Howard L. Weiner - US grants

Multiple sclerosis is a presumed autoimmune disease of the central nervous system. In the past three years we have studied immunoregulatory T-cells in MS using monoclonal antibodies and have found perturbations of suppressor/cytotoxic cells that appear to correlate with disease activity. In addition, we have found that various forms of immunotherapy may affect the course of multiple sclerosis; specifically, the use of short courses of high dose cyclophosphamide, which presumably acts by destroying immunocompetent lymphocytes. The current proposal is an extension of these studies, applying T-cell cloning techniques and limiting dilution analysis for a more sophisticated analysis of T-cell immunoregulation in multiple sclerosis. In addition, immunologic studies in patients currently undergoing a variety of immunotherapeutic regimens will be continued. Investigation of immunoregulatory T-cells in MS will include the following: 1) limiting dilution analysis of T-cells in peripheral blood and cerebrospinal fluid in order to correlate T-cell phenotype with functional characteristics (viz., cytotoxic, suppressor and helper function); 2) analysis of white matter reactive clones in peripheral blood and cerbrospinal fluid to a series of defined white matter antigens; 3) investigation of autoreactive T-cell clones; 4) analysis of T-cell activaton antigens; and 5) immunohistochemical analysis of lymphocytes in multiple sclerosis plaques. Clinical studies will involve investigation of the effect of other immunotherapeutic regimens on immune parameters in MS. This proposal represents a revised competitive renewal as follows: Clinical studies treating multiple sclerosis patients with monoclonal anti-T12 antibody have been deleted from this revised proposal and new data and investigations related to T-cell activation antigens, limited dilution analysis of T-cell clones, and autoreactive T-cells have been incorporated.

Multiple sclerosis is a presumed autoimmune disease of the central nervous system. In the past three years we have studied immunoregulatory T-cells in MS using monoclonal antibodies and have found perturbations of suppressor/cytotoxic cells that appear to correlate with disease activity. In addition, we have found that various forms of immunotherapy may affect the course of multiple sclerosis; specifically, the use of short courses of high dose cyclophosphamide, which presumably acts by destroying immunocompetent lymphocytes. The current proposal is an extension of these studies, applying T-cell cloning techniques and limiting dilution analysis for a more sophisticated analysis of T-cell immunoregulation in multiple sclerosis. In addition, immunologic studies in patients currently undergoing a variety of immunotherapeutic regimens will be continued. Investigation of immunoregulatory T-cells in MS will include the following: 1) limiting dilution analysis of T-cells in peripheral blood and cerebrospinal fluid in order to correlate T-cell phenotype with functional characteristics (viz., cytotoxic, suppressor and helper function); 2) analysis of white matter reactive clones in peripheral blood and cerbrospinal fluid to a series of defined white matter antigens; 3) investigation of autoreactive T-cell clones; 4) analysis of T-cell activaton antigens; and 5) immunohistochemical analysis of lymphocytes in multiple sclerosis plaques. Clinical studies will involve investigation of the effect of other immunotherapeutic regimens on immune parameters in MS. This proposal represents a revised competitive renewal as follows: Clinical studies treating multiple sclerosis patients with monoclonal anti-T12 antibody have been deleted from this revised proposal and new data and investigations related to T-cell activation antigens, limited dilution analysis of T-cell clones, and autoreactive T-cells have been incorporated.

This is an application for partial funding of a Conference on Autoimmunity, being held under the auspices of the Federation of American Societies for Experimental Biology (FASEB), from July 3 - July 8, 1988 at the Vermont Academy, Saxton's River, Vermont. Participation will be limited to 155 scientist applicants, who will be selected on the basis of their expertise and interests as most likely to contribute to a stimulating and productive environment. The meeting will focus on latest developments related to our understanding of autoimmunity. The conference will consist of eight scientific sessions each with four to five speakers and a discussion leader. In addition there will be two poster sessions during the week and a dinner speaker. The major session topics will be as follows: 1) Molecular mechanisms of T Cell Activation; 2) Genetics of Autoimmune Diseases; 3) Immunoregulatory mechanisms; 4) Immunopathogenic mechanisms in Autoimmune Endocrine Disease; 5) Cellular Mechanisms of Autoimmunity; 6) Models of Viral Induced Autoimmunity; 7 and 8) New Approaches to Immune Intervention. The conference will present a comprehensive view of both basic immune mechanisms related to autoimmunity, immunopathogenic mechanisms involved in the autoimmune process, and immune intervention. It will provide a forum in which both senior and younger scientists from diverse backgrounds will discuss autoimmunity. The FASEB Conference on Autoimmunity has been held every two years at Saxton's River and has proven to be an important vehicle for scientific dialogue related to autoimmunity.

The specific objective of this contract is to determine the optimal frequency for performing magnetic resonance imaging in untreated multiple sclerosis patients. The Contractor shall demonstrate that the recommended frequency of imaging will minimize missing important changes that may occur, avoid duplication of studies, and permit monitoring of disease activity, and quantification of progression in an expeditious and cost- effective manner.

This is a double-blind, placebo-controlled one year study of efficacy/safety of administration of bovine myelin to relapsing-remitting M.S. patients, in reducing frequency of M.S. attacks.

Experimental autoimmune encephalomyelitis (EAE) has long been studied as a model for multiple sclerosis. A major focus of research in this model has been the investigation of immunologic mechanisms to suppress the disease process. Oral tolerance is a classic mechanism for the induction of antigen specific peripheral immunologic tolerance, and we have been studying the suppression of the EAE by the oral administration of myelin basic protein (MBP) and myelin antigens. Such studies have direct clinical relevance as there currently are ongoing trials at our institution treating patients with early relapsing remitting multiple sclerosis via oral tolerization to myelin antigens. We have previously shown that oral administration of MBP leads to the generation of CD8+ T cells that adoptively transfer protection and suppress in vitro antigen specific responses. This effect appears to be mediated by an antigen nonspecific factor tentatively identified as TGF-beta which is secreted by CD8+ cells after being triggered by specific antigen. The specific aims of the revised proposal are as follows: 1) What are the characteristics of the cells involved in suppressing EAE following oral administration of MBP (in vitro studies)? 2) What are the in vivo mechanisms of bystander suppression? 3) What are the in vivo cellular mechanisms involved in generating oral tolerance? 4) Investigation of suppression in the relapsing Lewis rat EAE model and in adoptively transferred EAE.

The NOD mouse has been studied as a model for insulin dependent diabetes mellitus (IDDM). A major focus of research in this model has been the investigation of immunologic mechanisms to suppress the disease process. Oral tolerance is a classic mechanism for the induction of antigen specific peripheral immunologic tolerance, and we have been studying the suppression of experimental autoimmune diseases in a number of animal models, with a primary focus on experimental autoimmune encephalomyelitis (EAE). The oral administration of myelin basic protein (MBP) suppresses clinical and histologic manifestations of EAE and leads to the generation of CD8+ T cells that adoptively transfer protection and suppress in vitro antigen specific responses. The CD8+ T cells mediate their effect by the secretion of TGFbeta after being triggered by specific antigen. In the NOD mouse, we have found that oral tolerization to insulin delays the onset and reduces the incidence of diabetes over a one year period in animals administered 1 mg of porcine insulin orally twice a week for 5 weeks and then weekly until one year of age. As expected, orally administered insulin had no metabolic effect on blood glucose levels. The severity of lymphocytic infiltration of pancreatic islets was also reduced by oral tolerization to insulin. Furthermore, spleen cells from orally tolerized animals adoptively transfer protection against diabetes demonstrating that active cellular mechanisms are generated by oral insulin administration which suppress disease. These results raise the possibility that immunity to insulin may play a pathogenic role in diabetes in the NOD mouse and that oral tolerization to insulin may provide a new approach for the treatment of autoimmune diabetes. The present grant proposal will investigate basic mechanisms involved in suppression of diabetes by oral tolerization to insulin. The specific aims of the proposal are as follows: 1) Which portions of the insulin molecule trigger suppression of diabetes via oral tolerization? 2) Can protection of NOD mice from diabetes be enhanced by alternate dosing schedules or the use of adjuvants? 3) What are the characteristics of the cells generated which mediate suppression? 4) What are the in vivo mechanisms associated with disease suppression? 5) Does oral administration of other pancreatic antigens suppress diabetes?

The purpose of this protocol is to develop new laboratory tests in order to both diagnose and follow the course of multiple sclerosis patients. These tests could potentially lead to new treatments for multiple sclerosis.

We have previously studied oral tolerance to myelin basic protein (MBP) in the Lewis rat model of EAE. We have demonstrated that feeding MBP generates regulatory cells that suppress EAE by the release of TGF-beta after being triggered by the fed antigen. More recently, we have found that orally administered antigen also generates T cells that are of the Th2 type in that they secrete IL-4 after being triggered by the fed antigen. We have also found that depending on the dose of antigen fed, active suppression or clonal anergy is generated and can be demonstrated according to cytokine patterns. We have now initiated studies of oral tolerance in murine models of EAE which will allow better immunologic characterization of the mechanisms of oral tolerance. The following specific aim will be addressed: 1) In MBP-TcR transgenic mice, to what degree does active suppression, clonal deletion and clonal anergy occur following orally administered antigen? Three potential mechanisms of antigen driven peripheral tolerance can result following orally administered antigen. Investigations using MBP-TcR transgenic animals will allow a delineation of which of these pathways occur and the factors that influence one pathway over the other. We hypothesize that low dose oral antigen is processed through a unique mucosal pathway to activate regulatory T cells in Peyer's patches whereas high dose oral antigen leads to clonal anergy via systemic delivery of antigen following passage through the gut. The MBP-TcR animals will also allow us to determine to what degree clonal deletion occurs following oral antigen and whether anergy occurs locally in the gut in association with oral tolerance. 2) What are the relative roles of TGF-beta, IL-10 and IL-4 in mediating oral tolerance? Orally administered antigens generate regulatory cells which act by secreting suppressive cytokines following antigen specific triggering. The relative role of these cytokines in oral tolerance will be investigated using anti-cytokine antibodies administered in vivo, cytokine transgenic animals, and cytokine deficient animals. 3) What are the roles of costimulatory molecules in the induction of oral tolerance? A fundamental question related to oral tolerance is why orally administered antigen preferentially activates cells producing IL-4, IL-10 and TGF-beta. We hypothesize that T cells producing these cytokines require costimulatory signals qualitatively different from those required for Th1 cells and that the mucosal pathway of antigen presentation provides signals for activating Th2 and TGF-beta T cells, but anergize Th1 cells. This will be investigated using monoclonal antibodies to B7 and other costimulatory molecules and in B7 deficient mice. In summary, the studies outlined will help provide a basic understanding of the mechanisms of oral tolerance in murine experimental models.

This program project is in response to RFAAI-93-10 "Mechanisms of Oral Tolerization and Immunization." The purpose of the program project is to understand basic mechanisms of oral tolerance to autoantigens both in animal models and human disease states. The investigators have performed a series of experiments demonstrating that oral tolerance is effective in treating autoimmune diseases in animals which led them to conduct a series of human trials in multiple sclerosis and rheumatoid arthritis that demonstrated positive immunologic and clinical effects of oral tolerization. Though oral tolerization is a long-recognized method of inducing peripheral immune tolerance, the basic mechanisms underlying oral tolerance as applied to the treatment of human autoimmune disease remain to be defined. The theme of the program project is to define which mechanisms of peripheral immune tolerance as related to active suppression, clonal anergy, clonal deletion occur following oral tolerance both in animals and in man. Project 1 will focus on the role of cytokines IL-4, IL-10, and TGF/beta in the mediation of oral tolerance of that affect the initiation of oral tolerance using cytokine transgenic and knockout animals. The requirement for co-stimulatory molecules in initiating oral tolerance will be studied. The factors which lead to anergy vs. active suppressive will be investigated in MBP-TcR transgenic animals. Project 2 will characterize the T cells mediating oral tolerance, and investigate differential epitope recognition. This will be done by generating T cell clones and hybridomas from orally tolerized animals. In addition, immune effects following neonatal administration of antigen via the oral route will be studied. Project 3 will study oral tolerance to autoantigens in humans, determining the degree to which active suppression, clonal anergy and clonal deletion occur in multiple sclerosis patients orally tolerized to myelin antigens and in rheumatoid arthritis patients orally tolerized to type II collagen. In summary, the program project presents and integrated approach for the study of basic mechanisms of antigen-driven peripheral immune tolerance induced by orally administered autoantigens in animals and humans. The program project encompasses both basic science and clinical immunologic investigations that will increase our understanding of the mechanisms of oral tolerance so that they may be ultimately applied to human disease states.

The purpose of this study is to determine whether daily doses of LINOMIDE for 48 weeks have an beneficial effect on multiple sclerosis.

DESCRIPTION (Adapted from the Investigator's abstract): The application is a revised competitive renewal application of a grant in its 11th year which has focused on the study of immune dysregulation in multiple sclerosis (MS). Over the past ten years, the investigator has studied T-cell mechanisms in MS using functional and phenotypic analysis of T-cells, T-cell cloning and immune function related to treatment of the disease. In future years, Dr. Weiner wishes to study abnormal IL-12 and IFN-g regulation in MS. MS is postulated to be a TH1 type cell mediated autoimmune disease: IFN-g induces MS relapses and IFN-g is in the inflammatory lesions in the nervous system. Nonetheless, the regulation of IFN-g production in MS and its relationship to the clinical course are not defined. The investigator has found that anti-CD3 induced IFN-g is increased in MS and is not regulated by IL-10. In the revised application, new observations are reported linking IL-12 to increased IFN-g secretion in MS and an in vitro system has been developed which will allow the investigation of mechanisms associated with increased IL-12/IFN-g in MS. Furthermore, an initial link between response to therapy and decreased IFN-g production has been found, and a defect in IL-10 regulation of IFN-g production has been identified. The aims of the revised application will address the following questions: 1) what is the cell type that produces IFN-g? 2) what is the cell type that produces IL-12? 3) what are the ligand receptor interactions required for IL-12/IFN-g production? 4) what is the mechanism of defective regulation of IL-12/IFN-g secretion by IL-10? 5) what is the linkage of raised anti-CD3 induced IL-12/IFN-g to cytokine profiles of antigen specific cells? 6) is there a genetic linkage of increased anti-CD3 induced IL-12/IFN-g in first degree relatives? 7) is there any link between these findings and disease type and stage in patients followed serially and in response to therapy?

Oral tolerance is a long held observation that oral antigen results in systemic hypo-responsiveness. Recently, there is increased interest in oral tolerance following the demonstration that orally administered antigen can suppress several animal models of autoimmune disease. In addition, the oral administration of autoantigen is being tested in human autoimmune diseases and in transplantation. Nonetheless, a great deal remains to be learned about the basic mechanisms that underlie immune responses in the gut associated lymphoid tissues (GALT). Effector mechanisms of immune tolerance induced by oral antigen have been defined and relate to dose of protein fed. Low doses of protein favor induction of regulatory T cells that secrete Th2 cytokines and TGF-beta whereas higher doses induce anergy or deletion. The inductive events, however, that govern the generation of oral tolerance are not well understood and investigation of the inductive events in oral tolerance is the focus of this grant application. Based on our preliminary data we hypothesize that multiple factors contribute to the induction of regulatory cells in the GALT that mediate low dose oral tolerance including the unique micro-environment (cytokine milieu) in the gut, specialized antigen presenting cells (most likely B cells) that handle incoming antigen and the predominant expression of B7.2 on GALT antigen presenting cells. We also propose that these factors promote low affinity interactions between peptide-MHC-TCR leading to preferential generation of TGF-beta and Th2 responses. Feeding higher doses results in deletion or anergy in the GALT and at higher doses antigen leaks in the bloodstream and induces systemic tolerance. In our investigations, we will study altered peptides, which vary in their affinity for the TCR or MHC and will provide unique tools to address the issue of peptide-MHC-TCR affinity in the induction of oral tolerance. This together with studying oral tolerance in B-less mice and altering the expression of costimulatory molecules (B7.1 or B7.2) will allow us to define the role antigen, antigen presenting cells and costimulatory molecules in the inductive limb of oral tolerance. Thus the three specific aims of our proposal are: 1) How does the affinity of a peptide for binding to MHC II or the TCR affect the induction of regulatory cells following oral antigen administration? 2) What is the role of B cells in the induction of oral tolerance? and 3) What is the role of costimulation in the induction of oral tolerance? Addressing these questions will provide basic information regarding mechanisms of the induction or oral tolerance and immune responses in the gut associated lymphoid tissue.

The ultimate goal of immunotherapy in multiple sclerosis (MS) is to find immunologically specific, relatively non-toxic forms of therapy. Currently available immunomodulatory therapies have demonstrated the ability to modify disease outcomes in patients with relapsing remitting disease. Our understanding of the immunopathogenesis of MS implicates T cell activation as an important step in the pathogenesis of this disease. The CD40-CD40L pathway of T cell co-stimulation plays a role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE) the animal model of MS. Furthermore, CD40 was found in MS brain and evidence for the involvement of this co-stimulatory pathway in MS pathogenesis is accumulating. Treatment of animals with anti-CD40L suppresses EAE. initial safety studies in humans with anti-CD40L showed that this treatment could safely be administered to patients. Thus, our primary objective in this study is to evaluate in a pilot study two doses of anti-CD40L therapy versus placebo in patients with MS. Safety will be initially investigated in 5 individual patients who are treated with a single dose of anti-CD40L and evaluated by clinical and MRI criteria as well as safety blood tests. Dose requirements will be investigated in a randomized double-blind placebo controlled 2 dose study. The outcome measures will be progression on clinical disease scales, number of relapses, and time to sustained progression. Secondary outcome measures will include T2 lesion volume on MRI and number of GD enhancing lesions.

This is a randomized trial of two standard approaches to therapy of patients with multiple sclerosis remaining active despite therapy with beta-interferons. One arm receives cytoxan plus solumedrol plus continued beta-interferons, while the other receives only solumedrol plus beta-interferons. There will be 20 patients at BWH and 20 at MGH, stratified for disease severity assessed by number of gadolinium lesions.

The goal of this program is to use specific myelin antigens to alter differentiation of autoreactive T cells, and to study the cellular and molecular mechanisms by which myelin antigens or altered forms of myelin antigens may shift the balance of pro-inflammatory cytokines from responding autoreactive T lymphocytes. To this end, three projects have been constructed: Project 1 (Kuchroo, V.K. Project Leader) will study the mechanism by which altered peptides of myelin proteolipid protein can either inhibit or enhance the autoimmunity of the CNS using the EAE model. In this project native encephalitogenic and altered peptide ligand (APL) specific TCR transgenic mice will be utilized to understand the mechanism by which APLs can shift the cytokine balance. Project 2 (Weiner, H.L Project Leader) will study the effect of feeding myelin antigens, altered forms of myelin antigens or co-polymer (Copaxone ) on the regulation of EAE. In this project Dr. Weiner will study the effect of changing the affinity of the peptides that have been altered either at the MHC residue or at the T cell contact residue, and determine the effect of these altered peptides in inhibiting or enhancing oral tolerance. In addition, this project will study the mechanisms of induction of regulatory T cells (Th3 type cells that produce TGF-beta) that are induced following oral administration of antigen, and also will study enhancement by oral tolerance by oral cytokines (IL-4, IL-10, IFN- tau). Project 3 (Hafler, D.A. Project Leader) will examine the effect of APLs in the induction of regulatory cytokines from human MBPp85-99 reactive T lymphocytes. This project examines whether APLs induce a new population of Th2 autoreactive cells or if they differentially signal pathogenic T cells and alter their differentiation. In addition, this project will examine whether Copaxone acts as an APL for MBP reactive T cell using combinatorial peptide libraries. The use of combinatorial libraries will also be used to define preferential antigens recognized by Copaxone reactive T cells. Projects 1 & 3 will have a consortium with Dr. S. Burakoff's laboratory at Dana Farber Cancer Institute, Boston to study the intracellular signaling events in T lymphocytes following crosslinking of TCRs with cognitive versus APLs. An immunopathology core will support projects 1 and 2. The three interdependent projects address a common theme: development of antigen specific, non-toxic therapy for the treatment of the CNS autoimmune disease-MS. Study of basic mechanisms in animal models and immune responses in MS patients as outlined above will help understanding towards reaching this goal.

Oral tolerance is a long held observation that oral antigen results in systemic hypo-responsiveness. Recently, there is increased interest in oral tolerance following the demonstration that orally administered antigen can suppress several animal models of autoimmune disease. In addition, the oral administration of autoantigen is being tested in human autoimmune diseases and in transplantation. Nonetheless, a great deal remains to be learned about the basic mechanisms that underlie immune responses in the gut associated lymphoid tissues (GALT). This section of the program project grant will investigate the role of antigen affinity and cytokines in oral tolerance in the EAE model. Effector mechanisms of immune tolerance induced by oral antigen have been defined and related to dose of protein fed. Low doses of protein favor induction of regulator T cells that secrete Th2 cytokines and TGF-beta whereas higher doses induce anergy or deletion. The nature of the antigen and its interaction with the GALT has not been well studied. In addition, an understanding of the importance of Th2 cytokines in regulating oral tolerance and their use as enhancers has not been studied. Finally, we have described a new subset of T cells termed Th3 cells that are associated with oral tolerance and the nature of these cells has not been well characterized. We propose to study the factors that promote low affinity interactions between peptide-MHC-TCR leading to potential generation of TGF-beta and Th2 responses. In our investigations, we will study altered peptides (APLs), which have been generated by altering TCR or MHC contact residues of the encephalitogenic MPB and PLP peptides; these APLs will provide unique tools to address the issue of strength of signal in the induction of oral tolerance. We also have available MBP and PLP TcR transgenic mice to undertake these studies. Thus the three specific aims of our proposal are: 1) What is the effect of native, altered and cross-reactive peptides on oral tolerance? 2) What are the mechanisms associated with the induction of TGFbeta secreting (Th3 type) cells? And 3) What are the mechanisms associated with enhancement of oral tolerance by orally administered cytokines? Addressing these questions will provide basic information regarding mechanisms of the induction of oral tolerance and immune responses in the gut associated lymphoid tissue that will have implications for the use of oral antigen to treat autoimmune diseases such as multiple sclerosis.

immunopathology therapy; immunopathology diagnosis; clinical trials; biomarker; clinical research;

PROJECT SUMMARY (See instructions): The Administrative Core will be responsible for providing scientific administration and coordination, fiscal oversight and administrative support for this program project, keeping the Program a highly integrative and interactive consortium. The Core will coordinate travel arrangements and teleconference interactions for Program scientific group meetings among investigators at the Brigham and Women's Hospital and New York University School of Medicine. This will include annual program meetings for investigators to share data, plan future studies, and discuss how the Program will be optimally managed to enhance scientific communication and further collaboration. A similar effort will also be implemented for the Scientific Advisory Board. The Core will coordinate the administrative aspect of annual progress reports and renewal of subcontract agreements, and will liaison between the grant offices of each institution and the grant and contract officer at the Brigham and Women's Hospital. Dr. Howard L. Weiner will direct the Core with the support of the administrative coordinator, Ms. Shari Ori. The specific administrative functions of Core A are as follows: 1) Coordination of the scientific multi-tiered core with the four PPG projects; 2) Facilitate optimal interactions among all participants of the PPG; 3) Oversee educational commitments and opportunities for fellows and students; 4) Provide a measure of oversight for PPG through the Scientific Advisory Board; 5) Oversee internal quality control of ongoing research; 6) Management of day-to-day program activities and contractual agreements; 7) Oversee allocation of funds; and 8) Maintain fair, effective communication and cooperation among program investigators including resolution of disputes.

DESCRIPTION (provided by applicant): Our laboratory has been interested in vaccination strategies for non-infectious diseases using the mucosal immune system, an approach which is less invasive and more physiological than parenteral administration and which is clinically applicable. In addition, we have begun a series of studies to investigate immune mechanisms of Alzheimer's disease both in animal models and in humans. Immunizing transgenic APP mice which develop beta-amyloid (Abeta) deposition resembling plaques in Alzheimer's disease (AD) results in a decrease of amyloid burden. In a clinical trial using this approach, some patients developed a neurological complication consistent with meningoencephalitis. A case report in humans showed the presence of microglia immunoreactive for Abeta in the regions lacking Abeta deposits and investigators have reported a local reduction of senile plaques in a neocortical region affected by incomplete ischemia in a case of AD with stroke. In studies in which we investigated the ability to induce experimental allergic encephalomyelitis (EAE) in PDAPP mice we unexpectedly discovered that animals with EAE had clearance of amyloid from the brain. Furthermore, this was independent of antibody as we observed it in B-cell deficient animals and appeared related to the induction of activated microglia that led to the amyloid clearance. This led to a series of experiments in which we were able to demonstrate clearance of amyloid by nasal vaccination with glatiramer acetate (a drug approved by the FDA for the treatment of relapsing forms of multiple sclerosis) combined with a proteosome based adjuvant that has been shown to be safe in humans as part of a flu vaccine. Thus, we have discovered in animals a novel immune mechanism to clear amyloid in an antibody independent fashion using two drugs shown to be safe in humans. In the present grant proposal we will investigate the mechanism by which this occurs and the ability to use this treatment in a preventative paradigm. The specific aims of the proposal are: 1. What is the role of microglia in clearance of beta-amyloid in vivo. In this aim, we will investigate mechanisms by which our vaccination strategy activates endogenous microglia that in turn lead to efficient amyloid clearance. We will employ both in vitro and in vivo systems to isolate cell population. 2. What is the role of T-cells in beta-amyloid's clearance in APP Tg mouse model? In this aim, we will investigate the role of different T-cells subsets in activation of microglia towards amyloid clearance. 3. Immune approaches for long term mucosal immunotherapy to reduce Abeta deposition. In this aim, we will investigate long term mucosal strategy to prevent and treat APP Tg mice using unique mucosal adjuvant combination given nasally and their ability to enhance amyloid clearance.

Oral tolerance is classic method by which one can induced immunologic tolerance and it is now recognized to be an active immunologic process mediated by multiple mechanisms including active suppression, anergy, and deletion. Low doses of oral antigen administration favor induction of regulatory T cells whereas higher doses induce anergy or deletion. Over the past decade there has been increasing interest in oral (and mucosal) tolerance with the demonstration that orally administered antigen can suppress several animal models of autoimmune disease includingEAE. Furthermore mucosal antigen administration may have even broader biologic implications as a therapeutic modality for neurologic diseases including Alzheimer's disease and stroke. Human trials of oral tolerance have given mixed results and a great deal remains to be learned about the basic mechanisms that underlie immune responses in the gut associated lymphoid tissues (GALT) and both the inductive and effector mechanisms associated with oral tolerance. We will continue our investigation of oral tolerance as one of the projects in the program project grant and will address the following questions: 1. What is the role of T-bet transcription factor in the induction of mucosal tolerance? T-bet is a major regulator of Thl differentiation and we have found that T bet -/- animals have a phenotype analogous to that observed in low dose oral tolerance. We will investigate the role of T-bet in the induction of the different mechanisms of oral tolerance. 2. What is the role of T-bet and TIM molecules in TGF-0 producing Th3 type regulatory cells? We have shown that the gut is a unique environment that leads to the induction of TGF-p (Th3 type) secreting regulatory cells and cells which express LAP on their surface. We will investigate T-bet and TIM molecules in Th3 clones derived from the mesenteric lymph nodes of low dose orally tolerized animals. In addition, we have generated a TGF-P transgenic mouse in which the production of TGF-|3 is inducible in T cells, driven by the IL-2 promoter. These mice will be used to investigate the effector function of TGF-P secreting regulatory cells. 3. What are the mechanisms associated with enhancement of oral tolerance to glatiramer acetate and MOG by novel mucosal adjuvants that polarize towards Thl vs. Th2 responses . Mucosally active adjuvants may be crucial for the induction of immune responses of significant strength to be effective as immune therapy. We will investigate unique adjuvants that polarize towards Thl/Th2-Th3 responses when given orally in the investigation of glatiramer acetate, a immunomodulatory drug currently approved for the treatment of MS, and MOG an encephalitogenic myelin antigen.

DESCRIPTION (provided by applicant): (30 lines): This is a revised application. Oral tolerance is an important immunological phenomenon that refers to the suppression of immune responses to an antigen that has been previously administered by the oral route. It is believed to have evolved as a mechanism to avoid hypersensitivity reactions to food antigens. The mechanisms involved in oral tolerance induction have been described as active suppression by regulatory T cells, anergy and deletion. Some factors, such as age, are able to affect and modulate oral tolerance induction. Neonates are less susceptible and aging impairs the susceptibility to oral tolerance induction. Our groups have described that aging impairs oral tolerance induction but continuous feeding of the antigen is able to overcome this impairment. Moreover, aging affects oral tolerance induction but not its maintenance in mice. Other groups have shown that aging affects Peyer's patches function but the relationship between this and oral tolerance induction is still elusive. In this project our aim is to address the role of aging in oral tolerance induction using BALB/c mice at 4 different ages (6-8 weeks, 28-32 weeks, 53 weeks and 100 weeks). Oral tolerance will be induced either by single feeding or by continuous feeding and an experimental model of food allergy will also be used to test tolerance induction. We will address the following questions:1) How the immunological changes brought about by aging interfere with oral tolerance;2) How oral tolerance induced in young animals is maintained throughout their lives;3) Why continuous feeding is able to induce oral tolerance in aged animals. Our working hypothesis is that oral tolerance is impaired in aged animals due to a defect in de novo generation of regulatory T cells (Tregs) in the gut. We will test this hypothesis by flow cytometry analysis of Tregs (Th3, Tr1, CD4+CD25+ Foxp3+ and LAP+ T cells) and by the measurement of regulatory cytokines in the gut of young and aged mice. The ability of antigen presenting cells from aged versus young mice to induce Tregs in vitro will also be tested. This application has been revised in order to clarify the aims and to focus our analysis on the role of aging in the generation of Tregs. With the increasing number of elderly worldwide, studies on the impact of aging in natural immunological processes such as tolerance to dietary protein will help care for this population. (3 sentences): The decline in oral tolerance induction to food proteins during aging raises concern about possible allergic reactions in the older individuals upon oral exposure to novel dietary proteins. With the increasing number of elderly worldwide, studies on the impact of aging in these immune processes will help care for this population.

DESCRIPTION (provided by applicant): Multiple sclerosis is an autoimmune disease thought to be caused by a dysregulation between effector T cells, including Th1/Th17 T cells, and regulatory T cells. The factors associated with MS including disease onset, relapses and progression are poorly understood, although epidemiological studies have suggested a role for diet, seasonal variation, infection, and genetic factors. We hypothesize that MS may be linked to the gut microbiome, as it is now becoming recognized that the gut microbiome may play a key role in shaping the immune repertoire and the balance between various effector and regulatory T cell populations. Nearly 80% of all T lymphocytes of the body are compartmentalized to the gut associated lymphoid tissues (GALT. It is becoming increasingly recognized that the gut may have a major influence on the systemic immune system. Although to date, the interface between the gut microbiome has not been extensively studied, relationships between the gut microbiome and autoimmune illness have been found. Most studies have been carried out in inflammatory bowel disease, though the effect of gut microbiota is not restricted to local autoimmune processes. The fecal microbiota in patients with rheumatoid arthritis differs from healthy controls. Studies on the microbiome and MS have been few. Japanese investigators suggest that H. pylori is a potential protective factor against MS in Japanese populations and Bifidobacteria have been reported decreased in MS. In the EAE mouse model of MS, probiotic administration of lactobacillus reduces disease activity and depletion of microflora using antibiotics in inbred SJL and C57BL/6 mice impaired the development of EAE, and that this protection was associated with a reduction of proinflammatory cytokines, potentially suggesting a role for induction of peripheral tolerance through alterations of gut commensals. They also reported positive effects on EAE in animals treated with gut components orally. We believe an investigation of the gut microbiome in MS is timely, and have established collaboration with the Broad Foundation which has a major program related to the Human Microbiome Project. We will address the following Specific Aims: 1. Do patients with relapsing remitting MS have differences in the gut microbiome compared to healthy controls? 2. Do patients with secondary progressive MS have differences in the gut microbiome compared to healthy controls and to relapsing-remitting MS? 3. Is there a link between immune signatures as measured by antigen arrays and other immune measures in the blood of MS patients (oxysterols, osteopontin, heat shock proteins) and the gut microbiome? In summary, we believe that the investigation of the gut microbiome in MS fits with the R21 mechanism as it is exploratory and novel and seeks to break ground towards new directions and applications. It applies for the first time an important newly developing technology to gain potential basic insights into an autoimmune disease whose underlying pathogenesis remains unknown. PUBLIC HEALTH RELEVANCE: Multiple sclerosis (MS) is a disease of the nervous system that affects young adults. The body's immune system plays a key role in MS and it is now recognized that the large number of bacteria in the gut can affect the immune system. We will be studying the gut flora in MS patients to determine if the gut flora is different in MS patients and is in some way linked to the disease.

SUMMARY/ABSTRACT MicroRNAs are a growing family of conserved molecules involved in normal biological processes including cell growth and differentiation and pathological conditions such as cancer. MiR-124 is expressed specifically in the CNS but not other tissues and plays an important role in the regulation of the genes involved in differentiation of neurons. We found that miR-124 was expressed only in normal CNS-resident macrophages, but not in inflammatory macrophages or normal macrophages isolated from blood, spleen, bone marrow, peritoneal cavity or liver. We also found that overexpression of miR-124 deactivated inflammatory macrophages and converted them into microglia-like cells. Further analysis demonstrated that miR-124 inhibited macrophage activation by targeting CEBP¿, a transcription factor responsible for the differentiation of myeloid linage cells. We applied these new basic findings related to miR-124 to EAE which serves as a model of MS. We found that microRNA 124 (miR-124) is expressed in microglia cells but not in peripheral macrophages isolated from mice with EAE. Furthermore, we found that i.v. injection of liposomes containing miR-124 markedly suppressed clinical EAE and inhibited the infiltration of encephalitogenic T cells and inflammatory macrophages into the CNS. Based on these findings we hypothesize that brain-specific miR-124 mediates phenotype switching of inflammatory macrophages into a non-inflammatory phenotype, which results in suppression of autoimmune inflammation. Our data suggest that miR-124 provides a new avenue to understand basic mechanisms of microglia/monocyte biology and provides a novel therapeutic approach for diseases such as MS. We will address these specific aims in our proposal AIM 1: How does miR-124 affect the phenotype and function of macrophages? We will investigate how transfection of M¿ with miR-124 in vitro affects 1) polarization of macrophages into M1 or M2 types and secretion of pro- and anti-inflammatory cytokines; 2) the ability of M¿ to stimulate or inhibit proliferation and cytokine production by autoimmune T cells. AIM 2 : What is the role of miR-124 in promoting quiescence of microglia cells in the CNS microenvironment? We found that co-culture of macrophages with astroglial and neuronal cell lines deactivated macrophages and induced expression of miR-124 in these cells. We will utilize this co-culture system to investigate factors that result in upregulation in the expression of miR-124 in microglia/macrophages in CNS. AIM 3: What are the mechanisms of EAE suppression by peripheral administration of miR-124? We investigated which cell types are affected by i.v. injection of miR-124 and found that miR-124 directly affects macrophages but not DCs, or T and B cells. We will investigate whether suppression of EAE is related to induction of regulatory subsets of myeloid cells or T cells in vivo and will investigate the treatment of relapsing and progressive EAE mouse models with miR-124. 1

DESCRIPTION (provided by applicant): A major health problem relates to progressive cognitive decline with age and the threat of Alzheimer's disease (AD). We postulate, based on provocative new data, that the brain's innate immune system plays a central role in successful aging and resistance to AD. The role of the innate immune system in aging and AD has received limited study and is poorly understood. This is due in part to confusion regarding microglial cells and how they relate to the peripheral inate immune system. We discovered unique biomarkers and microRNA/gene signatures for resident microglia in the brain vs. recruited monocytes in both mice and humans. This advance presents a unique opportunity to determine the functions and dysfunctions of innate immune cells in brain aging and the development of AD. Hypothesis to be tested: We hypothesize that changes in the innate immune system with age play a major role in the development of diseases of aging that affect the human brain, including AD. We have termed this the Innate immunity hypothesis of brain aging. If our hypothesis is correct, it will change our understanding of the functions of microgli and macrophages in healthy aging and in AD and provide new therapeutic opportunities to treat AD by targeting these unique cell populations. We have established a comprehensive research strategy to test the key components of our hypothesis both in animal models and in aging human subjects. Aim 1. Investigate resident microglia/recruited macrophages in aging and AD mouse models. Using our unique markers that distinguish resident microglia from infiltrating monocytes, we will determine the gene/microRNA profiles of these two populations during aging and AD progression and study their interaction with A? oligomers. Aim 2. Target microglia and recruited monocytes as a treatment for AD in animal models. We will target cells of the innate immune system to treat animal models of AD based on our characterization of these cells. We will utilize our unique antibodies to microglia/ monocytes, antigomirs to unique microRNAs, and ligands of the TAM system. Aim 3. Investigate brain- resident microglia and monocytes during human aging and in AD. We will study two unique human cohorts. 1. We will investigate pathologic specimens from an aging brain cohort in collaboration with David Bennett (Rush Memory and Aging Project). We will perform immunopathological characterization of microglia and recruited monocytes in healthy aged brains and those with typical AD and correlate changes in microglia with changes that occur in aging. 2. We will investigate the relationship between A? deposition in the brains of an aging human population (measured by PiB imaging) and the innate immune system in the peripheral blood. We will characterize monocytes in the blood from an aging cohort (under NIH study by Dr. Reisa Sperling at Brigham and Women's Hospital) in whom PiB imaging and cognitive assessment is being performed. This will provide a direct link between the peripheral innate immune system and brain changes with aging. PUBLIC HEALTH RELEVANCE: There is decrease in brain function with aging that leads to cognitive decline and in some instances Alzheimer's Disease (AD). We hypothesize that the innate immune system plays a major role in aging and the development of AD. We will study this relationship to develop treatments to prevent AD and cognitive decline with aging.

DESCRIPTION (provided by applicant): This application is in response to an NIH FOA inviting applications for projects to identify and qualify extracellular RNA (exRNA)-based biomarkers derived from human body fluids to diagnose and monitor disease progression and response to therapy. We propose to identify and validate candidate circulating miroRNAs in well-defined multiple sclerosis (MS) patient populations. Studies using existing human biospecimen collections were strongly encouraged by the NIH FOA and for the UH2 phase we will take advantage of a unique resource termed CLIMB (Comprehensive Longitudinal Investigation of Multiple Sclerosis at Brigham and Women's Hospital). CLIMB is a cohort of over 2000 MS patients that began enrollment 12 years ago and includes yearly blood samples, clinical exams and quantitative MRI imaging. We present preliminary data that we can indeed measure circulating miRNAs in our MS cohort and have found links to disease stage, response to therapy and disability. For the UH3 phase we have identified valuable sources of blood samples from both academic and pharmaceutical collaborators that will be used to validate our UH2 findings and provide the basis for the use of circulating miRNAs in the clinic. SPECIFIC AIMS FOR UH2 PHASE: 1) Identify miRNAs that will act as diagnostic biomarkers by comparing MS patients to healthy controls, patients with other neurological diseases and patients with other autoimmune diseases. 2) Identify disease stage miRNA biomarkers by comparing RRMS patients, SPMS patients and PPMS patients. 3) Identify prognostic miRNA biomarkers by determining the change in each miRNA over two years and assessing which miRNAs are sensitive to long-term change in disease status. 4) Identify treatment response miRNA biomarkers by comparing the baseline miRNA expression in responders and non- responders to treatment. 5) Identify disability miRNA biomarkers by correlating miRNAs with EDSS and MRI measures of disease status. SPECIFIC AIMS FOR UH3 PHASE: Circulating miRNAs identified in the UH2 phase will be extensively studied and we will address the following two aims: 1. Validation of miRNA biomarkers identified in the UH2 phase in well defined independent patient cohorts. 2. Determine which miRNAs identified in the UH2 phase can be used as preclinical disease biomarkers. We will take advantage of well characterized serum samples from a number of sources including: The SUMMIT International MS Consortium, the NIH sponsored CombiRx clinical trial, the Biogen Idec sponsored IMPACT trial, the Swedish EIMS epidemiologic investigation of MS, the Canadian Pediatric Demyelinating Disease Study and the GEMS (Genes and Environment MS study).

DESCRIPTION (provided by applicant): This is a revised PPG application to build a better understanding of T cell mechanisms in autoimmunity that has stemmed from the discovery of critical regulators of both T effector and T regulatory cells. We have discovered that IL-27 and the transcription factor aryl hydrocarbon receptor (AhR) play central roles in immune responses in animals and humans and these pathways impinge on all aspects of immune function, both adaptive and innate and include Tregs, Th17 cells and dendritic cells. Little is known about these new pathways, which appear crucial in CNS inflammation and have direct relevance to diseases such as multiple sclerosis. The primary goal of this program project grant (PPG) is to identify molecular mechanisms of how IL-27 inhibits effector T cells and induces regulatory Tr1 cells with a focus on two transcription factors cMaf and AhR, both of which are induced by IL-27 in responding Tr1 cells. The investigation of the inter-relationship of cytokines and transcription factors in both animal models and MS patients will allow a better basic understanding of immune abnormalities in MS and allow both the development of more specific immunomodulatory therapy and a mechanistic understanding of current therapies being used to treat MS. IFN¿, a widely used first line therapy in MS has been shown to act in part by the induction of IL-27, underscoring the importance of studying this pathway in CNS autoimmunity. This PPG brings together four investigators, each with unique strengths and expertise to investigate this new area. Project 1: (Kuchroo) Transcriptional regulation of effector and regulatory T cells in EAE; Project 2: (Littman) Transcriptional network analysis of Tr1 cells. Project 3: (Quintana) Role of AhR in the control of the innate immune response during the development of EAE. and Project 4: (Weiner) Role of IL-27 and AhR in the regulation of human effector and regulatory T cells in healthy subjects and MS patients. The revised PPG will establish a Multi-tiered Expression Analysis Core consisting of two components: Project 2 will undertake expression analysis using RNA-seq and ChlP-seq technologies and Core B will undertake multiplex expression analysis using the latest digital Nanostring technology to analyze the expression of transcripts in subsets and rare populations. The core will service all projects. An Administrative Core will coordinate the various administrative aspects of the PPG and a Scientific Advisory Board has been assembled to provide scientific oversight.

PROJECT SUMMARY (See instructions): MS is a chronic autoimmune disease of the CNS in which pro-inflammatory T helper 17 (Thi7) cells are increased. Tri type regulatory T cells (Tregs) are important in peripheral tolerance, act via IL-10, and are deficient in MS. The basic immune mechanisms underlying abnormalities in Teffector/Tregulatory networks in humans and patients with MS are not well understood. We found an important role for IL-27 and the aryl hydrocarbon receptor (AhR) in these networks and the focus of this portion of the Program Project Grant is to investigate these pathways in humans and MS patients. In this revised application we have included the study of other disease controls and the study of cells within the CNS. We will address the following aims: Aim 1: Role of IL-27 in the regulation of Th17 and Tr1 cell differentiation in humans. IL-27 plays an important role in inducing IL-10 from murine and human T cells. In addition, we found that AhR activation induces regulatory Tri-like cells both in mice and humans. Furthermore, we found that IL-27 derived Tri cells produce IL-21 which acts as a autocrine growth factor for both Tri and Th17 cells in mice. We will investigate the role of AhR/cMAF signaling in the differentiation of human Tri cells triggered by IL-27 or IL- 27 plus TGFB. We will also study the production and role of IL-21/1L-2 in IL-27 or IL-27 plus TGFB induced Tri cells. Aim 2: Role of AhR in innate immunity. We have shown that AhR induces tolerogenic DCs in mice that promote regulatory T cell differentiation and that IFN-p increases expression of IL-27 in murine DCs which increases the expression of AhR in murine T cells. We will investigate the effect of AhR activation in DCs in humans and determine the synergistic effect of AhR activation in the presence of IFN-p on the function and activation of human mDCs. Aim 3: IL-27 and AhR in Th17/Tr1 cells and DCs in Multiple Sclerosis. Both AhR and IL-27 signaling are involved in induction of Tri cells and tolerogenic DCs. We will investigate AhR /IL-27 signaling and how it influences Th17 responses in MS, its role in the differentiation of naive T cells to Tri cells and in the induction of tolerogenic DCs in MS patients. We will also investigate how these pathways differ in the various stages of MS and how are they are affected by response to immunotherapy in MS patients.

DESCRIPTION (provided by applicant): The suppression of the immune system is one of the major obstacles in the treatment of glioblastoma (GBM). Regulatory immune cells, such as FoxP3+ T lymphocytes and suppressive myeloid cells play a significant role in this process. LAP+ CD4+ T lymphocytes were recently described as superior regulatory cells in colorectal cancer and we found that other LAP+ immune cells may also have regulatory functions. Latency-associated peptide (LAP) is a derivative of the TGF-?ene and TGF-?s strongly implicated in GBM pathogenesis. Based on TCGA (The Cancer Genome Atlas) data, we found that TGF-?mRNA expression negatively correlates with human patient survival. We also found that different LAP+ immune cells infiltrate intracranial GBM, including subsets of both ? and ??T cells, macrophages and dendritic cells in a mouse model. Treatment of mice bearing sub-cutaneous GBM with anti-LAP antibodies led to elimination of GBM growth and was associated with a reduction of FoxP3+ and LAP+ T cells and up-regulation of the Th1 and cytotoxic T cell responses. Based on these observations, we hypothesize that cells expressing LAP suppress GBM-specific immunity and neutralization with anti-LAP antibodies will activate a tumoricidal immune response. In this project, we propose to study the role of neutralizing antibodies against LAP in overcoming the immunosuppression associated with GBM. Our specific aims are: Specific Aim 1: Investigate the immunosuppressive role of LAP in GBM. We propose to 1) determine the levels of LAP expression on immune cells in normal and GBM conditions in mouse and human; 2) define the phenotype of LAP+ immune cells infiltrating intracranial GBM isolated at different stages of disease progression in mouse and human; 3) perform functional analysis of LAP+ regulatory immune cells isolated from intracranial GBM. Specific Aim 2: Evaluate the therapeutic potential of anti-LAP antibodies in the treatment of a GBM intracranial mouse model. We propose to 1) characterize the effects of anti-LAP antibodies on the immune response in naive mice and mice bearing intracranial GBM; 2) assess the therapeutic value of anti-LAP antibodies in the experimental GBM models.

DESCRIPTION (provided by applicant): Neuro-inflammation and immunosuppression have a significant impact on glioblastoma (GBM). Macrophages infiltrate brain tumors, undergo modulation by the GBM microenvironment and promote the suppression of tumor-specific immunity. Signaling through the ligand-activated transcription factor Aryl Hydrocarbon Receptor (AHR) has strong effects on the regulation of the immune response and has been recently implicated in the suppression of GBM-specific immunity. However the biological mechanisms by which AHR regulates the immune response to GBM and the potential of AHR as a therapeutic target for GBM are unknown. We found that AHR controls the recruitment of inflammatory macrophages, also called glioma- infiltrating macrophages, to GBM. Moreover, we found that the specific deletion of AHR in macrophages significantly slows GBM growth. Based on these findings, we hypothesize that the AHR modulates glioma-infiltrating macrophages that suppress GBM-specific immunity. In this project, we propose to study the role of AHR in GBM-infiltrating macrophages. Our specific aims are: Specific Aim 1: Investigate the mechanism by which AHR controls immunosuppressive macrophages in GBM. We propose to 1) study the differential contribution of AHR signaling in macrophages and microglia on GBM immunosuppression; 2) investigate the transcriptional effects of AHR signaling in macrophages and microglia in GBM. Specific Aim 2: Treatment of a GBM model by targeting AHR with nanoparticles carrying AHR inhibitors. We propose to 1) investigate the effects of nanoparticles loaded with an AHR inhibitor on glioma- infiltrating macrophages in vitro; 2) determine the therapeutic effects of nanoparticles in an experimental GBM model.

DESCRIPTION (provided by applicant): One of the major efforts currently being explored to understand the immune system in health and disease is investigation of the human microbiome. Numerous studies in animal models of MS implicate the microbiota of the gut in triggering CNS autoimmunity. We have had a long-term interest in the gut immune system and we reembarked on a pilot study to determine if differences in the gut microbiome existed in MS patients and whether any changes in the gut microbiome occurred in association with treatment. We found a significant increase in abundance of organisms from the Archaea kingdom in MS and changes in the gut microbiome associated with treatment. To our knowledge, this is the first demonstration of changes in the gut microbiome in MS. The focus of our investigations are to expand the numbers of patients studied, include patients with the progressive form of the disease, to study patients in a longitudinal fashion before and after treatment, and to perform immunologic investigations to understand the manner in which Archaea affects immune function in MS. We hypothesize that changes in the gut microbiome are linked to susceptibility and immune changes that occur in MS as well as response to treatment. Aim 1. Investigate the gut microbiome in untreated MS subjects including CIS, relapsing- remitting and secondary progressive MS. Aim 2. Investigate the effect of treatment on the gut microbiome in MS. Aim 3. Investigate the linkage of Archaea in the gut microbiome of MS patients to immune function. To perform our studies, we will take advantage of the large cohort of well characterized MS patients followed at the Partners MS Center as part of the CLIMB longitudinal observational study and the Center for Clinical and Translational Metagenomics at the Brigham and Women's Hospital.

DESCRIPTION (provided by applicant): This application is in response to an NIH FOA inviting applications for projects to identify and qualify extracellular RNA (exRNA)-based biomarkers derived from human body fluids to diagnose and monitor disease progression and response to therapy. We propose to identify and validate candidate circulating miroRNAs in well-defined multiple sclerosis (MS) patient populations. Studies using existing human biospecimen collections were strongly encouraged by the NIH FOA and for the UH2 phase we will take advantage of a unique resource termed CLIMB (Comprehensive Longitudinal Investigation of Multiple Sclerosis at Brigham and Women's Hospital). CLIMB is a cohort of over 2000 MS patients that began enrollment 12 years ago and includes yearly blood samples, clinical exams and quantitative MRI imaging. We present preliminary data that we can indeed measure circulating miRNAs in our MS cohort and have found links to disease stage, response to therapy and disability. For the UH3 phase we have identified valuable sources of blood samples from both academic and pharmaceutical collaborators that will be used to validate our UH2 findings and provide the basis for the use of circulating miRNAs in the clinic. SPECIFIC AIMS FOR UH2 PHASE: 1) Identify miRNAs that will act as diagnostic biomarkers by comparing MS patients to healthy controls, patients with other neurological diseases and patients with other autoimmune diseases. 2) Identify disease stage miRNA biomarkers by comparing RRMS patients, SPMS patients and PPMS patients. 3) Identify prognostic miRNA biomarkers by determining the change in each miRNA over two years and assessing which miRNAs are sensitive to long-term change in disease status. 4) Identify treatment response miRNA biomarkers by comparing the baseline miRNA expression in responders and non- responders to treatment. 5) Identify disability miRNA biomarkers by correlating miRNAs with EDSS and MRI measures of disease status. SPECIFIC AIMS FOR UH3 PHASE: Circulating miRNAs identified in the UH2 phase will be extensively studied and we will address the following two aims: 1. Validation of miRNA biomarkers identified in the UH2 phase in well defined independent patient cohorts. 2. Determine which miRNAs identified in the UH2 phase can be used as preclinical disease biomarkers. We will take advantage of well characterized serum samples from a number of sources including: The SUMMIT International MS Consortium, the NIH sponsored CombiRx clinical trial, the Biogen Idec sponsored IMPACT trial, the Swedish EIMS epidemiologic investigation of MS, the Canadian Pediatric Demyelinating Disease Study and the GEMS (Genes and Environment MS study).

PROJECT SUMMARY/ABSTRACT: Microglia are resident myeloid-lineage cells in both the CNS and in the eye and function in the maintenance of normal tissue. Retinal microglia can become activated and/or dysregulated during disease, and thus affect disease progression in retinitis pigmentosa. Understanding the biology of microglia is a challenge due to absence of markers and molecular microglia signatures. Recently, we identified a homeostatic molecular microglia signature which provides new tools for investigating retinal microglial biology and the possibility of targeting retinal microglia for the treatment of retinitis pigmentosa. Using our new microglial markers, we investigated microglia in the rd1 murine models of RP. We found increased numbers of resident microglia but no infiltration of monocytes in the retinal. Most importantly, we found that intravitreal transfer of microglia from animals with RP into normal animals, resulted in photoreceptor loss. Consistent with this, intravitreal transfer of retinal microglia from normal animals into animals with RP reduced photoreceptors loss. We hypothesize that in RP, microglia proliferate and acquire a cytotoxic phenotype mediated by intrinsic activation of the TREM2-APOE pathway which suppresses microglia homeostatic molecular properties and leads to uncontrolled chronic inflammation and photoreceptors damage. Treatments aimed to target microglia by suppressing the TREM2-APOE pathway is associated with activation of both the TGF? pathway and MERTK which abrogates the inflammatory microglial phenotype and restores retinal microglial homeostatic properties. This provides a new direction for studying RP and development of novel therapies that target microglia. We believe that an innovative feature of our approach is that it is a mutation- independent approach that applies to RP independent of the genetic mutation. In addition, there is a translational aspect to the proposed work as we will investigate human eyes with our recently described microglial antibodies. We will address the following specific aims: Aim 1. Identify molecular pathways affected in retinal microglia in mouse models and human RP. Aim 2. Target the TREM2-APOE-SPP1 pathway to inhibit MGnD-cytotoxic microglia in rd mice. Aim 3. Restore M0-homeostatic microglia via TGF?1-MERTK signaling in rd mice.  

PROJECT SUMMARY/ABSTRACT This is a revised application. Neuroinflammation has been identified as a major contributing factor to the pathogenesis of Alzheimer?s disease (AD). Studies showing activated microglia surrounding A? plaques in the postmortem AD brain suggest significant involvement of inflammatory pathways in disease progression. PET imaging of AD patients with radio labeled tracers for microglial activation has confirmed these findings. Neuroinflammation involvement of microglia has also been observed in animal models used to study AD. However, the investigation of microglia in AD has been hampered by the lack of specific molecules for understanding microglial phenotype and function and equally as important there is the lack of therapeutic approaches that can target microglia and neuroinflammation. We hypothesize that neuroinflammation is an important target for developing new treatments for AD and that by dampening microglial neuroinflammation with our novel approach (nasal anti-CD3) we will be able to treat AD. We found that nasal anti-CD3 induces an anti-inflammatory immune response that attenuates neuroinflammation in brain-resident microglia cells. In new preliminary data, we found that nasal anti-CD3 improved short-term memory in an AD mouse model. We have found that nasally administered anti-CD3 localizes to cervical lymph nodes where it induces IL-10 secreting T cells that then migrate to the brain and suppress neuroinflammation in microglia. Thus, nasal administration of anti-CD3 is a unique and unexplored target for treating neuroinflammation in AD. The objective of this proposal is to obtain investigate the potential of nasal anti-CD3 to dampen microglia neuroinflammation as a treatment strategy in AD. Aim 1. Investigate the effect of nasal anti-CD3 on microglia phenotype and phagocytic function in aging. Nasal anti-CD3 will be given to 24-month old mice to assess the effect of treatment on in vivo microglia phagocytic function and restoration of microglia homeostasis. We will also measure the induction of IL-10- secreting T cells in the periphery of these mice, which will serve as an easily measured biomarker and on the role of IL-10 on mediating the effect of nasal anti-CD3 on microglia function in aging. Furthermore, we will use IL-10Rflox/floxCX3CR1Cre conditional knockout mice, which do not express the IL-10 receptor on microglia to investigate the role of IL-10 in microglia phagocytic properties as well as its ability to degrade Ab plaques. Aim 2. Investigate the effect of nasal anti-CD3 in the 3xTg AD model. We will perform detailed behavior analysis and neuropathology on 3xTg AD mice treated with nasal anti-CD3 to determine the biological effect and therapeutic potential of anti-CD3. Moreover, we will investigate the effect of nasal anti-CD3 treatment on microglial neuroinflammation in this mouse model of AD using high throughput RNA sequencing (Smart-Seq2).

PROJECT SUMMARY ALS is a neurodegenerative disease affecting motor neurons with limited treatment options and a median survival of 3-5 years. We submit a significantly revised proposal that addresses the comments of the reviewers and provides new preliminary data. Our hypothesis is that the intestinal microbiome and its metabolites play an important role in in ALS by modulating peripheral and CNS immunity and by affecting ALS disease pathways. Our hypothesis is strengthened by a recent paper in Nature by Blacher showing an important role of the gut microbiome and metabolites in ALS. In new preliminary data we show: 1) Antibiotics that worsen survival also downregulate microglia homeostatic genes while upregulating inflammatory genes. 2) Changes in the microbiome in 68 ALS patients vs. 61 healthy controls (largest microbiome study to date), including a decrease in butyrate producing bacteria E. rectale and R. intestinalis, are robust when controlled for ALS clinical confounders. 3) Administering these bacteria or Akkermansia reverses SOD1-disease associated transcriptional changes in the spinal cord; including Fus, Oxr1, and Smn1, and protein degradation (Ubqln1). 4) Transferring SOD1 microbiota to WT mice modulates microglia pathways involved in ALS related to RNA processing (Fus), protein degradation (HSPa1b and USP2) and lysosomal transport (CD68 and Lyz2). We believe there is compelling evidence to support the investigation of the microbiome in ALS. We will address these aims: AIM 1. Which microbial components are associated with protection in SOD1 and TDP-43 models? We will deplete the microbiota with specific low-dose antibiotics, orally administer Akkermansia components and a unique micro-RNA and identify brain, serum, and stool metabolites associated with disease protection. AIM 2. Which human microbiota components contribute to disease pathogenesis? It is unknown whether the ALS microbiota can also drive disease pathogenesis. We will transfer microbiota from patients with ALS to the SOD1 and TDP-43 models and measure motor function and survival time. We will identify microbial populations, functions, and metabolites that are associated with protection or worsening of disease. We will also confirm and expand our findings in the ALS microbiome in a newly recruited cohort. AIM 3. Investigate the immune mechanisms by which the gut microbiota modulates disease progression in ALS animal models. We will sort microglia, monocytes, and T cells from mice treated with individual antibiotics, colonized with ALS microbiota, or specific bacterial strains and characterize transcriptional signatures by RNAseq. Utilizing WT mice to investigate how the microbiota affect microglia, we will transfer specific microbes identified associated with ALS and screen for changes in genes involved in ALS pathogenesis.