Cornelia Bergmann - US grants

[unreadable] DESCRIPTION (provided by applicant): The major cell types responsible for maintaining humoral immunity are antibody secreting cells (ASC). Following peripheral infections, ASC preferentially migrate to bone marrow (BM), where they differentiate into long lived sessile plasma cells (PC) dedicated to immunoglobubulin (Ig) secretion. However, ASC are also drawn to inflammatory sites, where they can persist for prolonged periods similar to the BM. Intrathecal antibody (Ab) synthesis is well documented in humans during infections associated with neurological complications and the demyelinating disease multiple sclerosis (MS). Although antibody may contribute to pathology, local secretion within the central nervous system (CNS) may also be protective in controlling neurotropic viruses. However, B cell migration to the CNS, and local survival are poorly understood. The overall goal of this proposal is to identify factors mediating ASC homing, differentiation and maintenance in the CNS following acute encephalomyelitis induced by neurotropic coronavirus. We have shown that virus specific ASC (vASC) peak in the CNS after infectious virus is cleared and their retention at high frequencies prevents viral recrudescence. The Specific Aims are to 1) characterize differentiation and specificities of ASC within the CNS; 2) identify signals regulating preferential vASC migration into the CNS; 3) determine the relative role of CNS localized Ag versus chemokines in regulating ASC retention and 4) demonstrate that BAFF is the major survival factor for ASC within the CNS. Using a novel transgenic mouse, termed Blimpgfp/+, a combination of flow cytometry and ELISPOT techniques will characterize GFP+ ASC populations unique to the CNS and their potential to differentiate into sessile PC. The role of virus induced chemokines as major signals for CNS ASC recruitment into the CNS are assessed using partial bone marrow chimeras as well as chemokine inhibition. Immunization with a tracer Ag will monitor recruitment and retention of 'bystander' ASC to the CNS following viral infection. Understanding the regulation of humoral immunity associated with persistent CNS infection will reveal novel insights into intrathecal ASC survival during persistent infections of the human CNS, i.e. measles virus, rubella virus, JC virus, and HIV. Their protective role in a model of viral persistence associated with limited ongoing inflammation may distinguish them from detrimental events prevailing during chronic inflammatory autoimmune diseases. [unreadable] [unreadable] [unreadable]

T cell mediated immune responses within the central newous system (CNS) are beneficial by clearing infectious agents, but are also detrimental by activating resident cells and destroying tissue, leading to immunopathogy. A vital force in triggering T cell effector function both during virus and auto-Ag induced inflammation is recognition of cognate antigen (Ag) presented by major histocompatibility antigens (MHC) within the CNS. Strict regulation of MHC expression within the CNS is thus a critical determinant in disease progression. This proposal examines MHC dependent T cell regulation during acute and persistent viral infection established by the neurotropic JHM virus, a member of the coronaviridae. Despite control of acute infection by T cells, the consequences are ongoing chronic demyelination associated with virus persistence in the form of noninfectious RNA. The tropism of this virus for a variety of CNS cell types provides a unique model to analyze the contribution of individual glial cells to Ag presentation and T cell stimulation. Our overall hypothesis is that interactions with distinct glial cell populations result in heterologous outcomes with respect to CD8 + T cell activation, suwival and pathogenesis. Aim 1 examines the role of type I versus type II interferons in inducing MHC class I and class II expression on glial cell subsets during acute virus infection in vivo. Aim 2 tests the hypothesis that class I/CD8+ T cell interactions are critical in augmenting class II expression on microglia via secretion of IFN-gamma. This is based on the observation that class I expression precedes class II expression on resident CNS cells. Aim 3 evaluates the ability of glial cell subsets to stimulate CD8+ T cells during acute infection. Relative Ag presentation capacities of oligodendrocytes, astrocytes and microglia during distinct stages of inflammation will be assessed using highly sensitive virus specific T cell hybridomas in and T cell transfers in vivo. Aim 4 addresses the correlation between viral persistence, Ag presentation, and IFN-gamma/RANTES secretion in mediating retention of T cells within the CNS. Transgenic technology, specifically mice expressing green fluorescent protein (GFP) in distinct glial cells, and flow cytometry will be used to characterize interactions between T cells and distinct glial cells in vivo. The goals of this proposal are to delineate contributions of individual glial cell types in regulating CD8+ and CD4+ T cells in a MHC-dependent manner. Specifically the propensity of oligodendrocytes and astrocytes to interact with CD8+ T cells in an Ag specific manner in vivo will be revealed. Lastly, the mechanisms regulating T cell secretion of cytokine/chemokine during persistence, thus potentially contributing to chronic demyelination, will be defined.

flow cytometry; biomedical facility;

T cell mediated immune responses within the central newous system (CNS) are beneficial by clearing infectious agents, but are also detrimental by activating resident cells and destroying tissue, leading to immunopathogy. A vital force in triggering T cell effector function both during virus and auto-Ag induced inflammation is recognition of cognate antigen (Ag) presented by major histocompatibility antigens (MHC) within the CNS. Strict regulation of MHC expression within the CNS is thus a critical determinant in disease progression. This proposal examines MHC dependent T cell regulation during acute and persistent viral infection established by the neurotropic JHM virus, a member of the coronaviridae. Despite control of acute infection by T cells, the consequences are ongoing chronic demyelination associated with virus persistence in the form of noninfectious RNA. The tropism of this virus for a variety of CNS cell types provides a unique model to analyze the contribution of individual glial cells to Ag presentation and T cell stimulation. Our overall hypothesis is that interactions with distinct glial cell populations result in heterologous outcomes with respect to CD8 + T cell activation, suwival and pathogenesis. Aim 1 examines the role of type I versus type II interferons in inducing MHC class I and class II expression on glial cell subsets during acute virus infection in vivo. Aim 2 tests the hypothesis that class I/CD8+ T cell interactions are critical in augmenting class II expression on microglia via secretion of IFN-gamma. This is based on the observation that class I expression precedes class II expression on resident CNS cells. Aim 3 evaluates the ability of glial cell subsets to stimulate CD8+ T cells during acute infection. Relative Ag presentation capacities of oligodendrocytes, astrocytes and microglia during distinct stages of inflammation will be assessed using highly sensitive virus specific T cell hybridomas in and T cell transfers in vivo. Aim 4 addresses the correlation between viral persistence, Ag presentation, and IFN-gamma/RANTES secretion in mediating retention of T cells within the CNS. Transgenic technology, specifically mice expressing green fluorescent protein (GFP) in distinct glial cells, and flow cytometry will be used to characterize interactions between T cells and distinct glial cells in vivo. The goals of this proposal are to delineate contributions of individual glial cell types in regulating CD8+ and CD4+ T cells in a MHC-dependent manner. Specifically the propensity of oligodendrocytes and astrocytes to interact with CD8+ T cells in an Ag specific manner in vivo will be revealed. Lastly, the mechanisms regulating T cell secretion of cytokine/chemokine during persistence, thus potentially contributing to chronic demyelination, will be defined.

The purpose of the administrative Core (Core A) is to provide administrative support to the Program Director, to the Principle Investigators (Pi's) ofthe individual projects, and to the scientific personnel ofthe Projects and Cores. Core A will provide a key administrative and organizational role that will enable the Principle Investigators, their staff and the scientific cores to focus on experimental and scientific efforts. The specific tasks of Core A will be to: a) facilitate interactions between Program Investigators, Scientific Advisors and administrative personnel;b) to plan and coordinate the meetings between the Pi's and their staff;c) to plan and coordinate the internal scientific advisory committee interactions with the Pi's and their staff;d) to plan and coordinate travel for the external scientific advisory committee members, visiting scientists, the Pi's and their profession staff;e) to assist in assembling, obtaining and maintaining multi-user and individual IACUC protocols that cover the efforts included within this program;f) to facilitate resource sharing including assistance in timely execution of material transfer agreements;and g) to assist the Principle Investigators in preparation of progress reports, financial reports and manuscripts for publication.

PROJECT SUMMARY (See instructions): PROJECT 1 Viral infections targeting glia and neurons commonly cause encephalitis and have high mortality. Long term neurological dysfunction potentially resulting from altered cell function in persistently infected cells or immune pathology are common. Type I interferons (IFNa/(3) provide the first line of host defense against viral spread to and within the CNS. However, little is known about the ability of resident CNS cells to mount innate responses during acute or persistent CNS infections. This proposal addresses how distinct IFNa/p responses in microglia, astrocytes and oligodendrocytes contribute to pathogenesis following infection with a non lethal, demyelinating neurotropic coronavirus. Despite the concerted efforts of IFNa/p and adaptive immune cells to prevent mortality, virus is incompletely cleared, resulting in viral RNA persistence in the CNS. The overall hypothesis tested is that narrowly focused innate signals by oligodendrocytes constitute a protective mechanism for their survival and function in maintaining myelin, at the cost of persistent infection. Induction of IFNa/p by microglia and/or astrocytes is critical to induce an antiviral state in an autocrine and paracrine fashion. Aim # 1 will establish whether CNS cell types with highly specialized function, e.g. oligodendrocytes, rely on other cell types to signal the presence of invading pathogens. A dependence of IFNa/p signaling by individual cell types for protection will be confirmed by conditional abrogation of IFNa/p signaling in oligodendrocytes, astrocytes and microglia. Aim #2 will analyze mechanisms of RNAseL mediated protection against virus induced demyelination. It is based on novel findings of significantly enhanced demyelination and mortality of virus infected RNAseL deficient mice, despite effective viral control. Aim #3 will reveal whether persistently infected oligodendrocytes are immunologically silent, or trigger sufficient innate responses to directly or indirectly sustain local inflammation and demyelination. The overall approaches rely on a combination of RNA expression analysis in glial populations isolated directly from the infected CNS, immunohistochemical analysis, and chimeric mice to distinguish between innate affects on resident versus infiltrating cells. By defining mechanisms of innate immune regulation within the CNS during both acute and persistent infection, these studies will benefit our understanding and manipulation of factors critical for both anti viral function and amelioration of pathology manifested by demvelination.

DESCRIPTION (provided by applicant): The long term goals of this new program are an understanding of persistent viral infection and central nervous system (CNS) demyelinating disease. To this end, this program represents a multidisciplinary approach to defining mechanisms of viral persistence and myelin loss using a well defined murine model of demyelination induced by the neurotropic JHM strain (MHV-4) of mouse hepatitis virus (JHMV). This model provides a means to understand the interactions between a pathogen and its natural host that result in demyelination during acute and persistent CNS infection. The host response is competent to control infectious virus. However, a persistent CNS infection without detectable infectious virus is associated with chronic ongoing myelin loss. The pathological alterations within the CNS during viral persistence have numerous similarities to multiple sclerosis, the most prevalent human demyelinating disease. This program is unique, comprising a core of investigators addressing fundamental questions of viral persistence and immune responses, both as protective mechanisms and as inducers of demyelination. Project 1 focuses on the pro-inflammatory and anti-inflammatory effects of innate immunity. New data suggest that the limited capacity of oligodendroglia to respond to innate signals protect from oligodendroglial dysfunction and myelin loss. This project uses newly developed techniques and novel transgenic mice to demonstrate the unique response of oligodendroglia during both acute viral encephalomyelitis and viral persistence. Project 2 explores the unknown area of T cell retention and homeostasis within the CNS. The role of CNS resident and infiltrating cells in presenting viral antigen as well as the potential for cross priming are defined using a variety of transgenic and bone marrow chimeric mice. Project 3 analyzes the role of regulatory T cells and the anti-inflammatory cytokine IL-10 in CNS viral persistence and demyelination. This project uses a novel transgenic mouse which allows definition of the role of regulatory T cells during viral persistence and ongoing demyelination. Data obtained from these projects will provide novel insights into the mechanisms regulating viral persistence and demyelination as well as the CNS as a target for viral persistence. Importantly, it will provide valuable information on the interactions of specific CNS cells involved in viral persistence and demyelination and the cellular and soluble mediators of the host immune response.

DESCRIPTION (provided by applicant): The central nervous system (CNS) is a major target for acute encephalitic viral infections, as well as a reservoir of latent/persisting viruses. While effective immune control of persisting viruses in immunocompetent individuals is reflected by the absence of overt neurological deficits or pathology, this balance is highly tenuous. Studies supported by this award were the first to demonstrate that intrathecal antibody secreting cells (ASC) were critical to control CNS viral persistence even despite early T cell mediated control and presence of anti-viral serum antibody (Ab). We have also defined the regulation of ACS recruitment into the CNS by identifying the crucial chemokine receptor and chemokine. The reliance on local ASC for prolonged Ab output during neurotropic coronavirus infection is a critical observation, as it provides a potent non lytic mechanism of sustained immune control applicable to numerous neurotropic infections. Indeed, a vital local protective role of ASC is supported by other experimental CNS infections, particularly by RNA viruses such as Sindbis, Semliki Forest, and Rabies viruses. Intrathecal Ab synthesis is also well documented in humans during infections associated with neurological complications and the demyelinating disease multiple sclerosis (MS). However, virtually nothing is known about the origin, maintenance, and relevance of ASC in the CNS or other specialized microenvironments. The overall goal is to define the regulation of protective, yet minimally destructive ASC within the CNS, to combat infections where virus replicates in the CNS in the absence of overt blood brain barrier damage or inflammatory signals. The Specific Aims are to 1) determine the role of CNS resident cells in mediating ASC entry and localization within the CNS; and 2) determine the relative contribution of migratory ASC versus circulating, non Ab secreting memory B cells in maintaining ASC within the CNS. In situ hybridization combined with confocal microscopy in Aim 1 will determine the spatial relationship between chemokines, viral antigen, ASC localization and the microvasculature. The results will further reveal a central role of astrocytes in regulating ASC recruitment. Aim 2 uses transgenic mice in which germinal center derived ASC and memory B cells are phenotypically marked, to specifically test the role of virus specific B cells in contributing to ASC maintenance during chronic CNS infection. Adoptive transfers will define whether the initial burst of ASC in the CNS is sufficient to sustain local protective capacity. Non of these parameters have been studied during viral or autoimmune encephalomyelitis. The ability to trace ASC and Bmem using transgenic markers provides a versatile innovative approach to dissect humoral immunity in a non-lymphoid organ prone to persisting infection. The contribution of peripherally activated B cells rather than lymphoid tissue neogenesis, in sustaining intrathecal humoral responses during persistent infections associated with limited ongoing inflammation will be beneficial for understanding protective responses during human CNS infections caused by measles virus, rubella virus, JC virus, and HIV.

DESCRIPTION (provided by applicant): Antibody (Ab) production and the presence of B cells within the central nervous system (CNS) is well documented in humans with the demyelinating disease multiple sclerosis (MS) and those afflicted by neurotropic infections. Their role in MS is currently unclear. However, detrimental humoral responses are implied by ongoing immune activation due to ectopic B cell follicle formation, as well as improvement in MS patients treated with anti-CD20 monoclonal Ab rituximab to reduce circulating B cells. Antibody secreting cells (ASC) are also detrimental if Ab are cross-reactive with, or directly target, self antigens. By contrast, during viral CNS infections intrathecal humoral responses are associated with protective functions. Locally produced anti-viral Ab coincide with sustained immune control within the CNS without overt pathology implicating a potent non lytic anti-viral role. Furthermore, the potential danger of losing control of clinically inapparent persisting viruses became apparent by development of progressive multifocal leukoencephalopathy following rituximab treatment during therapy for rheumatoid arthritis and MS. Despite the biological significance of humoral immunity in diverse settings of neuroinflammation, how Ab production in the CNS is sustained and which B cell populations and environmental factors support differentiation of ASC remain poorly characterized. This proposal uses a neurotropic coronavirus model of acute and persistent infection associated with demyelination to define the interactions between peripheral and CNS humoral responses in supporting CNS Ab production. The overall goal is to broaden insights into treatment options for inflammatory diseases such as MS, without provoking emergence of endogenous viruses, as well as targeting acute viral encephalitis. Three Specific Aims are pursued. Aim 1 will define the contribution of peripheral lymphoid tissue derived B cells in replenishing and maintaining ASC within the CNS. Results will reveal whether all B cells within the CNS are germinal center derived, and whether ASC migrating to the CNS differentiate within the CNS to become long lived ASC. Furthermore, the unexplored role of non Ab producing memory B cells (Bmem) to CNS humoral immunity will be defined. The role of antigen (Ag) and CD4 T cells in promoting B cell differentiation to sessile ASC within the CNS will be determined in Aims 2 and 3, respectively. Approaches are based on immunization of transgenic mice in which germinal center derived B cells are marked by fluorescence to isolate ASC and Bmem for adoptive transfer. Their CNS migration, differentiation and protective capacity will be monitored in recipients defective in Ab production. The influence of cognate Ag is examined by variations in donor B cell specificities, and inflammatory insult. T cell help will be assessed b CD4 T cell ablation and supplementation approaches. The results will for the first time define the parameters regulating both protective and pathogenic responses associated with B cells during human autoimmunity and CNS infections.

? DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is a debilitating disease of the central nervous system (CNS). The loss of the protective myelin shielding axons is the hallmark pathology associated with MS. The focal nature of the white matter plaques in the CNS of MS patients, and following experimental autoimmune encephalitis (EAE) and viral induced demyelination all support the concept that a highly evolutionarily conserved regulatory mechanism(s) prevents spread of damage to adjacent uninvolved white matter areas. However, the mechanism preventing plaque expansion undefined. A novel system is described in which viral infection in mice deficient in interleukin 10 (IL-10) results in enhanced viral clearance and reduced plaque formation. However, well after is virus is eliminated from the CNS, plaques dramatically enlarge. In contrast to other rodent models, this proposal uses a viral infection in which demyelination is independent of macrophages, IL-17, or self-reactive T cells. Importantly, the data will identify the mechanism preventing expansion of focal demyelination and directly tests the emerging concept of active communication between astrocytes and microglia in regulating demyelination. This model system will allow definition of the cells secreting IL-10 and the cell which receives the signal restraining focal myelin injury. Preliminary data indicate that Foxp3+ regulatory T cells (Treg) are the source of IL-10 limiting demyelination. The first aim will be accomplished by specific depletion of Treg from the CNS by infusion of diphtheria toxin using an osmotic pump approach to supply toxin intraventricularly after resolution of acute infection and confirmed by ablation of IL-10 secretion only from Treg. The second Aim will define the cell within the CNS which is the recipient of the IL-10 signaling that limit tissue damage. This will be accomplished by infecting mice in which the IL-10 receptor has been specific ablated in astrocytes, the only cell type expressing the receptor during chronic demyelination. Demyelination in this model is independent of bone marrow derived macrophages, therefore gene profiling of cells purified from the infected adult CNS will be used to define the interaction of IL-10 with astrocytes and the subsequent alteration in microglia activity. These data will provide the first evidence for a temporally distinct pathological event within the CNS that is vira induced, but distinct from the acute event and demonstrate that limiting the expansion of lesions within white matter are dependent upon the astrocyte response to the anti-inflammatory cytokine IL-10.

DESCRIPTION (provided by applicant): Autoimmunity is an attack on the host carried out by its own immune system. Genetic and environmental agents, i.e., infectious agents, are thought to be major contributing factors in both the induction of autoimmunity and in precipitation of relapse events. Significant attention has been given to possible mechanisms of autoimmune activation following infection. However, little is understood concerning the mechanisms that prevent autoimmunity, especially following an infection that produces pathology similar or identical to an autoimmune disease. In this proposal we will use a viral infection of the central nervous system (CNS) that induces significant demyelination, a hallmark of human multiple sclerosis (MS) and murine experimental autoimmune encephalitis (EAE), without progressing to autoimmunity. The virus used is the glial tropic JHM strain of mouse hepatitis virus (JHMV). This infection of the murine CNS produces a nonfatal encephalomyelitis. The host's immune system clears infectious virus from the CNS but is unable to affect sterile immunity, resulting in a persistence viral infection confined to the CNS. Demyelination is a hallmark finding associated with both the acute infection and, importantly, the persistent infection. Following viral induced demyelination autoreactive T cells are not present during the window of maximal demyelination, but only during viral persistence, after resolution of the infection and re-established blood brain barrier integrity. Nevertheless there is no evidence of an autoimmune response associated with these self reactive T cells. This proposal examines the mechanisms regulating both the induction and suppression of autoreactive T cells. Our overall hypothesis is that extensive viral induced demyelination following acute infection induces a milieu within the CNS which suppresses the effector function of self reactive T cells. This proposal defines the kinetics of activation and CNS retention of self reactive T cells secreting interferon gamma (IFN-?) interleukin (IL)-17, and IL-9 and defines the requirement for demyelination in the activation of self reactive T cells. The concept that suppression of self reactivity is a residual effect of acute viral encephalomyelitis is tested by adoptive transfer of encephalitogenic Th1, Th9 and Th17 cells into persistently infected mice. Finally a novel transgenic mouse which allows depletion of regulatory T cells within the CNS during viral persistence will prove that regulatory T cells prevent expression of self reactive T cell effector function. Using both virus specific reagents and tools developed to understand EAE regulation these data will provide a mechanistic understanding of the regulation self reactive T cells induced by a viral infection. In addition, the data will provide the first demonstration of a viral induced mechanism suppressing effector function specifically within the target tissue, preventing autoimmune attack by self reactive T cells.

Abstract Multiple Sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) characterized by demyelination and axonal loss. Demyelinating lesions are defined by cellular infiltrates composed predominantly of T lymphocytes and two distinct types of myeloid cells, namely CNS infiltrating bone marrow- derived macrophages (BMDM) and resident microglia. Understanding their interplay is essential to MS pathogenesis, as T cell-produced cytokines and chemokines promote myeloid cell populations to produce toxic factors and strip myelinated axons, culminating in tissue damage. However, how T cells instruct myeloid cells, as well as the relative contributions of BMDM and microglia to tissue damage and disability, are unclear. Specifically how Th1 and Th17 cells, both found in MS patients, affect BMDM and/or microglia to express toxic functions, as well as strip and phagocyte myelin, are still poorly understood. The overall goal of this proposal is to define in vivo mechanisms that regulate microglia and BMDM to actively participate in the demyelinating process. Using a unique murine virus encephalomyelitis model in which microglia mediate demyelination in the absence of BMDM and Th17 response, this proposal will define how distinct T cell functions specifically promote microglia-mediated demyelination. This model provides a unique tool to dissect how Th1 versus Th17 cells regulate microglia effector functions during demyelination. Based on our preliminary data, we hypothesize that strict Th1 conditions drive microglia to mediate demyelination, while Th17 response can modify this effect by altering microglial pathogenic functions and promoting BMDM-mediated demyelination. Aim 1 will test how microglial responsiveness to T cell-derived IFN-? regulates their demyelinating function independent of BMDM. More specifically, this aim will define the contribution of microglia oxidative burst to myelin damage, as well as determine whether TREM-2 modulation of microglial phagocytic functions can contribute to demyelination. Aim 2 will reveal how Th17 cells alters microglia effector functions and myelin damage, in the presence or absence of BMDM, and whether this effect is directly dependent upon IL-17 and/or GM-CSF secretion. Gene array analysis will also characterize phenotypic markers associated with the pathogenic versus protective functions of microglia in a distinct inflammatory environment. By revealing how microglia respond to prominent T cell cytokines, this proposal will provide new insights into the direct contribution of microglia to lesion formation in MS patients, potentially leading to new therapeutic targets.