Noel R. Rose - US grants

Two newly developed experimental systems of organ-specific autoimmune disease will be used to study the age-related changes in T cell regulation of murine autoimmunity. One method is based on induction of autoimmune disease in selected strains of mice by neonatal thymectomy (NTx). In the other method, athymic nude mice are reconstituted with particular T cell subsets from spleens of H-2 matched euthymic mice. Thyroiditis, gastritis with macrocytic anemia, and oophoritis are produced by both methods, depending upon the strain of mice used. In both experimental systems, regulatory T cell subsets seem to play a primary role in maintaining self-non-self discrimination.

During the past four years, H-2 linked control of the autoimmune response of mice to murine thyroglobulin (MTg) was worked out in detail. During the next grant period, we plan to examine the role of non-H-2 genes, especially those concerned with immunoglobulin synthesis, define the idiotype population engaged in this autoimmune response, identify the idiotypes that are related to the pathogenic manifestations, correlate the production of the major pathogenic idiotypes with particular populations of anti-idiotypes and regulatory T-cells, and develop methods for arresting production of the harmful idiotypes. Ultimately the studies of murine thyroiditis will be correlated with findings in human cases of chronic lymphocytic thyroiditis.

The etiology of human disease such as insulin-dependent diabetes mellitus, multiple sclerosis, idiopathic thrombocytopenic purpura, chronic aggressive hepatitis and post measles encephalitis have all been related to possible infection followed by autoimmune reactions. The availability of heart-specific autoantibodies generated in the course of Coxsackie B3 infection of certain strains of mice provides us with a unique opportunity to evaluate the oft-stated hypothesis that autoimmunity contributes significantly to post-infectious myocarditis. With these autoantibodies we can identify and isolate the target antigens and test their ability to engender an autoimmune response under experimental conditions. Initially, we will characterize the tissue reactivity of the CB3-induced autoantibodies by immunofluorescence analyses. Characterized antisera will be used in the identification of the heart specific autoantigens. These antigens will be solubilized from heart homogenates and sarcolemmal-subsarcolemmal vesicle preparations and then electrophoresed on acrylimide gels. Once identified by immunodetection of Western blotted myocardial antigens, heart specific serological reagents (i.e., xenoantiserum, alloantiserum and monoclonal antibodies) will be prepared from these enriched antigens. The reactivity of these serological reagents will be compared with the heart specific autoantibodies (antiserum and monoclonal antibodies) from CB3 infected animals. We will examine the mechanisms for initiating the post-infectious autoimmunity using these serological reagents and purified autoantigens. In vitro and in vivo experiments will determine whether an autologous antigen, a cross-reacting antigen or an anti-id antibody are the major stimuli of the post-infectious autoimmune myocarditis. Experiments will be designed to study disorders of the normal immunoregulatory mechanisms involved in autoimmune myocarditis. These investigations can be directly related to clinical post-infectious myocarditis and lead to more rational modes of prevention or treatment of these cardiopathies.

The long term goals of this project are to delineate the mechanisms of autoimmune disease. The murine experimental autoimmune thyroiditis model of an organ specific autoimmune disease will be used in this study. There are two specific aims in this proposal. 1. During organ specific autoimmune disease, self-tolerance is lost and autoantibodies to indiginous antigens arise through, as yet, an unknown mechanism. There are two possible pathways through which auto Ab may arise. In the germline immunoglobulin variable gene repertoire, there exist autoreactive V genes. Pathologic autoantibodies may be derived from these autoreactive, germline encoded V genes. Alternatively, autoreactive antibodies could arise from non-autoreactive germline V genes as a result of post- immunization somatic mutation. The first pathway could be explained totally by a loss of immunoregulation without the need of an antigen driven maturation process. The second pathway would necessitate an the presence of antigen to drive maturation, however, the process may be initiated by a unrelated antigen. We therefore, plan to clone, using recombinant DNA technology, the V genes of anti-mouse thyroglobulin (MTg) Ab developing during the disease process. The selection of V genes used will be compared to that of "natural" Ab that are autoreactive with MTg. The results from these experiments could point to one these pathways and give insight into the processes which induce autoimmune disease. 2. In the EAT model, disease can be easily induced by immunization with mouse thyroglobulin. The specific determinants involved in this induction process are not known. By chemical or enzymatic cleavage of MTg, we propose to define specific peptide sequences which are pathogenic or which can induce protective immunosuppression. Using the DNA probes derived from the amino acid sequence of these peptides, the location of these sites within the MTg molecule will be determined. Furthermore, we will attempt to distinguish determinants associated with development of disease from those which are involved in inducing autoantibody production alone. These finding would further our understanding of the nature of autoantigenic determinants.

Human dilated cardiomyopathy is responsible for approximately 10,000 deaths in the United States annually. Endomyocardial biopsy has shown that 20-30% of cardiomyopathy patients have active myocarditis. Our studies of murine Coxsackievirus B3(CB3) myocarditis have revealed many similarities between the lats-phase myocarditis in mice and human myocarditis. Both share similar pathology patterns of inflammation and are associated with circulating heart-specific autoantibodies. The disparity between the severe cardiac dysfunction in myocarditis, and the small amount of myocyte necrosis, highlights the need to define the pathogenic mechanisms responsible for myocarditis. Recently, we have discovered that a major myocardial autoantigen in murine myocarditis was the heavy chain of cardiac myosin. We have thus developed a second model of autoimune myocarditis by immunizing mice with purified cardiac myosin. Immunization with cardiac myosin produces severe myocarditis similar in appearance, time course and genetic predisposition pattern to late-phase myocarditis induced by CB3. Our hypothesis is that late-phase, post-CB3 myocarditis is an autoimmune response to unique epitopes on cardiac myosin heavy chain. It is our goal to use both murine models of myocarditis to elucidate the initiation and pathogenesis of autoimmune myocarditis. The work proposed herein will initially determine the sequence of events leading to myosin-induced autoimmune myocarditis, with respect to both the roles of antibody and T-cell-mediated immunity. We will then compare the immunopathology and antibody specificity of the two models of myocarditis. Passive transfer with antibody and adoptive transfer of myocarditis with T cell populations will be performed in both models. Since immunization with cardiac myosin produces severe myocarditis while immunization with skeletal myosin does not, the epitopes unique to cardiac myosin will be investigated. Finally, we will determine whether cardiac antigens other then myosin heavy chain may contribute to the development of autoimmune myocarditis. The addition of a simple model of myocarditis due to a known specific cardiac antigen allows us the unique opportunity to define the relative roles of humoral and cell-mediated immunity in myocarditis. lt is anticipated that these studies will form a rational basis for the development of successful therapies for myocarditis and possibly prevent the development of post-viral cardiomyopathy.

Funds are requested to support the 1991 Second International Symposium on Immunologically Mediated Heart Disease, to be held from 5-8 May 1991 at the Airlie House, Airlie, Virginia. This symposium will gather an international group of scientists and clinicians actively engaged in cardiac immunology research. Research in the field of immune-mediated myocarditis recently has yielded unique insights into the pathogenesis of cardiomyopathy. A scarcity of animal models of cardiomyopathy and the clinical impact of over 200,000 new cases each year in the United States highlights the substantial contributions which the study of myocarditis can make towards the understanding of the basic mechanisms of heart failure. The primary goals of this symposium include the assimilation of progress in animal models of myocarditis and cardiomyopathy with particular attention to mechanisms of cardiac dysfunction produced by the interaction of circulating lymphokines (cytokines) and immunoglobulins on cardiac membrane receptors and ion channels. Direct and indirect viral contributions to cardiac injury leading to cardiomyopathy as well as the development of strategies for new non-invasive diagnostic approaches will also be highlighted. The symposium will invite speakers from other fields in order to promote new research strategies for the 1990's. In addition, the proceedings of the Symposium will be published by Springer-Verlag thereby allowing this information to be widely available to those interested in this interdisciplinary approach to the study of cardiac immunology and cardiomyopathy.

Myocarditis is an important cause of heart failure among adolescents and young adults. While about 33 percent of myocarditis patients recover, the remainder may develop chronic dilated cardiomyopathy which accounts for 25 percent of all heart failures in North America. Coxsackievirus B3 (CB3) infections have been implicated in about 40 percent of myocarditis cases. Antiviral drugs and immunosuppressive therapies have given mixed results, requiring further research in order to develop effective therapies. Studies of Coxsackievirus-induced myocarditis in mice have provided valuable information about the role of the adaptive, acquired immune response to CB3 in the development of autoimmune myocarditis. Recently, there has been renewed interest in how the innate immune response to infection may affect the development of adaptive immunity. However, very little research has been conducted on how the early, innate immune response to Coxsackievirus infection effects the development of autoimmune myocarditis. The aim of this proposal is to delineate the role of "early" cytokines and immune cells involved in innate immunity in the development of acute and chronic CB3-induced myocarditis. We hypothesize that the initial innate immune response to CB3 determines whether the adaptive immune response leads to the later development of autoimmune myocarditis.

Myocarditis is an important cause of heart failure among adolescents and young adults. While about 33 percent of myocarditis patients recover, the remainder may develop chronic dilated cardiomyopathy which accounts for 25 percent of all heart failures in North America. Coxsackievirus B3 (CB3) infections have been implicated in about 40 percent of myocarditis cases. Antiviral drugs and immunosuppressive therapies have given mixed results, requiring further research in order to develop effective therapies. Studies of Coxsackievirus-induced myocarditis in mice have provided valuable information about the role of the adaptive, acquired immune response to CB3 in the development of autoimmune myocarditis. Recently, there has been renewed interest in how the innate immune response to infection may affect the development of adaptive immunity. However, very little research has been conducted on how the early, innate immune response to Coxsackievirus infection effects the development of autoimmune myocarditis. The aim of this proposal is to delineate the role of "early" cytokines and immune cells involved in innate immunity in the development of acute and chronic CB3-induced myocarditis. We hypothesize that the initial innate immune response to CB3 determines whether the adaptive immune response leads to the later development of autoimmune myocarditis.

DESCRIPTION (provided by applicant): Myocarditis is a major cause of sudden death in people under 40 years of age. While some myocarditis patients recover, many of them progress to dilated cardiomyopathy, an often fatal condition and a frequent reason for cardiac transplantation. Many cases of myocarditis are associated with an autoimmune process in which cardiac myosin is a major autoantigen. The mechanisms leading to the immune-mediated damage to the heart, particularly the role of cytokines, are not fully elucidated. We propose to study these mechanisms using the murine model of cardiac myosin-induced experimental autoimmune myocarditis (EAM) previously established in our laboratory. The goal of the present proposal is to delineate the mechanisms by which IL-4 and IFN-gamma, prototypic Th2 and Th1 cytokines respectively, influence the disease process. Our interest in these two cytokines is based on our preliminary findings which indicate that IL-4 has a disease-promoting role in EAM, whereas IFN-gamma limits the disease. Specific aims 1 and 2 will address the role of IL-4 in EAM and the mechanisms by which IL-4 promotes disease. Specific aims 3 and 4 will examine the role of IFN-gamma and the mechanisms by which IFN-gamma limits disease. We are planning to achieve these specific aims by blocking cytokines with specific antibodies, using cytokine and cytokine receptor knock out mice, and transferring disease with antibodies and subsets of T cells. Disease outcomes will be assessed by gross and histologic examination of the hearts. In vivo assessment will include echocardiography and pressure-volume analysis. Our preliminary findings contrast with the prevailing opinion that organ-specific autoimmune diseases are driven by a Th1 response and are ameliorated by a Th2 response. The proposed study will help us understand how Th1 responses may limit and Th2 responses may promote organ-specific autoimmunity. It will also provide us with a basis for designing new therapeutic interventions in patients with myocarditis.

[unreadable] DESCRIPTION (provided by applicant): Myocarditis is an important cause of heart failure among adolescents and young adults. While about 33% of myocarditis patients recover, the remainder may develop chronic dilated cardiomyopathy which accounts for 25% of all heart failures in North America. Coxsackievirus B3 (CB3) infections have been implicated in around 40% of myocarditis cases. Antiviral drugs and immunosuppressive therapies have given mixed results, requiring further research in order to develop effective therapies. Studies of CB3-induced myocarditis in mice have provided valuable information about the role of the adaptive, acquired immune response to CB3 in the development of autoimmune myocarditis. Although the adaptive T cell response to CB3 is known to play a role in the development of myocarditis, little is understood on how the early, innate immune response to CB3 infection effects the development of autoimmune disease. The aim of this proposal is to characterize the innate immune response to CB3 infection in order to determine the factors that lead to autoimmune disease. We hypothesize that the early innate immune response to CB3 infection determines the later adaptive immune response leading to the development of autoimmune myocarditis. To investigate this hypothesis, we propose three specific aims: (i) to examine the role of early innate immune cytokines, (ii) the role of early innate immune cells, and (iii) the role of complement in the development of autoimmune myocarditis. The findings generated by these studies will aid in understanding the development of autoimmune disease initiated by infection.

DESCRIPTION (provided by applicant): Myocarditis is an important cause of heart failure among adolescents and young adults. While about 33% of myocarditis patients recover, the remainder may develop chronic dilated cardiomyopathy, which accounts for 25% of all heart failures in North America. CB3 infections have been implicated in around 4 % of myocarditis cases. Studies of CB3-induced myocarditis in mice have provided valuable information about the role of the adaptive, acquired immune response to CB3 in the development of autoimmune myocarditis. Recently we have examined the role of the innate immune response to CB3 infection on the development of myocarditis and have found significantly increased levels of acute viral and autoimmune myocarditis in males. Although the adaptive T cell response to CB3 is known to play a role in exacerbating disease in males, little is understood about the underlying mechanisms that determine the sex-based difference. The goal of this application is to characterize the immune and hormonal differences between the male and female immune response to CB3 infection in order to determine the factors that lead to increased disease in males. We hypothesize that the early hormonal and cytokine environment after CB3 infection determines the later adaptive immune response that results in increased myocarditis in males. To investigate this hypothesis, we propose two Specific Aims: (1) to characterize the differences between the male and female immune response to CB3 infection, and (2) to identify the hormonal factors that influence the immune response to CB3 infection resulting in increased severity of myocarditis in males. The findings generated by these studies will aid in understanding the sex- based differences in severity in this model of infection-induced autoimmune disease.

DESCRIPTION (provided by applicant): Myocarditis is a major cause of sudden death in people under 40 years of age, and many of these cases are associated with an autoimmune process[1, 2]. Like other autoimmune diseases, the fundamental causes and mechanisms of pathogenesis of myocarditis are not understood. To study the mechanisms of autoimmune myocarditis and autoimmune diseases in general we have developed a murine model of experimental autoimmune myocarditis (EAM) induced by cardiac myosin [3]. This model demonstrates that there are strong genetic influences to susceptibility to myocarditis, offering a fresh avenue into understanding the pathogenesis of this autoimmune disease [8]. The EAM model is unique and worthy of study apart from other autoimmune disease models because it shows greater influence of non H-2 genes, and shows an unusual male influence. In preliminary work we have demonstrated that loci on murine chromosomes 6 and possibly 1 and 4 are involved in susceptibility. Two of these loci (Chr. 1, Chr.6) interact and are also implicated in other autoimmune disease such as diabetes [7]. Furthermore, the Chr. 1 locus includes CTLA-4, an immunologically important gene, which we have previously demonstrated to regulate the pathogenesis of imyocarditis. The Chr.6 locus, which functions primarily in males, overlaps with loci that are important in thymocyte homeostasis and apoptosis [5]. We propose to conclusively establish the genetic findings which will be the foundation of future positional cloning and perform functional studies of polymorphisms in CTLA-4 which may lead to differential susceptibility to myocarditis. Preliminary experiments also indicate that these genetic loci act through hematopoietic tissues. We propose to solidify these findings and investigate through which specific cell types in the immune system these genetic loci operate. We build on our preliminary data to propose hypothesis-driven aims to identify host genes that control susceptibility and characterize their mechanisms of action in a murine model of autoimmune myocarditis.

DESCRIPTION (provided by applicant): Continued training in The Molecular and Cellular Bases of Infectious Diseases (MCBID) is proposed for 8 PhD students and 3 postdoctoral fellows selected from large pools of highly qualified applicants. The training program is uniquely situated in the Molecular Microbiology and Immunology Department (MMI) within the Johns Hopkins Bloomberg School of Public Health. The 29 training faculty have a wide range of experience and expertise in viruses, bacteria and parasites causing human disease and in the vectors and environmental factors associated with emergence and transmission of these pathogens. The training program has been funded since 1994 and has produced scientists working in many areas of academia and government on problems related to infectious diseases, vaccine development and the public's health. The goal of the MCBID training program is to provide students with both a firm foundation in the basic disciplines necessary for the study of infectious diseases and a perspective that will enable them to apply their knowledge creatively to public health problems. Each student is expected to complete 1) a series of required courses in the basic disciplines of cell and molecular biology, biochemistry, and immunology, 2) courses in virology, bacteriology, parasitology, and disease ecology, 3) courses in research ethics and public health perspectives, and 4) elective courses relevant to thesis topic and long-term career goals. Elective courses are chosen from among courses available in MMI, other departments in the School of Public Health, or in other Divisions of the University. Students will also complete 3 11-week laboratory rotations during the first year. Student progress is monitored by a Thesis Advisory Committee and the Graduate Program Committee. The goals of the postdoctoral training program are 1) to provide focused training in those areas of the molecular and cellular basis of infectious diseases in which program faculty have special expertise;2) to provide an opportunity for doctoral degree holders trained in more traditional environments to broaden their exposure to problems of public health importance and to evaluate their career goals in terms of public health issues;and 3) to prepare the PDF for an independent career in the biological sciences. RELEVANCE : This program is highly relevant to national interests in the areas of emerging infectious diseases, as it trains students and postdoctoral fellows broadly not only in both the molecular aspects of pathogen biology and disease pathogenesis, but also in the ecology of disease emergence and the role of vectors in pathogen transmission.

DESCRIPTION (provided by applicant): Eosinophilic myocarditis is a rare form of myocarditis with a poorly understood pathogenesis and an exceptionally poor prognosis. Patients with eosinophilic myocarditis present with a rapid, acute onset, and commonly progress to heart failure. We have developed several models of eosinophilic experimental autoimmune myocarditis (Eo-EAM) using either IL5CD2 transgenic mice or mice deficient in both IFN? and IL17A (IFN?-/-IL17A-/-) and immunizing them with myocarditogenic peptide in CFA. Both strains of mice develop severe myocarditis with more than 30% of their heart infiltrate comprised by eosinophils. Based on these observations, we propose the overarching hypothesis that Th2-driven inflammation, especially eosinophilic infiltration, contributes significantly to the most severe forms of immune-mediated heart disease. We propose to investigate the mechanisms underlying the pathogenesis of eosinophilic myocarditis by identifying the most critical cells and mediators that could be targets for a potential treatment. First, in Aim 1, we will examine the induction of eosinophilic autoimmune responses at the level of innate immunity. We hypothesize that early production of IL4 and IL13 by cells activated during the innate immune response leads to autoaggressive Th2 differentiation in the pathogenesis of eosinophilic EAM. We will employ transfer experiments to determine the ability of dendritic cells (Subaim 1.1), basophils (Subaim 1.2), and nuocytes (Subaim 1.3) to induce Eo-EAM. Additionally, we will use in vivo depletions to establish the necessity of these cells in the potentiation of Eo-EAM. In Aim 2, we will study determinants of eosinophil trafficking to the heart and the effectiveness of blocking such pathways to prevent eosinophilic myocarditis. Using IL5-/- mice, we have established that the recruitment of eosinophils to the heart during myocarditis is IL5-independent. In this Aim, we will investigate whether the CCR3 (Subaim 2.1) or eicosanoid lipid derivative pathways (Subaim 2.2) contribute to trafficking of eosinophils to the heart. In Aim 3, T cell decision-making in the pathogenesis of eosinophilic myocarditis will be examined. First, we will determine if IFN¿ and IL17A control development of eosinophilic myocarditis through Th2 responses (Subaim 3.1). To farther examine the sufficiency and necessity of Th2 differentiation for Eo-EAM development, we will employ IL4R¿F709 mice, which carry a point mutation in the IL4 receptor essentially rendering the signaling pathway hyperactive (Subaim 3.2). In Aim 4, we will explore possible therapeutic effects of blockade of one of the major products that eosinophils release upon activation, eosinophil peroxidase (EPO). Dissecting the mechanisms by which different cell types and cell mediators drive eosinophilic myocarditis development can lead to greater understanding of the pathogenesis of eosinophilic myocarditis and suggest ways in which this life-threatening disease can be prevented or treated.

DESCRIPTION (provided by applicant): Continued training in The Molecular and Cellular Bases of Infectious Diseases (MCBID) is proposed for 8 PhD students and 3 postdoctoral fellows selected from large pools of highly qualified applicants. The training program is uniquely situated in the Molecular Microbiology and Immunology Department (MMI) within the Johns Hopkins Bloomberg School of Public Health. The 29 training faculty have a wide range of experience and expertise in viruses, bacteria and parasites causing human disease and in the vectors and environmental factors associated with emergence and transmission of these pathogens. The training program has been funded since 1994 and has produced scientists working in many areas of academia and government on problems related to infectious diseases, vaccine development and the public's health. The goal of the MCBID training program is to provide students with both a firm foundation in the basic disciplines necessary for the study of infectious diseases and a perspective that will enable them to apply their knowledge creatively to public health problems. Each student is expected to complete 1) a series of required courses in the basic disciplines of cell and molecular biology, biochemistry, and immunology, 2) courses in virology, bacteriology, parasitology, and disease ecology, 3) courses in research ethics and public health perspectives, and 4) elective courses relevant to thesis topic and long-term career goals. Elective courses are chosen from among courses available in MMI, other departments in the School of Public Health, or in other Divisions of the University. Students will also complete 3 11-week laboratory rotations during the first year. Student progress is monitored by a Thesis Advisory Committee and the Graduate Program Committee. The goals of the postdoctoral training program are 1) to provide focused training in those areas of the molecular and cellular basis of infectious diseases in which program faculty have special expertise; 2) to provide an opportunity for doctoral degree holders trained in more traditional environments to broaden their exposure to problems of public health importance and to evaluate their career goals in terms of public health issues; and 3) to prepare the PDF for an independent career in the biological sciences. RELEVANCE : This program is highly relevant to national interests in the areas of emerging infectious diseases, as it trains students and postdoctoral fellows broadly not only in both the molecular aspects of pathogen biology and disease pathogenesis, but also in the ecology of disease emergence and the role of vectors in pathogen transmission.