Bethany B. Moore - US grants

Prostate cancer is the second leading cause of malignancy-related mortality in males in the United States. While carcinogenesis is a complex process that involves multiple stages of transformation, clinically significant tumor growth and metastasis are dependent upon angiogenesis. This neovascularization allows new blood vessels to form which supply the tumor with oxygen and nutrients necessary for growth. Evidence suggests that net tumor-derived angiogenesis is determined by an imbalance in the expression of angiogenic and angiostatic factors in the local milieu of the tumor. We hypothesize that in part, net tumor- derived angiogenesis is determined by an imbalance in the expression of angiogenic as compared to angiostatic CXC chemokines. Our preliminary data demonstrates that IL-8 is a common angiogenic CXC chemokine which is constitutively overexpressed by prostate cancer cell lines. Furthermore, the expression of IL-8 positively correlates with prostate cancer growth in a SCID mouse model. In addition, the expression of the angiostatic CXC chemokine IP-10 is inversely correlated with prostate cancer growth and metastasis. Using a variety of molecular and cellular techniques, experiments are designed to examine the effect of neutralizing the angiogenic IL-8 and/or overexpressing the angiostatic IP-10 CXC chemokines on growth of prostate cancer cell lines in SCID mice. Specifically we will abrogate angiogenic CXC expression by stable transfection of appropriate anti-sense IL-8 vectors. Conversely, we will enhance angiostatic or angiogenic CXC expression by stable transfection or adenoviral infection of appropriate IL-8 or IP-10 cDNA constructs. We can confirm the specificity of these changes using neutralizing Abs to CXC chemokines in bioassays for angiogenesis as well as in SCID mice. The genetic dysregulation of these chemokines will be examined and characterized in prostate cancer cell lines. We will identify what transcriptional and post-transcriptional mechanisms account for the dysregulated overexpression of angiogenic CXC chemokines. These studies should lead to new insights into the biology of prostate cancer growth and metastasis, and pave the way to adapt adjuvant therapies for the treatment of this widespread disease.

DESCRIPTION (provided by applicant): Pulmonary fibrosis is a disordered response to lung injury that involves damage and/or loss of alveolar epithelial cells (AECs), inflammation, fibroblast proliferation, and excessive deposition of extracellular matrix. Fibroblasts are key target cells in fibrotic responses, and much is known about their capacity for activation and matrix secretion. However, there is a growing appreciation that fibrogenesis involves inadequate generation of, or responses to, suppressive signals that ordinarily control fibroblast responses. AECs are an important source of such suppressive signals. This proposal focuses on AEC-derived prostaglandin E2 (PGE2) as a crucial down-regulator of fibrotic responses. Fibrotic stimuli can cause the loss of PGE2 secretion from AECs. The suppressive actions of PGE2 on fibroblasts are mediated by four distinct prostaglandin E2 (EP) receptors. Fibrotic alterations to EP2 and/or EP4 receptor expression and/or signaling could abrogate suppressive PGE 2 actions on fibroblasts. Thus, the hypothesis to be tested in this proposal is that AEC generation of PGE2 and fibroblast responses to PGE2 via EP2/EP4 receptors are critical determinants of AEC-fibroblast interactions governing fibrotic responsiveness, and that both may be impaired by fibrotic insults. This hypothesis will be tested by 1) Examining PGE2 production from AECs isolated from saline- and Neomycin-treated mice. 2) Examining the regulation and signaling of EP2 and EP4 receptors on fibroblasts purified from saline- and Neomycin-treated mice. 3) Examining the ability of AECs to suppress fibroblast proliferation and collagen synthesis during the disease course, and determining the functional significance of PGE2 secretion in mediating these outcomes. 4) Determining the fibrotic response of EP2-/-and EP4-/- mice in vivo. 5) Determining whether AECs, via secretion of PGE2, can inhibit the transformation of fibroblasts into myofibroblasts. These investigations suggest a novel role for AEC-derived PGE2 as an inhibitor of fibrotic reactions in the lung, and suggest that manipulations to increase PGE2 secretion and or enhance suppressive EP2/EP4 signaling in fibroblasts will have therapeutic benefit in the treatment of this devastating disease.

[unreadable] DESCRIPTION (provided by applicant): Bone marrow (BM) transplantation (BMT) is the treatment of choice for many malignancies and specific inherited disorders. Unfortunately, infections such as Pseudomonas aeruginosa (P. aeruginosa) occur frequently even post-engraftment. Little is known about the mechanisms responsible for the increased risk of lung infections in BMT patients. We have shown that mice undergoing syngeneic BMT are more susceptible to pulmonary infection with P. aeruginosa than are non-transplanted mice despite complete donor leukocyte reconstitution. We found defects in the ability of alveolar macrophages (AMs) and polymorphonuclear leukocytes (PMNs) from BMT mice to phagocytose and/or kill bacteria and release tumor necrosis factor-a (TNFa). In addition, AMs and PMNs from BMT mice produce up to 125-times more prostaglandin E2 (PGE2) than cells from control mice. PGE2 is able to limit AM phagocytosis and to limit AM and PMN bacterial killing via interactions with the E prostanoid 2 (EP2) receptor. Thus, PGE2 is an attractive candidate to play a central role in the dysfunctional activity of AMs and PMNs from BMT mice. In vivo confirmation comes from our studies where pharmacologic blockade of PGE2 production (using indomethacin) restores lung host defense against P. aeruginosa post-BMT. These data have led to our hypothesis that over-production of PGE2 in the lung post-BMT, acting via EP2 receptor signaling, suppresses lung phagocyte function leading to impaired pulmonary host defense against P. aeruginosa. Our current studies will determine whether increased production of granulocyte-macrophage colony-stimulating factor (GM-CSF) post-BMT leads to increased production of PGE2 and whether the donor vs. host origin of the lung phagocytes or the BMT-conditioned lung environment contributes to impaired host defense. Finally, we will explore the role that the phosphatase and tensin homolog on chromosome ten (PTEN) and IL-1 receptor-associated kinase (IRAK)-M play as mediators of the PGE2-induced suppression. Using molecular, cellular and animal modeling strategies, we will address the following aims: Aim 1) To determine the role of increased GM-CSF in determining PGE2 elevation and impaired host-defense post- BMT; Aim 2) To determine whether the origin of the AMs (donor vs. host) dictates function post-BM; Aim 3 To determine whether the BMT lung microenvironment is inhibitory to AM function; Aim 4) To determine the roles that PTEN activation and/or IRAK-M elevation play in mediating the PGE2-induced immunosuppression post-BMT. This work provides mechanistic insight into the persistent immunosuppression seen post-BMT and provides a proof of concept for the development of new therapeutic strategies (neutralization of GM- CSF, inhibition of PGE2 synthesis/signaling and pharmacologic inhibition of PTEN activation) which may result in effective new treatments to prevent pulmonary infections in patients post-stem cell transplant. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Idiopathic pulmonary fibrosis (IPF) is a disease that culminates in progressive loss of pulmonary function and deposition of extracellular matrix (ECM). Additionally, many IPF patients will experience an acute worsening of symptoms due to known causes (e.g. infection) or of idiopathic nature termed "acute exacerbations". Circulating, bone-marrow-derived cells known as fibrocytes (identified by shared leukocyte and mesenchymal markers) may be regulators of IPF pathogenesis. Fibrocyte numbers are increased in patients with IPF and murine fibrosis studies have demonstrated that fibrocytes are 1) recruited to the lung by chemokines 2) adoptive transfer of fibrocytes worsen disease and 3) fibrocyte recruitment correlates with infectious exacerbations. Thus, absolute numbers of fibrocytes, or particular fibrocyte phenotypes, may serve as novel biomarkers in IPF. The IPF network (IPFnet) is the parent organization that will oversee the PANTHER trial which is a multi-center, randomized, double-blind placebo controlled trial to investigate the efficacy of prednisone + azathioprine + N-acetylcysteine (NAC) or NAC alone versus placebo in IPF patients with mild- moderate disease (130 patients in each arm followed up to 67 weeks). The second trial is the STEP trial to test sildenafil in IPF patients with severe disease over a 12 week period (85 patients in both the treatment and placebo arms). All these patients will be well characterized for diagnosis, physiology, disease progression and adverse events. The studies proposed in this grant would take advantage of the large and diverse IPF patient populations enrolled in these trials and the abundant clinical/physiological data collected over time on each patient to perform longitudinal and cross-sectional studies to characterize fibrocyte numbers and phenotypes in an effort to provide mechanistic correlations into the natural history of IPF and response to therapy. In addition, we will compare IPF fibrocyte phenotypes to those obtained from disease-specific control patients including patients with chronic obstructive pulmonary disease (COPD) and scleroderma as well as normal volunteers. We hypothesize that absolute numbers of fibrocytes, or distinctive fibrocyte phenotypes (chemokine receptor expression, collagen synthetic capacity, secretion of pro-fibrotic mediators or proliferation) will correlate with severity of disease at entry, rate of progression of disease, response to therapy and occurrence of acute exacerbations. The IPFnet steering committee recognizes the value of these mechanistic studies and fully supports this application. Aim 1) To determine whether absolute numbers of fibrocytes or alterations in fibrocyte phenotype differentiate between normal, COPD, scleroderma and IPF subjects. Aim 2) To determine whether changes in fibrocyte numbers or alterations in fibrocyte phenotype(s) predict severity of disease, changes in physiology or response to therapy in IPF and control patients. Aim 3) To determine whether changes in absolute numbers of alterations in fibrocyte phenotype(s) correlate with acute deteriorations or differentiate between acute exacerbations of known and unknown cause. Statement of Relevance Fibrocyte phenotypes have been validated as biomarkers in murine models of fibrosis. Our proposed studies would be the first to provide longitudinal and cross sectional analyses of fibrocyte phenotypes as biomarkers in human fibrotic lung disease. (End of Abstract)

Project Summary/Abstract Pulmonary fibrosis may result from dysregulated wound healing responses to sequential lung injuries. Recruitment of circulating bone-marrow-derived mesenchymal precursors (fibrocytes) is crucial for a fibroproliferative host response post-injury. Fibrocytes contribute to extracellular matrix (ECM) generation and promote fibrosis through the secretion of profibrotic/proinflammatory factors. A focal alveolar epithelial cell (AEC) injury is believed to be the initiating event of the fibrotic process. The etiologic agents of lung injury are unknown but latent viral infections, especially by members of the herpesvirinae have been associated with idiopathic pulmonary fibrosis (IPF). Viral infection might influence fibroproliferative responses via lysis of parenchymal lung cells or by inducing alterations in the function(s) of resident or recruited cells. Infection of mice with MHV-68 (a murine gammaherpesvirus) results in both lytic and latent infection of AECs and mimics human infection with Epstein-Barr virus (EBV). Our preliminary data demonstrate 1) MHV-68 infection augments fluorescein isothiocyanate (FITC)-induced lung fibrosis when given both prior to or after the fibrotic insult. 2) Fibrocytes are recruited to the lung in response to MHV-68 infection. 3) MHV-68 infection results in the generation of cysteinyl leukotrienes (cys LTs) which can induce migration and activation of fibrocytes. 4) MHV-68 can infect fibrocytes and enhance their proliferation. 5) The additional alveolar injury induced by FITC, induces a dysregulated cytokine and eicosanoid response which favors fibrocyte proliferation, differentiation and ECM deposition. We hypothesize that increased fibrosis following MHV-68 infection/FITC injury is the result of enhanced recruitment, proliferation and differentiation of fibrocytes. Recruitment and proliferation are increased due to releases of cys LTs and CC chemokines by resident cells of the lung. Differentiation is enhanced by production of altered ratios of fibrocyte stimulatory and protective molecules by parenchymal and recruited cells. Completion of the following specific aims will provide new information regarding mechanisms that lead to generation of or exacerbation of pulmonary fibrosis. Aim 1) To determine the kinetics and role of alveolar MHV-68 infection in augmentation of FITC-induced pulmonary fibrosis;Aim 2) To determine whether MHV-68 infection alters the secretion of pro- or anti-fibrotic mediators by AECs, alveolar macrophages (AMs), interstitial macrophages (IMs) and B cells and to perform correlative studies in human AMs infected with EBV;Aim 3) To determine whether MHV-68 infection recruits more fibrocytes to the lung, whether MHV-68 or EBV infection alters fibrocytes and fibroblasts and to determine the role of cys LTs in MHV-68-induced recruitment of fibrocytes and augmentation of fibrosis. Project Narrative/Relevance The experiments proposed in this application will provide mechanistic insight into the role that viral infections may play in predisposing people to the development of fibrosis. Research will also address the role that viral infections play in exacerbating disease in patients with established fibrosis. Finally, this work will explore the therapeutic potential of anti-leukotriene strategies to limit viral-induced exacerbations of fibrosis.

DESCRIPTION (provided by applicant): Hematopoietic stem cell transplantation (HSCT) is used to treat a variety of defects and malignancies, but its usefulness is limited by pulmonary infections. Infectious complications can occur both in allogeneic and autologous transplant settings and susceptibility to infection remains elevated despite hematopoietic reconstitution. To better understand the innate immune deficiencies that characterize HSCT, we developed a murine model of Pseudomonas aeruginosa infection post-bone marrow transplant (BMT). Following myeloablative conditioning, mice which have fully restored hematopoietic compartments remain highly susceptible to P. aerugionsa lung infection. We have previously demonstrated that this increased susceptibility is related to elevated transcription of cyclooxygenase 2 (COX-2) which leads to overproduction of prostaglandin E2 (PGE2) in alveolar macrophages (AMs) and neutrophils (PMNs) and subsequent impaired innate immune function. PGE2 signaling in AMs and PMNs post-BMT critically impairs both opsonized and non-opsonized phagocytosis of P. aeruginosa by AMs, but our results demonstrate an imperative role for non- opsonized phagocytosis in limiting acute infection. Importantly, our murine studies have shown inhibition of COX-2 post-BMT restores lung innate immunity. In this renewal application, we present preliminary data that COX-2 elevations post-BMT are associated with demethylation of the COX-2 gene. We also show that AMs from BMT mice have a different miRNA expression profile which likely influences AM function. One key change noted in the BMT AMs is diminished expression of a key scavenger receptor (MARCO) which mediates uptake of non-opsonized bacteria pre- and post-BMT. In addition, our preliminary results suggest that alveolar epithelial cells play important roles in promoting innate immune functions of AMs and the process of BMT impairs epithelial cell functions in this regard. In fact, inhibition of macrophage innate immune functions may be limited to the lung post-BMT. Finally, we provide evidence that human HSCT patients also display elevations in COX-2, and we will explore whether the mechanisms and treatments that we have characterized in the murine model are also relevant in the human transplant setting. The overall hypothesis of the renewal application is that stem cell transplantation alters epithelial cells and results in epigenetic, miRNA, and scavenger receptor dysregulation of AMs which impair host defense against bacterial pathogens. We will test this hypothesis with the following specific aims. Aim 1) To determine whether DNA hypomethylation or miRNA alterations contribute to increased COX-2 expression and decreased TNF¿ production noted in AMs post-BMT; Aim 2) To determine whether BMT induces functional alterations in the expression profile of AM scavenger receptors and how PGE2 influences their individual expression and function; Aim 3) To explore the role of BMT alveolar epithelial cells (AECs) in limiting AM function and to determine whether the inhibition of innate immunity post-BMT is lung specific and related to TGF¿; Aim 4) To determine whether AMs from human HSCT patients overexpress COX-2 and PGE2 and have altered scavenger receptor profiles, the mechanisms by which this occurs and to test the therapeutic effect of cyclooxygenase or EP2 inhibition on innate immune function.

DESCRIPTION (provided by applicant): Periostin Regulation of Lung Fibrosis Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disorder of the lung that is characterized by the accumulation of myofibroblasts and the deposition of extracellular matrix leading to respiratory failure. Unfortunately, the disease is fatal and there are no effective therapies other than lung transplantation. The most potent profibrotic mediator studied to date is transforming growth factor (TGF) b and TGFb is elevated in many models of organ fibrosis. Unfortunately, TGFb is a difficult therapeutic target as total loss of TGFb or TGFb signaling can cause devastating autoimmune inflammation and mortality. As such, there is great interest in identifying downstream mediators of TGFb signaling which may be better targets for therapeutic intervention to treat fibrotic disorders. Recently, the TGFb-regulated matricellular protein, periostin, a molecule which has been studied in asthma, atherosclerosis and cancer, has been implicated in the pathogenesis of interstitial fibrotic lung disease. We and others have shown that periostin is increased in cells and lung tissue of IPF patients and that elevated levels of circulating periostin in IPF patients predict declines in lung function. Additionally, we and other have demonstrated that periostin-/- mice are protected from bleomycin-induced fibrosis. We recently demonstrated that periostin can induce mesenchymal cell proliferation, collagen expression and ability to close a scratch wound. Blockade of periostin interactions with the avb3 and avb5 integrins via the administration of the OC-20 monoclonal Ab could partially reverse periostin-mediated wound closure and partially blocks the development of bleomycin-induced fibrosis when administered during the fibroproliferative phase of the disease. Our published results using bone marrow chimeric mice indicate that both structural and hematopoietic sources of periostin are required for development of bleomycin-induced fibrosis. Preliminary data show that periostin may induce myofibroblast survival via the induction of the anti- apoptotic proteins (survivin, X-linked inhibitor of apoptosis (XIAP) and Bcl-2). Periostin is also elevated in aged mice and may contribute to the enhanced susceptibility of aged mice to gammaherpesvirus-induced fibrosis. Our revised studies are aimed at verifying the ability of periostin to promote fibrosis in two additional animal models. We also have proposed studies to elucidate the role that circulating fibrocytes and fibrocyte-derived periostin may play in regulating fibrotic development in multiple models. TGFb and periostin reciprocally regulate each other; however, the fact that periostin-deficient mice are viable and have relatively few health issues suggests that this molecule may be highly amenable to therapeutic targeting. To further explore this possibility, we will perform mechanistic studies to understand the reciprocal regulation of these mediators and to understand how periostin influences lung mesenchymal cell behavior. Finally, we will measure periostin in the lung and the changes in circulating periostin levels over time in IPF patients and determine the correlations this biomarker may have on disease progression. We hypothesize that the matricellular protein, periostin, promotes the development and progression of pulmonary fibrosis and may serve as a biomarker for disease progression. We will mechanistically explore this hypothesis in the following specific aims. Aim 1) To determine whether periostin regulates the development of fluorescein isothiocyanate-induced pulmonary fibrosis or gammaherpesvirus-induced fibrosis in aged mice Aim 2) To determine the contribution of fibrocytes and fibrocyte-derived periostin in the development of lung fibrosis in animal models Aim 3) To determine the molecular mechanisms via which periostin and TGFb regulate each other and the function of lung mesenchymal cells Aim 4) To determine whether periostin levels in plasma or bronchoalveolar lavage fluid of IPF patients correlate with disease progression

DESCRIPTION (provided by applicant): This is a competing application for continued support of an NIAID sponsored multi-disciplinary program for Research Training in Experimental Immunology at the University of Michigan School of Medicine beginning its 20th year. Faculty preceptors with interests in immunology and immunological mechanisms of disease have been selected from the Departments of Pathology, Internal Medicine, Microbiology and Immunology, Pediatrics, Neurology and Surgery. Our senior preceptors direct active, highly regarded laboratories with exemplary records of securing extramural funding. These preceptors have outstanding records in mentoring graduate students and post-doctoral fellows. In addition, we have added some highly regarded new investigators and have established mechanisms to ensure that these new preceptors have ample guidance in becoming accomplished mentors themselves. The program is directed by Dr. Steven Kunkel who has led the program for the last 10 years. Beginning this year, the co-director will be Dr. Bethany Moore who also serves as the Director of the Immunology Graduate Program at the University of Michigan. Drs. Kunkel and Moore will work closely with the T32 advisory committee and the Immunology graduate student affairs committee to advise trainees, monitor curriculum and resolve any issues that arise. The T32 will continue to sponsor several major activities: 1) a weekly seminar for students and post-doctoral fellows to present journal club or works-in progress, 2) a visiting professor monthly seminar where invited guest speakers present their research and spend a day interacting with program faculty and trainees, 3) research colloquium courses which provide in depth training in experimental immunology and special topics relating to translational immunology and 4) a new i-club which will provide additional journal club, career development and networking opportunities for our trainees. In addition, the program provides training in research responsibility and ethics and is committed to continuing to recruit and train talented mentees from underrepresented populations to facilitate the creation of a diverse and talented pool of researchers for the next generation. Support is requested for 6 pre-doctoral and 2 post-doctoral trainees, the same as has been supported previously. This T32 program has been essential to the formation of the Immunology graduate program at the University of Michigan and our success is best measured by the outstanding publication and presentation records of our recent trainees and the excellent post-doctoral and academic positions they earn as they progress.

? DESCRIPTION (provided by applicant): Hematopoietic stem cell transplantation (HSCT) is used to treat a variety of genetic defects and malignancies, but its usefulness is limited by pulmonary infections. Infectious complications can occur both in allogeneic and autologous transplant settings and susceptibility to infection remains elevated despite hematopoietic reconstitution. To better understand innate immune deficiencies that characterize HSCT, we developed a murine model of bacterial infection post-syngeneic (syn) bone marrow transplant (BMT). We have previously shown that these mice are more susceptible to infection with Pseudomonas aeruginosa even after the hematopoietic system is reconstituted. We identified the upregulation of cyclooxygenase-2 (COX-2) and the overproduction of PGE2 as major contributing factors to the impaired innate immune function in these mice. We identified that alveolar macrophages (AMs) and neutrophils (PMNs) had defects in innate immune functions such as phagocytosis, bacterial killing and cytokine secretion. In addition, the profile of scavenger receptors on AMs were altered post-BMT, with loss of macrophage receptor with collagenous structure (MARCO), a critical receptor for recognition of P. aeurignosa. We determined that PGE2 signaled via elevated E prostanoid 2 (EP2) receptors on these AMs to inhibit their functions. Importantly, our murine studies have shown that pharmacologic or genetic inhibition of COX-2 post-BMT restores lung innate immunity and AM function against P. aeruginosa. These results are exciting because they suggest inhibition of PGE2 signaling can be a therapeutic to improve host defense post-transplant. However, there are systemic problems with a therapeutic strategy that globally blocks all prostaglandin synthesis. Thus, one aspect of this proposal will be to test a newly developed EP2 antagonist (PF-044148948) which we have obtained from Pfizer. We believe this will be a much more specific and effective therapeutic to block the inhibitory PGE2 signaling. The application also seeks to provide insight into the following unanswered questions. 1) Why is COX-2 elevated post-BMT leading to overproduction of PGE2? 2) Do these same innate immune defects characterize allogeneic (allo) BMT? 3) Do these defects post-BMT make mice more susceptible to Gram positive infections (like Streptococcus pneumoniae) as well as Gram negative ones? 4) Can we determine whether impairment of autophagy is one mechanism for impaired killing post-BMT? 5) Are the defects we have noted in our murine model also present in human HSCT patients? Our overall hypothesis is: BMT conditioning induces transforming growth factor (TGF) ß secretion from lung epithelial cells. This augments miR-29b expression to block synthesis of DNA methyltransferases (DNMTs) causing hypomethylation of COX-2 leading to PGE2 overexpression in AMs. Furthermore, PGE2-EP2 induced alterations in autophagy impair host defense against P. aeruginosa and S. pneumoniae post-BMT and we speculate that host defense post-BMT can be improved via treatment with a COX inhibitor, an EP2 antagonist or via induction of autophagy. These hypotheses will be explored in the following specific aims. Aim 1) To determine if syn BMT and allo BMT mice are more susceptible to P. aeruginosa and S. pneumoniae infection and if susceptibility is related to PGE2 signaling via EP2 in mice and man Aim 2: To determine whether TGFß-induced miR-29b causes COX-2 hypomethylation to increase PGE2 production post-BMT Aim 3: Aim 3: To determine if autophagy is impaired in syn and allo BMT AMs, to determine whether this is related to PGE2- EP2 signaling and the importance of autophagy to host defense post-BMT

Abstract: Hematopoietic stem cell transplantation (HSCT) is a curative option for the treatment of numerous malignancies and inherited genetic disorders; however, the success of the procedure is hampered by numerous complications. Pulmonary complications cause significant morbidity and mortality following HSCT and most notably include infections, idiopathic pneumonia syndrome (IPS), bronchiolitis obliterans (BOS) and cryptogenic organizing pneumonia (COP). These conditions are characterized by acute and chronic inflammation and can include tissue fibrosis, severely damaging lung function. While more common following allogeneic HSCT, IPS, BOS and COP have also been reported as a complication of autologous HSCT and their diagnosis is defined, in part, by the absence of infection. However, accumulating evidence suggests these lung pathologies could represent immunopathology that develops as a consequence of a previous viral infection, or possibly due to an occult infection. Importantly, herpesviruses were recently identified as the most common occult infection in patients with IPS. We have developed a murine model in which syngeneic bone marrow transplant (BMT) mice that are fully reconstituted with donor-derived cells develop severe lung pathology characterized by interstitial pneumonitis, vasculitis and fibrosis following infection with murine gamma herpesvirus-68 (?HV-68). This lung pathology is well established at 21 days post-infection when lytic viral infection has been cleared and the virus has established latency. Our published and preliminary findings suggest the process of BMT alters the phenotype of lung dendritic cells (DCs). DCs from BMT mice overproduce IL-1?, IL-6, TGF? and IL-23. These BMT-induced DC alterations lead to the skewing of the CD4 response to a Th17, rather than a Th1 response post-infection with ?HV-68. The consequence of this persistent skewing is the development of IL-17-dependent pneumonitis and fibrosis. In addition, we have evidence that adoptive transfer of DCs from infected control mice into BMT mice can restore Th1 cell priming and limit development of lung immunopathology focusing our current studies on trying to understand how the process of BMT impairs the function of lung DCs. Preliminary data suggest that lung DCs in BMT mice are characterized by reduced autophagy and impaired expression of the Notch ligand, delta like ligand 4 (DLL4). Reduced autophagy could explain the overproduction of IL-1? if DCs in BMT mice are unable to clear activated inflammasomes. Notch signaling is highly context-dependent and can both enhance and inhibit T cell signaling. Thus, the overall goal of the proposed research is to mechanistically understand how DCs are altered following HSCT in ways that promote pathologic rather than protective immune responses to respiratory viruses. Most importantly, we will determine whether these molecular alterations that characterize murine DCs post-BMT are also evident in DCs from human patients experiencing lung dysfunction post-HSCT. Our hypothesis is that HSCT results in reduced autophagy and impaired DLL4 expression in lung DCs in response to ?HV-68 infection, and that this alteration in DCs promotes pathologic Th17 rather than protective Th1 responses. We will address this hypothesis in the following specific aims. Aim 1) To identify the relevant DC population responsible for priming ?HV-68-specific T cell responses and determine whether autophagy is impaired in these DCs post-BMT and ?HV-68 infection Aim 2) To determine if BMT DCs are characterized by defective Notch ligand expression or altered costimulatory receptors Aim 3) To determine the translational relevance of these findings by determining whether the responses are specific to ?-herpesviruses, and whether similar DC phenotypes are noted in patients post-HSCT Completion of these specific aims will be significant for several reasons. This work will solidify the critical role that latent or occult infection play in causing ?idiopathic? lung pathology post-HSCT and will provide novel information regarding the mechanistic alterations that occur in DCs post-transplant. Most importantly, this work will identify and test strategies to restore DC function post-BMT and will provide translational proof that these phenotypes characterize human DCs post-HSCT.

DESCRIPTION (provided by applicant): Influenza infections are a leading cause of death in the United States and worldwide, with 50 000 of deaths occurring annually in the United states alone. Although many patients succumb to the primary viral infection, a significant number of influenza-related deaths are attributable to the development of secondary bacterial infections. Why the host is more susceptible to bacterial infections post-influenza is poorly understood. A better understanding of how the host responds to sequential influenza + bacterial infection is necessary in order to develop therapies to improve outcomes. Using a murine model of sequential pulmonary influenza and methicillin-resistant Staphylococcus aureus (MRSA) or Streptococcus pneumoniae infection, we have found that these mice have decreased survival and impaired anti-bacterial responses. We also showed that host responses to post-viral pneumonia are characterized by impaired macrophage autophagy responses, and impaired macrophage phagocytosis and killing of bacteria in the lungs. These findings correlate with significant upregulation of interferon (IFN)? and upregulation of miR 155 when compared to mice infected with either pathogen alone. Thus, we hypothesize that impaired innate immunity against MRSA in the setting of post-influenza pneumonia is due to IFN?-induced upregulation of miR 155 which in turn blocks protective autophagy responses and prevents bacterial phagocytosis and killing. We will address these goals with the following specific aims. Aim 1) To determine the role of IFN? in regulating miR 155 expression, host defense, cytokine production, and macrophage autophagy following infection with influenza alone, MRSA/Streptococcus pneumoniae alone or sequential infection. This aim will characterize these outcomes in single and sequential infections, will test responses in chimeric mice generated using wild-type and IFN? receptor-/- mice and will test a therapeutic approach to block IFN? using a neutralizing mAb. Aim 2) To determine whether the impaired host defense noted during post-viral pneumonia is due to a failure of lung macrophages to upregulate protective autophagy responses This aim will determine whether autophagy is impaired in lung macrophages post- sequential infection compared to single infection, whether this is correlated with miR 155 inhibition of DAPK1 and whether enhancement of autophagy using rapamycin/Tat Beclin-1 can improve host defense by increasing bacterial phagocytosis and killing. Aim 3) To identify the mechanism that miR-155 uses to regulate macrophage recruitment, autophagy and to impair the anti-bacterial host-defense. This aim will characterize expression of miR 155 during post-viral pneumonia and explore inhibition of autophagy during post-viral pneumonia as a result of miR 155 inhibition of DAPK1. The ability of miR 155 to regulate bacterial phagocytosis and killing also will be explored. Additionally, transgenic mice will be generated using floxed miR155 and CD11b-Cre mice enabling cell type specific removal of miR155 from macrophages further providing mechanistic insight regarding regulation of autophagy by miR 155 expression on macrophages.

The overarching theme for our research program over the past 20 years has been to better understand the etiology and pathogenesis of lung injury, repair and remodeling, with a particular interest in how the immune response shapes pathologic and homeostatic processes in the lung. Our laboratory has contributed seminal studies related to 1) chemokine-mediated angiogenesis in cancer and lung fibrosis, 2) eicosanoid regulation of lung fibrosis, 3) eicosanoid regulation of innate immunity in the setting of hematopoietic stem cell transplantation (HSCT), 4) chemokine regulation of lung fibrosis, 5) fibrocyte functions in lung fibrosis, 6) matricellular protein regulation of lung fibrosis, 7) proteomic, microbiome and other biomarker studies in lung fibrosis, 8) role of viral infections in ?idiopathic? lung fibrosis, 9) role of viral infections in mediating pneumonitis and fibrosis post-HSCT and 10) studies of secondary bacterial infection post-influenza. Our work has utilized animal models to carry out mechanistic studies and has utilized patient-derived materials to confirm relevant pathways, identify therapeutic targets and characterize novel biomarkers. Based on our previously published observations and novel preliminary data, our laboratory is broadly focused in 4 main areas. The first is to study innate immune signaling in regulation of secondary bacterial infections post-influenza. The second is to explore interactions between the lung microbiome and innate signaling receptors in the pathogenesis of lung fibrosis. The third is to explore the role of myeloid-specific heparin-binding epidermal-like growth factor (HB-EGF) signaling in regulation of lung fibrosis. The fourth is to further understand the viral etiology and pathogenesis of lung pneumonitis and fibrosis as a complication of HSCT. This outstanding investigator award mechanism will allow us to extend our studies in each of these areas and will allow for mechanistic understanding of the role of immune signaling in the pathogenesis of fibrosis, pneumonitis and lung injury, especially following viral infection. It will also allow our laboratory to complete proof of concept and validation studies needed in both animals and humans to advance new therapies to the clinics for treatment of lung diseases.

PROJECT SUMMARY/ABSTRACT Non-healing wounds in patients with Type 2 Diabetes (T2D) are a major cause of morbidity and mortality and are increasing at an alarming rate. Failure of wound healing in T2D patients represents the most common cause of amputation in the US with a 5-year mortality rate of nearly 50%. Thus, a critical need exists for understanding the wound healing defects in T2D in order to develop targeted therapies. We have utilized both genetic (db/db) and dietary (diet-induced obese) murine models of T2D as well as human wound tissue and blood samples collected from T2D patients to explore mechanisms of impaired wound healing. Our published and preliminary data point to a pivotal role for macrophage (M?) function in orchestrating appropriate wound healing and demonstrate that wound M?s in diabetic mice and patients with T2D are characterized by a persistent inflammatory state, impaired phagocytosis/killing and the over-production of the immunomodulatory lipid, prostaglandin E2 (PGE2). Our data demonstrate epigenetic regulation of key genes important for the production of PGE2, namely cytosolic phospholipase A2 (cPLA2) and cyclooxygenase-2 (COX-2), and show overexpression of PGE2 in M?s results in increased production of inflammatory mediators such as interleukin 1? (IL1?) and impaired host defense against bacterial pathogens that often colonize the wound bed. Our preliminary data are the first to identify that cPLA2/COX-2 and PGE2 are increased in diabetic wound M?s and that this pathway may be regulated by multiple epigenetic mechanisms, including DNA methylation and histone methylation, in both diet-induced and genetic models of diabetes. These results support our hypothesis that inhibition of the COX-2/PGE2 pathway in M?s is critical for resolution of inflammation and proper host defense that is required for effective wound repair. These results have led to our hypothesis that the COX-2/PGE2 pathway is epigenetically regulated and increased in diabetic wound M?s and this results in increased inflammation, impaired host defense and defective wound repair. Our data suggest that wound M? function may be restored via M?-targeted treatment of FDA-approved COX inhibitor(s), TGF? signaling receptors and/or the first-ever developed EP2-specific antagonist. To test our hypotheses, we will: Aim 1: Determine the regulation of cPLA2 to release AA and promote COX-2/PGE2 production in diabetic wound M?s. Aim 2: Determine whether TGF?-induced miR-29b causes hypomethylation of the COX-2 gene to increase COX-2 and PGE2 production in diabetic wound M? and evaluate the therapeutic efficacy of M? targeted COX-2 inhibition and TGF? receptor antagonists. Aim 3: Determine the M?-specific PGE2-mediated mechanism(s) that modulate inflammation, host-defense functions and fibroblast crosstalk in normal and diabetic wound tissue.