hemoglobin and key lipid mediators that accumulate in microparticles and supernatant fractions of leukoreduced red cells will be defined, and we will ascertain the effects of stored red cells and these mediators on platelets. We will determine whether FDA licensed manipulations, such as washing and/or rejuvenation, ameliorate the effects of stored red cells and supernatant on platelet function. Mediators that will be studied based on our preliminary and published data include: hemoglobin which alters platelet function; F2 isoprostanes that are markers of oxidative stress and cell damage and directly affect platelet function; and red cell-derived microparticles which stimulate platelets. Assessment of biologic effects on platelets will employ aggregation, spreading and bioactive/proinflammatory mediator release. We will focus our investigations on red cell supernatant mediators because our supporting studies demonstrate that supernatant removal reduces morbidity and mortality in patients with hematologic malignancies. Our compelling preliminary laboratory data demonstrate that even leukoreduced red cells accumulate large amounts of mediators (hemoglobin, isoprostanes and microparticles) in the supernatant that alter platelet function in vitro and if transfused may alter recipient platelet function and promote vascular disease and inflammation. In Aim 2, we will investigate the clinical importance of red cell storage time and infused mediators by correlating these with clinical and laboratory outcomes in critically ill patients. A prospective cohort study of 600 ICU patients will characterize the relationship of infused cell-free hemoglobin, isoprostanes, prostaglandins, and red cell storage time, with laboratory and clinical outcome measures of inflammation and hemostasis/thrombosis. Laboratory measures include CD40 ligand, C-reactive protein, D-dimer, blood cell count and differential. Clinical outcomes include organ failure, bleeding, thrombosis, time on ventilator and length of hospital stay. Overall, the proposed studies will determine whether the adverse effects of red cell transfusions are likely due to the stored red cells, to accumulated supernatant mediators, or to both. PUBLIC HEALTH RELEVANCE: Red cell transfusions are one of the most commonly used therapies in hospitalized patients. Transfusions are associated with increased complications, including lung failure, blood clots and heart attack. Our research will discover explanations for these effects, and we will devise inexpensive strategies to reduce these complications

DESCRIPTION (provided by applicant): This RC1 grant application is responsive to Challenge Area "04-Clinical Research" and specifically to the NHLBI Challenge Topic 04-HL-103: "Assess the role of leukocyte interaction with platelets, erythrocytes, and endothelium in the pathogenesis of heart, lung, and blood diseases." A significant knowledge gap is how the platelet communicates with other blood and vascular cells. Knowledge of this process could lead to improved disease management and biomarker development. The small anucleate platelet plays a seminal role not only in hemostasis, but also in diabetes and cardiovascular disease which affect millions of Americans. Further interest in the platelet is fueled by the fact that millions of units of platelets are transfused each year, sometimes with deleterious consequences. Platelets are now also recognized as key inducers of inflammation. Our laboratory discovered that platelets abundantly express the transcription factor peroxisome proliferator activated receptor-gamma (PPAR?) and that PPAR? ligands dampen platelet activation. Ligand activated PPAR? is a previously unrecognized target that attenuates unwanted platelet activation in patients with type 2 diabetes or cardiovascular disease. PPAR? is viewed as an anti-inflammatory transcription factor, which also functions via non-nuclear mechanisms. Our team recently discovered that PPAR? is released from platelets in microparticles (MPs). MPs are submicron membrane vesicles that contain bioactive proteins and mediators. PPAR? containing MPs are taken up by and dampen macrophage function. We also discovered that type-2 diabetics produce MPs that have abnormally low levels of PPAR? and are likely to stimulate inflammation rather than inhibit it. We propose the overall challenge and hypothesis that PPAR? in MPs influences other cells by a transcellular mechanism. To complete this challenge in 2 years we assembled an outstanding multidisciplinary team to complete 2 aims. Aim 1: Investigate platelet MPs containing PPAR? and determine their ability to influence key white blood cell and vascular cell functions. Purified platelets from normal and type-2 diabetics will be used to generate MPs in vitro under controlled conditions. We will study their ability to be taken up and influence blood monocytes/macrophages and blood vessel endothelial cells. Thus, we will identify a new form of cell-cell communication. Genetic systems and a preclinical mouse model will be used to study the role of PPAR?-containing platelet MPs in health and disease. Aim 2: Discovery and characterization of MPs containing PPAR? in the blood of normal humans and those with type-2 diabetes. We will determine the cellular sources in blood of PPAR? containing MPs. While some MPs will be of platelet origin (Aim 1), others could come from white blood cells or vascular endothelial cells. The patterns of MPs and cell of origin could be used as a biomarker of disease and response to therapy. This new information could be used to develop an "artificial MP" to deliver PPAR? to cells of choice leading to a new therapeutic paradigm in diabetes and cardiovascular disease. Type-2 diabetes and its consequences, particularly cardiovascular disease, affect millions of Americans. This project will study how small blood cells called platelets, which prevent bleeding, communicate with other blood and vascular cells in healthy and type-2 diabetic individuals. Platelets, when stimulated, release small parts of themselves that contain instructions that other cells take up and which then change their behavior. Understanding this new way of cell to cell communication will lead to new methods and biomarkers to detect disease and to new ways of delivering therapy to reduce the consequences of diabetes and other diseases. PUBLIC HEALTH RELEVANCE: Type-2 diabetes and its consequences, particularly cardiovascular disease, affect millions of Americans. This project will study how small blood cells called platelets, which prevent bleeding, communicate with other blood and vascular cells in healthy and type-2 diabetic individuals. Platelets, when stimulated, release small parts of themselves that contain instructions that other cells take up and which then change their behavior. Understanding this new way of cell to cell communication will lead to new methods and biomarkers to detect disease and to new ways of delivering therapy to reduce the consequences of diabetes and other diseases.

DESCRIPTION (provided by applicant): This R01 application responds to PAR-10-190: Vulvodynia - Systematic Epidemiologic, Etiologic or Therapeutic Studies. Our long-term goal is to develop an understanding of the vulvodynia pain mechanism leading to a mechanism-based disease classification and ultimately to a mechanism-based therapy. Our research team has reported a mechanistic connection between yeast products, regional fibroblast activation, pro-opiomelanocortins, and localized provoked vulvodynia (LPV). Fibroblasts are now recognized as more than structural cells as they not only respond to signals but can prodigiously produce many different biologic mediators, including those that promote pain. Fibroblasts also exhibit considerable regional specialization. We discovered that fibroblasts from the vulvar vestibule produce markedly elevated levels of pro-inflammatory, pro-pain mediators following activation with yeast cell wall products. In particular, heightened pro-inflammatory mediator responses are generated by fibroblasts from the vulvar vestibule of LPV-afflicted women. This may be related to single nucleotide polymorphisms (SNP) in the melanocortin-1 receptor (MC1R) that enhance inflammatory mediator production. We propose that the vulvar vestibule of all women possesses a unique inflammatory/pain-inducing responsiveness and that vulvodynia pain reflects an extreme but natural inflammatory phenomenon. We hypothesize that vulvodynia arises 1) in a region of the genital tract predisposed to inflammation, 2) in the presence of specific irritants such as yeast, that are 3) exacerbated by genetic predisposition. To significantly advance and impact the field, we have assembled a multidisciplinary team, experienced in LPV, fibroblast biology, and inflammation to achieve the following three aims. Specific Aim 1: To determine whether pro-inflammatory fibroblasts segregate to painful areas of the vulva. Using lower genital tract pain mapping, we will discover whether pro-inflammatory fibroblasts localize to painful anatomic regions in situ. Fibroblast strains will be developed from painful and non-painful areas of the vulva and their biosynthetic capabilities for pro-inflammatory and other mediators determined after exposure to key fibroblast activating cytokines. Specific Aim 2: To determine whether yeast or yeast products activate fibroblasts via Toll-like receptors (TLR) and whether specific MC1R SNPs modify that response. We will determine whether the LPV-afflicted patients carry a different pattern of yeast species and yeast load, and whether yeast cell wall products initiate, through toll-like receptors, a pro-inflammatory, pain-inducing response from fibroblasts derived from painful regions. Specific Aim 3: To determine whether pro-opiomelanocortin loss-of-function promotes vulvodynia. We will investigate whether loss-of-function melanocortin-1 receptor SNPs enhance site-specific fibroblast activation, and can be identified with a simple clinical measure, skin colorimetry. We will assess an anti-inflammatory melanocortin derivative with therapeutic potential for vulvodynia and investigate the underlying molecular mechanism(s).

DESCRIPTION (provided by applicant): The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that binds diverse synthetic and natural compounds. We recently discovered that treating human orbital fibroblasts with AhR ligands not only impairs TGFb-driven myofibroblast formation, but also inhibits expression of the GPI-anchored protein, Thy1. This finding is important because human orbital fibroblasts are heterogeneous and can be divided into two subsets based on their surface expression of Thy1. Orbital fibroblasts expressing Thy1 (Thy1+) have the potential to form myofibroblasts, unlike cells lacking Thy1. And, the conversion of activated orbital fibroblasts to form scar-producing myofibroblasts is a key sight-threatening and incurable pathological feature of Thyroid Eye Disease (TED). Myofibroblasts produce excessive amounts of alpha-smooth muscle actin, collagen and hyaluronan leading to a stiff, fibrotic orbit. TED occurs in more than half of patients with Graves' Disease. Chronic orbital inflammation in TED leads to extensive tissue remodeling causing pain, proptosis, and even blindness. Current treatments for TED such as corticosteroids, external beam radiation and surgery are aimed at the consequences of disease, rather than the etiology, and cause unwanted side effects including post-surgical morbidity. Our long-term goal is to understand the mechanisms underlying TED leading to mechanism-based therapies. As there is no good animal model of TED, our translational studies use primary human orbital fibroblasts that accurately reproduce the events in the TED orbit. Identification of molecules that blunt TED myofibroblasts is a pressing need, and AhR ligands (e.g. ITE, FICZ) are a promising class of small molecules for potential use in TED. Goals: Establish the therapeutic value and the fundamental mechanisms whereby AhR ligands attenuate myofibroblast formation and extracellular matrix accumulation in TED. We will also discover the underlying mechanisms whereby AhR ligands repress Thy1 expression and determine if Thy1 repression is required for the anti-scarring properties of AhR ligands. Organizing Hypothesis: AhR ligands attenuate human orbital myofibroblast production and connective tissue remodeling in TED. Specific Aim 1: Test the prediction that key small molecule AhR ligands block human orbital fibroblast-to-myofibroblast differentiation. Specific Aim 2: Determine the mechanisms whereby AhR ligands block human orbital fibroblast-to-myofibroblast differentiation. Specific Aim 3: Investigate how AhR ligands mediate orbital myofibroblast formation by regulating Thy1 expression. Significance: Discovering that AhR ligands have therapeutic value in treating TED is a major advance, establishing new candidate drugs and targets for TED therapies. We will identify the mechanisms underlying Thy1 expression, myofibroblast formation and extracellular matrix accumulation in TED, and translate this knowledge into clinically relevant and innovative approaches to attenuate or even cure orbital remodeling and scarring in TED.

DESCRIPTION (provided by applicant): An emerging concept is that resolution of inflammation is a dynamic process crucial in restoring homeostasis, thus preventing chronic inflammation and disease. Newly identified lipid mediators, derived from polyunsaturated fatty acids (PUFA) are now recognized as crucial players in resolving inflammation. These endogenous specialized proresolution mediators (SPM) constitute separate families including lipoxins, resolvins, protectins and maresins. SPM have newly discovered abilities to regulate cells of the immune system. The humoral response is essential for protection against microorganisms, however the role of SPM in humoral immunity and their effect on B cells has not yet been studied. This is an important knowledge gap. Our new pilot data in support of this R21 application show that SPM have an important ability to enhance mitogen and antigen-driven antibody responses. These results support our hypothesis that the lipid-derived SPM enhance the antibody response to infection by promoting B cell function, leading to improved immune memory and long-term protection. We propose to study the immuno-regulatory functions of SPM on human and mouse B cells and during the adaptive immune response against influenza, in an infection and vaccination pre-clinical model. Adjuvant or vaccine booster properties of SPM could increase vaccine efficacy and utility, permitting smaller doses when a vaccine is poorly immunogenic or in short supply. We propose two specific aims to begin to reveal the function of SPM on B cells. Aim 1: Characterize the ability of key SPM to stimulate human B cell antibody production. B cells will be activated with TLR and BCR ligands along with selected SPM. IgM and IgG antibody levels will be determined along with B cell phenotype, survival and proliferation analyses. We will study B cell memory responses using seasonal influenza-vaccinated human subjects. These studies will determine the effects of resolvins on memory and antibody-secreting B cell populations. Aim 2: Determine the capacity of lead SPM to stimulate protective antibody production in a pre-clinical mouse influenza virus vaccination and infection model. The functions of SPM during an adaptive memory response will be analyzed using a mouse influenza vaccination and infection model. Mice will be vaccinated using the trivalent seasonal flu vaccine, as well as influenza recombinant protein, hemagglutinin (HA), and complemented with or without SPM. IgM and IgG antibody titers will be measured along with the antibodies' inhibitory and neutralizing properties, reflective of a B cell-mediated adaptive response. The stimulatory properties of SPM will be further assessed by challenging immunized mice with live influenza viral strains, including the 2009 H1N1, and monitoring weight loss, survival, and viral titers.

DESCRIPTION (provided by applicant): Obesity has risen dramatically over the last 30 years. In the U.S. alone, 60 million people are defined as clinically obese. Especially concerning is the nearly epidemic rate of childhood obesity. Obesity occurs through excessive adipogenesis (formation of adipocytes) or through increases in established adipocyte size. Environmental toxicants termed obesogens disrupt the endocrine system and can increase adipogenesis. Tributyltin (TBT), dichlorodiphenyl-dichloroethylene (DDE), bisphenol-A diglycidyl ether (BADGE), and tetrabromobisphenol-A (TBBPA), have been reported to increase obesity. The mechanism(s) by which xenobiotic obesogens function to disrupt the endocrine system and increase rates of obesity requires active investigation. Our work focuses on the surface glycoprotein, Thy1 (CD90). Thy1 is an glycophosphatidylinositol (GPI)-anchored membrane protein expressed on subsets of neurons, stem cells and other cell types. We discovered that fibroblasts are heterogeneous for expression of Thy1 and that only Thy1+ fibroblasts can differentiate into scar-forming myofibroblasts and only Thy1-/low fibroblasts can differentiate int adipocytes. In this R21 application, we hypothesize that there is a direct role for Thy1 in preventing adipogenesis. Our supporting data show that depletion of Thy1 increases adipogenesis while over- expression of Thy1 impairs adipogenesis. These data support the concept that Thy1 is more than a marker and that Thy1 can function to modify cell fate. We also discovered that Thy1 expression is reduced in both human and mouse multipotent stromal cells (MSCs) after exposure to TBBPA, BADGE and TBT. Importantly, one mechanism by which obesogens may function is to reprogram the epigenetic code of MSCs to alter physiology. The Thy1 locus contains multiple CpG islands that can be controlled epigenetically and may be key targets of obesogens. Developmental exposure to obesogens may reprogram Thy1 expression levels and modify physiology long after exposure. Therefore, we hypothesize that environmental obesogens alter Thy1 expression to increase adipogenesis and obesity. To investigate this hypothesis we have developed the following aims. Aim 1: Test the hypothesis that environmental obesogens diminish Thy1 expression and activity in multipotent stromal cells to increase adipogenesis. Aim 2: Test the hypothesis that developmental exposure to obesogens reduces Thy1 expression and increases adipogenesis and obesity in vivo. The results of these studies will show for the first time that Thy1 expression is reduced by environmental obesogens and this reduction is key to the mechanism whereby obesogens increase adipogenesis and obesity.

DESCRIPTION (provided by applicant): Cigarette smoke is a profound inflammatory stimulus and the chronic inflammation caused by cigarette smoking contributes to multiple fatal diseases including cardiovascular disease, cancer, and COPD (chronic bronchitis and emphysema). The effects of smoking persist long after smoking cessation, and in the case of emphysema can even continue to worsen. This suggests that cigarette smoke interferes with the normal processes that resolve inflammation. It was previously believed that resolution of inflammation was a passive process; it is now known that resolution is an active process managed by specific pro-resolving lipid mediators (PRMs). These mediators, including lipoxins, resolvins, protectins and maresins, act by inhibiting pro-inflammatory signaling and inflammatory cell migration and promoting pro-resolving effector functions such as macrophage phagocytosis of apoptotic inflammatory cells and debris. One of these compounds is already in clinical trials for inflammatory eye disease. We have strong preliminary data that a PRM called resolvin D1 (RvD1), has specific anti-inflammatory and pro-resolving effects on human lung cells, and can inhibit acute cigarette smoke-induced inflammation and airspace enlargement in a mouse model. Our overall hypothesis is that pro-resolving lipid mediators will have profound anti-inflammatory and pro-resolving effects on both acute and chronic lung injury, and that treatment with pro-resolving mediators to promote resolution is a novel and important therapeutic goal for inflammatory diseases caused by cigarette smoking. To investigate this hypothesis we have proposed the following specific aims. Specific Aim 1. Determine PRMs with the greatest efficacy at promoting resolution of acute inflammation in vitro and in vivo and determine their mechanism of action using primary human lung cells and a mouse model of cigarette smoke-induced acute lung inflammation. Specific Aim 2. Determine changes in the PRM profile of human samples with smoke-induced chronic lung disease, and evaluate the ability and mechanism by which PRMs prevent and treat lung tissue destruction in a mouse model of chronic smoke exposure. These studies will show for the first time that pro-resolving mediators can be used to prevent inflammation and accelerate resolution/repair of lung injury due to both acute and chronic cigarette smoke exposure. Our results will pave the way for translational development of these exciting new compounds that have the potential to be the first ever effective therapies against human diseases of chronic inflammation and smoking.

? DESCRIPTION (provided by applicant): Highly active antiretroviral therapy (HAART) has significantly increased the life expectancy of HIV/AIDS patients. A serious side-effect is that HAART is associated with non-resolving inflammation and an increased risk for cardiovascular disease (CVD). Tobacco smoking also promotes CVD risk and is a major health issue for all individuals. It is of even greater concern in the HIV/AIDS population that is three times more likely to smoke (up to 70% smoke and they smoke intensely) compared to the general population (20% smoke). Platelets play a seminal role in cardiovascular-associated inflammation and thrombosis, and contribute to the etiology and pathogenesis of CVD. Tobacco smoke activates platelets. A major knowledge gap is that the combined effects of HAART and tobacco smoke on platelet activation and CVD risk are unknown. Our hypothesis is that HAART increases platelet dysregulation and HAART combined with tobacco smoke further promotes platelet dysregulation. The long term hypothesis and importance of this research proposal is that tobacco smoke places HIV-infected individuals undergoing HAART at even greater risk for developing comorbidities and mortality from platelet activation that causes CVD. Our goal is to perform essential basic in vitro human platelet function studies to determine the impact and mechanisms underlying the combined effects of tobacco smoke and key antiretroviral therapies on platelet activation. In Specific Aim 1, we will focus on the characterization of platelet dysregulation in nonsmoking and smoking HIV-infected volunteers undergoing ART. Specific Aim 2 will test key antiretroviral regimens identified in Aim 1 in order to characterize platelet dysregulation and begin to understand the underlying mechanisms. Completion of these R21 exploratory and developmental studies will provide the foundation to pursue future translational studies of the role of platelets in HIV and the design of new therapeutic interventions to reduce HIV-related CVD.

Organ fibrosis can occur in most body tissues, including the heart and lung, and it is a serious cause of morbidity and mortality world-wide. When tissues are damaged, they can either repair normally or they can repair with scarring (after a more severe or prolonged injury). The formation of scar tissue can be debilitating and even deadly when it occurs in vital organs such as lung and heart. Sadly, there are few available therapies for cardiac and pulmonary fibrosis. Thus, a major unmet need is to understand common pathologic processes involved in driving fibrogenesis, and to develop highly effective anti-scarring treatments. The objective of this RFA is to garner information from two or more organ systems to inform our understanding of common mechanisms of disease pathogenesis and to develop new therapeutics. Our grant application focuses on cardiac and lung scarring. We have discovered novel and common feedforward loops that we propose drive fibrosis in these and other organs. Our research team has published that Lactate Dehydrogenase A (LDHA) acts as an amplifier of lung fibrosis by increasing production of lactic acid, which lowers the local pH and activates latent TGF?, a potent pro-fibrotic cytokine. Separately, we were the first to report that Myocardin-Related Transcription Factor A (MRTF-A) drives myofibroblast differentiation of cardiac fibroblasts. We now have exciting data that links these two observations. Specifically, we have identified that MRTF-A drives LDHA expression via a previously unknown CArG box in the LDHA promoter. Here, we will test our overall hypothesis that MRTF-A and LDHA act in concert to form a pro-fibrotic feed-forward loop that amplifies tissue fibrosis in multiple organs by activating latent TGF?, via lactic acid-lactate production, and that pharmacologic inhibition of this feed-forward loop represents a novel therapeutic strategy for an otherwise untreatable pathologic response. Our Specific Aims to study fibrotic mechanisms in lung and heart are as follows. Specific Aim 1. Investigate the role of lactic acid/lactate and LDHA in promoting myofibroblast differentiation of lung and cardiac fibroblasts, and in driving pulmonary and cardiac fibrosis in vivo. Specific Aim 2. Investigate the role of MRTF-A as a common amplifier of fibrosis in lung and heart. Specific Aim 3. Evaluate novel small molecule inhibitors of LDHA and the MRTF/TGF-? pathway as potential common therapeutic agents for cardiac and lung fibrosis. These studies will provide key new mechanistic data describing a novel feed-forward loop that amplifies pro-fibrotic signaling in both heart and lung. Our findings will show that MRTF-A and LDHA act together to promote fibrosis via lactic acid-mediated activation of TGF-?. We will also provide critical proof-of concept data using cell culture and preclinical animal models that inhibition of the lactate metabolic pathway is a novel and viable therapeutic target in fibrosing diseases of lung and heart.

Abstract This Institutional National Research Service Award is designed to develop skilled investigators with research- oriented careers directed at solving basic, translational and clinical problems in lung disease. The structure of the program is based on the premise that training requires 1) a multidisciplinary approach, 2) a close relationship between the student/postdoc and mentoring team, and 3) a training environment with breadth and depth in basic, translational and clinical sciences. Training in this Pulmonary program spans disciplines that include cell and molecular biology, immunology, microbiology, toxicology, biochemistry, pulmonary and critical care medicine, infectious diseases and neonatology. Major research themes include: lung immunology, inflammation and infection; pulmonary fibrosis and remodeling; tobacco and biomass smoke and chronic obstructive pulmonary disease; and the health effects of air pollution. Four predoctoral and 4 postdoctoral trainees will be supported each year. Predoctoral students interested in lung research will be accepted for training through the Graduate Education in Biomedical Sciences (GEBS) programs, after they have passed their qualifying examinations. Predoctoral trainees will be supported for up to 3 years. Postdoctoral PhD trainees involved in lung-related research are also candidates for training, and are identified and recruited by members of this program. The MD/DO Candidates come from training programs in adult Pulmonary and Critical Care Medicine, Allergy/Immunology/Rheumatology, Neonatology, and Infectious Diseases. Only the top candidates will be considered based on potential success in and serious commitment to a pulmonary research-oriented career. During the current cycle of the training grant, we established and successfully recruited two MD trainees for a new research track. This track requires 4 years of subspecialty training with research training and support on this grant occurring in the final 2 years, and specific expectations of publications and research grant submission by the end of training. A direct research experience with research mentors forms the primary mechanism for training, supplemented by didactic courses, seminars, conferences, journal clubs, and instruction in research ethics, human and animal experimentation, and grant writing. All trainees have access to new Institution-wide programs in career development paths, grant writing, critical thinking, experiential learning and more. Program trainees will also develop research protocols and plans under the supervision of their mentoring team. This training program will help to meet the diverse need for scientific investigators in lung biology and disease in Academia, Government, and Industry. Research and expanded training by these trainees will enhance our understanding of how the respiratory system responds to injury and environmental challenges, and will improve public health by developing new and improved avenues for the prevention and treatment of lung disease.

The Focus: Thyroid eye disease (TED) is a disfiguring and sight-threatening autoimmune disease that involves inflammation and remodeling of the orbit. TED can present itself with mostly orbital fat (Type 1), orbital connective tissue (Type 2) or a combination of both. Here, we focus on how TED manifests itself as Type 1 disease. The Premise: We know that activated orbital fibroblasts produce high levels of inflammatory cytokines and form either connective tissue myofibroblasts or lipid rich inflammatory adipocytes. Our earlier work showed - that only human orbital fibroblasts that do not express a cell surface protein called Thy1 (Thy1 ) can differentiate into adipocytes when stimulated with an adipogenic medium. And, only those that express Thy1 (Thy1+) differentiate into myofibroblasts when provoked by pro-scarring agents like TGF?. Herein, we show that Thy1 presence has a direct role in diminishing orbital fibroblast fat accumulation. For example, deliberate - expression of Thy1 in Thy1 fibroblasts blocks their differentiation to adipocytes. Since adipogenesis requires the activation of the transcription factor PPAR?? we propose that Thy1 impairs PPAR? function. Adipogenesis can also manifest after thyroid stimulating hormone receptor (TSHR) signaling and our studies demonstrate that Thy1 and TSHR are inversely related. Goals: (a) test specific hypotheses about mechanistic and causal roles of Thy1 in TED, (b) determine the molecular mechanisms by which Thy1 expression dampens adipogenesis and inflammatory cytokine production. These mechanisms include [1] regulation of TSHR, [2] PPAR? activity and [3] modulation of post-transcriptional gene expression through key microRNAs. Organizing Hypotheses: Thy1 is more than a cell-surface marker in TED, as it is also a functional mediator of fibroblast fate in the disease. Thy1+ orbital fibroblasts have decreased PPAR? activity, decreased TSHR, and decreased miR-130a expression, which inhibit the fundamental processes in TED of orbital fibroblast adipogenesis and proinflammatory cytokine production. The proposed experiments will test corollaries of this hypothesis using primary human orbital fibroblasts and tissues; because there is no animal model that provides a consistent phenotype of fat accumulation in the orbit. Specific Aim 1: Test the hypothesis that orbital fibroblast Thy1 has anti-adipogenic and anti-inflammatory mechanisms of action by inhibiting PPAR? activity and regulating post-transcriptional gene expression. Specific Aim 2: Test the hypothesis that the mechanism by which Thy1 has its anti-adipogenic and anti-inflammatory effects is through the regulation of TSHR. Impact on the field: Discovering that Thy1 has a major biological function influencing eye disease is a major advance opening new areas for research and therapeutic intervention into the aberrant fat deposition and inflammation associated with TED. The inverse relationship that will be investigated between Thy1 and TSHR is the first demonstration that Thy1 can control TSHR. The results will lead to further research on how targeting of Thy1 and/or its anti-adipogenic mechanism of action could be used in more effective therapeutics.