Caroline Whitacre - US grants

This proposal presents an opportunity to lean more about genetic resistance to autoimmune neural tissue destruction. This study was prompted by an observation of clinical and histopathologic resistance to the induction of Experimental Allergic Encephalomyelitis (EAE) in a colony of Lewis (designated Le-R) rats. A subline of Le-R rats, in which greater than 99% of the population is EAE-resistant, was established by selective breeding. Other breeding experiments between Lewis (Le) and Le-R rats indicated that, at 7-8 weeks of age, resistance is a dominant, autosomal trait possibly mediated by only one or two genes. In contrast to EAE-resistant Brown Norway rats, Le-R rats develop cellular reactivity, as assessed by macrophage migration inhibition, for the critical disease-inducing determinant located within an encephalitogenic peptide derived from the myelin basic protein molecule. Although Le-R spleen cells failed to transfer disease to naive Le rats, Le-R recipients were susceptible to passive disease induction mediated by in vitro conditioned, sensitized Le spleen cells. These observations suggest that resistance is expressed during the development of disease-inducing effector cells from specific antigen-reactive cells. The experiments presented in this proposal are designed to investigate the resistance mechanism: 1) Is resistance immunologic? 2) Is resistance mediated by a mechanism similar to that of other EAE-resistant animals? Question 1 will be approached by constructing chimeras in which Le-R immune systems will be implanted into lethally irradiated Le rats. Question 2 will be approached by determining if mechanisms which mediate resistance in other EAE-resistant animals are operative in Le-R rats. The ultimate long-term goal of this project is to identify the Le-R gene(s) that mediates resistance, and to decipher its mechanism of action. This may be useful in developing immunotherapeutic approaches for treating human neurological diseases such as multiple sclerosis.

Although the etiology of multiple sclerosis (MS) is unknown, one of the central theories regarding its pathogenesis involves autoimmunity specific for myelin antigens. Experimental autoimmune encephalomyelitis (EAE), induced by myelin basic protein (MBP) and adjuvant, is studied as a model for MS since the two disease share clinical, histopathologic, and immunologic features. We have recently reported that the oral administration of MBP renders Lewis rats refractory to EAE. MBP-fed rats exhibit a profound decrease in clinical neurologic signs, significantly lessened CNS histopathologic changes, absent lymphocyte proliferative responses to MBP, and decreased serum anti-MBP antibody. We have accumulated much evidence supporting a profound reduction in MBP-reactive lymphocytes in orally tolerant rats. The goals of this application are the determination of whether MBP-reactivity lymphocytes are silenced in tolerant rats by clonal deletion, clonal anergy or an alteration in trafficking; the examination of the role of the intestinal epithelium in tolerance induction; the definition of the fine specificity of tolerance at the peptide level, and the applicability of this approach to an ongoing, chronic inflammatory process. Specifically, we propose: To examine mucosal and peripheral lymphoid tissues and the CNS for the presence of functional MBP-reactive T lymphocytes. These studies will utilize limiting dilution analysis (LDA) to detect IL-2 secreting MBP-specific T cells. Also, we propose To examine mucosal and peripheral lymphoid tissues and the CNS for the presence of lymphocytes containing mRNA for the MBP-specific T cell receptor. These studies, performed in collaboration with Dr. Ellen Heber-Katz, will employ northern analysis and address the question of whether specific MBP-reactive lymphocytes are deleted/inactivated or re- located in tolerant animals. Next, we propose To determine whether MBP- induced oral tolerance can be reversed by treatment of tolerant lymphocytes with IL-2, thus distinguishing between deletion and anergy mechanisms. Then, we will Examine a possible role for intestinal epithelial cells in the induction of oral tolerance to MBP since these cells constitutively express Ia, may lack costimulatory activity and are located at a critical position for contacting orally introduced antigen. Next, we propose To elucidate the fine specificity of MBP-induced tolerance by the oral administration of a nested series of peptides. We have shown that the 68- 88 peptide is tolerogenic when administered orally. These studies are directed at determining the minimal tolerogenic peptide within that region as well as the epitope specificity of oral tolerance. Finally, we propose To determine if the course of chronic relapsing EAE can be altered by the oral administration of MBP. These studies will use the mouse model of chronic relapsing EAE testing the therapeutic efficacy of oral MBP begun at various times throughout the disease course. Therefore, the ability of this therapeutic strategy, viz., orally administered myelin antigens, to alter the long-term course of a chronic inflammatory process such as MS can be tested.

The basis for the severe immune compromise seen in the acquired immunodeficiency syndrome (AIDS) is the profound human immunodeficiency virus (HIV)-induced depletion of helper lymphocytes bearing the CD4 molecule. With improved anti-retroviral therapies, it has become increasingly obvious that the currently used surrogate markers, CD4 lymphocytes number and serum p24 antigen level, are not sensitive enough to permit a rapid assessment of the therapeutic response, particularly in asymptomatic or mildly affected patients. We propose to assess the immune response to biologically relevant HIV peptides using a quantitative assay which is a sensitive measure of immunologic function, i.e., the limiting dilution analysis. With this approach, a minimal estimate of the frequency of HIV peptide-reactive lymphocytes can be determined, using the secretion of interleukin 2 (IL-2) as an endpoint. It is known that one of the first immune functions to decline following HIV infection is the ability of T lymphocytes to proliferate in response to soluble antigens such as tetanus toxoid. We have obtained preliminary data showing that detectable frequencies of tetanus-reactive lymphocytes are present in asymptomatic, ARC, and AIDS patients, even when the ability of their T lymphocytes are present in asymptomatic, ARC, and AIDS patients, even when the ability of their T lymphocytes to proliferate to tetanus is lost. We now propose to extend this quantitative approach to assessment of HIV-peptide reactive lymphocytes. First, the frequency of lymphocytes responding by IL-2 secretion to four synthetic gp160 peptides will be determined in seronegative controls, asymptomatic, ARC, and AIDS patients. Secondly, the frequencies of lymphocytes responding to these peptides will be followed longitudinally in an asymptomatic patients group and correlated with clinical course, CD4 number, serum HIV p24 level, virus culture and general cellular immune function. In this manner, the relevance of the HIV- specific cellular immune response as a predictor of disease progression or response to therapy can be adequately assessed.

Several lines of evidence argue that T cell-mediate autoimmunity plays a role in the pathogenesis of multiple sclerosis (MS). The precise nature of the antigen eliciting the autoimmune attack on the myelin sheath is not known, but in-vivo-activated, clonally expanded lymphocytes reactive with myelin basic protein (MBP) have been demonstrated in MS peripheral blood. Injection of MBP into susceptible strains of mice together with immunologic adjuvants results in the induction of experimental autoimmune encephalomyelitis (EAE), a chronic relapsing disease with extensive demyelination. EAE, induced by CD4+ cells of the Th1 type, is viewed as the best model currently available for testing of MS therapeutic regimens. We have reported that the oral administration of MBP exerts a profoundly suppressive effect on EAE in Lewis rats. Rats fed MBP exhibit a striking inhibition of EAE clinical neurologic signs, reduced CNS histopathologic changes, and suppressed T and B cell responses specific for MBP. Two mechanisms have been put forward to explain orally induced tolerance in the EAE model - clonal anergy and active suppression. In applying oral tolerance to the treatment of chronic disease like MS, it will be necessary to determine whether an ongoing autoimmune process can be suppressed after initiation of immunologic injury. We have obtained preliminary data indicating that orally administered MBP, given either before challenge or at the time of disease onset, is acute effective in suppressing relapses of EAE. Therefore, with our background in the oral tolerance field and new preliminary data relative to chronic, relapsing disease, we propose to address several aims relative to our underlying hypothesis that oral tolerance is mediated through clonal anergy or deletion and is capable of halting the progression of ongoing autoimmune disease. First, we will determine the relative efficacy of oral neuroantigen administration in relapsing EAE. In these studies, we will focus on the specificity of tolerance and the timing of tolerogen administration relative to disease using whole neuroantigens (MBP, PLP, myelin). Effectiveness of these neuroantigens at the time of disease appearance, or during a remission period or at the time of relapse will be tested. Second, we will determine the fine specificity of oral tolerance in the B10.PL mouse using MBP Peptides. Tolerogenicity of encephalitogenic as well as non- encephalitogenic peptides and MBP peptide analogs will be studied. Third, the mechanism(s) of suppression of relapsing EAE will be studied by examining suppressor T cells, clonal anergy, clonal deletion, and shifts in cytokine profile as possible candidates. These studies will be aided by the availability of a Valpha2/VBeta8.2 transgenic mouse. Therefore, at the conclusion of these experiments, the boundaries of the oral tolerance therapeutic approach in its alteration of the long-term course of a chronic inflammatory disease will be defined.

This proposal takes a novel approach to the etiology of secondary injury that occurs after neurotrauma. It investigates the role of the immune system in mediating delayed neuronal and myelin degeneration precipitated by traumatic spinal cord injury (SCI). The studies proposed here will utilize a highly characterized SCI model capable of producing lesions similar to those found in the clinical setting. This project will take advantage of an established consortium with existing expertise in immunological approaches, SCI, and cytokine analysis that may provide insights into the mechanisms of trauma-mediated secondary degeneration after spinal injury. It is the major hypothesis of this study that both intrinsic CNS inflammatory cells (microglia) and peripheral blood mononuclear cells (monocytes/macrophages, lymphocytes) are involved in the initiation of cytotoxic cascades which contribute to the production of specific CNS lesions, particularly in the white matter, for long time periods after SCI. Our approaches, using the Lewis rat strain, will allow us to: (1) define the nature and activation of cellular immune elements that may be involved in progressive tissue necrosis (immunocytochemistry/lectin histochemistry), (2) define the role of peripheral macrophages vs. activated parenchymal microglia in the secondary pathology of spinal cord trauma by selectively depleting peripheral macrophage populations in parallel with immunological and physiological assays, (3) estimate the effects of these depletion protocols on behavioral paradigms designed to assess functional recovery after SCI (behavioral paradigms designed for in the Ohio State Spinal Cord Injury Research Center), (4) correlate the presence of inflammatory infiltrates with the expression of various cytokines known to be involved in the injury process (measure mRNA by Northern blotting/quantitative RT-PCR before and after macrophage depletion), and (5) assess changes in spinal cord blood- brain barrier (BBB) integrity, patterns of lymphatic drainage, and peripheral lymphoid sensitization after SCI ([14C]-labeled alpha- aminoisobutyric acid (AIB), [14C]-labeled polyethylene glycol, and proliferation assays/adoptive transfer techniques respectively). In this manner, major questions about the role of the immune system in delayed traumatic injury may be answered and appropriate therapeutic regimens designed.

DESCRIPTION (Adapted from Investigator's abstract): Multiple sclerosis (MS) is characterized clinically by episodes of relapsing or chronic paralysis and the pathogenesis is attributed to T-cell-mediated inflammation of the CNS. Clinical, histopathological and immunological similarities between MS and experimental autoimmune encephalomyelitis (EAE) allow EAE to be used as a model for therapeutic approaches to MS. Inhibition of murine relapsing EAE with multiple oral administrations of myelin basic protein (MBP) to BP10.PL mice has been reported recently. Two mechanisms have been proposed for MBP-induced oral tolerance - active suppression and clonal anergy/deletion. Deciphering mechanisms of oral tolerance to MBP and testing tolerizing strategies for protection from EAE have been facilitated by the development of MBP T-cell receptor (TCR) transgenic (Tg) mice. These mice express a TCR specific for the immunodominant MBP epitope (NAc 1-11) on 95% of their CD4+ T-cells and are highly susceptible to EAE. The investigator has preliminary data showing that orally administered MBP not only protects MBP TCR Tg mice from EAE but also induces a profound reduction of Tg+ cells from the periphery. This is followed by the return of a Tg population with decreased functional capacity. This evidence supports the investigator's hypothesis that high dose oral administration of an autoantigen induces early deletion of antigen-reactive cells, followed by a return to the periphery of autoantigen-reactive cells rendered anergic. The investigator proposes to address several questions pertaining to the mechanism and ideal conditions for MBP-induced oral tolerance. The investigator will differentiate between immediate trafficking or deletion of MBP-specific Tg cells after MBP feeding. She will characterize the duration of MBP-induced oral tolerance. She will optimize conditions for oral tolerance to MBP. Therefore, at their conclusion these studies will have 1) defined the effects of feeding antigen on antigen-specific T-cells and 2) optimized oral tolerance for application to a chronic inflammatory disease.

DESCRIPTION (adapted from investigator's abstract): Psychological and behavioral states are known to exert a profound impact on the immune system, and can delay the onset of acute inflammation and healing of injured tissues. Previous research from our group and others has shown that psychological influences on immune functions can be highly subtle and diverse. Personality factors, attitudes and beliefs can lead to a down-regulation of immune function, and likely do so by an overlapping set of common mechanisms. While animal models may not approximate the intricacies of human psychology, they afford an important and necessary approach to understanding the mechanistic links between psychological states and health outcomes. Other projects of this proposal focus on healing of peripheral wounds, but from the vantage of psychological processes, a highly relevant issue is the impact of behavioral states on healing of brain injuries. After spinal cord injury, repair and regeneration of damaged tissues, both CNS and peripheral, is dependent on inflammatory processes. Life expectancy of spinal injured individuals has increased to a level that is consistent with the able bodied population. SCI survivors must learn to cope with the psychological stress of disability as well as the physiological challenges of rehabilitation, and these latter factors likely impact on recovery. In the present proposal, we will address the interrelationship between psychological stress, and wound healing in a clinically relevant model of SCI. A major hypothesis of this proposal is that physiological and psychological factors associated with SCI, and damage to sympathetic outflow will suppress inflammatory processes resulting in slowed or incomplete wound healing of both CNS and peripheral tissues. We will identify the time course of hypothalamic- pituitary-adrenal and sympathetic-adrenal medullary activation after SCI injuries in mice, which can then be correlated with measures of acute inflammation in wounds of the skin and spinal cord. Morphometric analysis of immunohistochemically-stained skin (above and below the level of SCI) and injured spinal cord sections will allow us to determine how altered immunological processes affect wound healing in the periphery and CNS. The addition of a psychological stressor (restraint stress), will further illuminate how stress may impact on wound healing and inflammation, especially under conditions where the immune system and CNS are uncoupled, as in spinal injury.

Aging and stress delay the onset of acute inflammation and subsequently healing of injured tissues. After spinal cord injury (SCI), repair and regeneration of damaged tissues, both CNS and peripheral tissues (e.g., surgical incisions, pressure sores), is dependent on inflammatory processes. As medical care has improved, the life expectancy of spinal injured individuals has increased to a level that is consistent with the able- bodied population. As such, SCI survivors must learn to cope with the physiological stress as disability, as well as the physiological challenges of aging. In the present proposal, we will address the interrelationship between aging, stress, and wound healing in a clinically relevant model of SCI. It is the major hypothesis of this proposal that aging and the stress associated with SCI will suppress inflammatory processes resulting in delayed or incomplete wound healing in the CNS. In this proposal, we will define baseline parameters of hypothalamic-pituitary-adrenal (HPA) axis activation (measurement of plasma CORT and ACTH) as a function of time post-injury in three ages of mice (young, adult, aged). These data will define the peak and duration of HPA activation, which can then be correlated with measures of acute inflammation in the spinal cord (e.g., cellularity, cytokines, chemokines). Morphometric analysis of immunohistochemically-stained injured spinal cord sections will allow us to determine how altered immunological processes affect wound healing in the CNS. Together, these analyses will help us to deter define the effects of aging and stress on repair and/or regeneration of the injured spinal cord.

DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS), which is more prevalent in women than men (2-3:1). During pregnancy, there is a decrease in the MS relapse rate, most prominent during the third trimester. After parturition, there is often an increase in the relapse rate. Therefore, within a 6 month time frame, there is a profound change in MS disease activity, with both improvement and disease worsening, providing an ideal window of opportunity for study. We are currently working in the area of sex differences in autoimmune disease, focusing on sex differences in susceptibility to experimental autoimmune encephalomyelitis (EAE), a model for MS. As shown in the preliminary results section, we have identified three differing patterns of disease in the B10.PL, SJL and C57BL/6 mouse strains, respectively, and we plan to utilize all three strains to explore the effects of pregnancy on EAE. We have obtained preliminary data showing that immunization of mice during mid-pregnancy or late-pregnancy suppresses the clinical signs of EAE. The goal of this application is to test the hypothesis that disease fluctuation occurring during and soon after pregnancy in MS and EAE are related to changes in the immune response and are influenced by sex and stress steroid hormones. The aims explore the effect of pregnancy on mice with chronic relapsing EAE and the mechanism of that disease modulation. Specifically, the aims are 1) to determine the effect of pregnancy on murine chronic relapsing EAE exploring the effects of pregnancy on three different EAE models differing in genetic background and extent of disease severity, 2) To probe the mechanism of pregnancy effects in EAE by examining the role of cytokine shifts as well as the influences of sex and stress steroid hormones, and 3) To examine the effect of pregnancy on the naive immune system relative to a biased and activated immune response. The long-term goal of this project is to gain a better understanding of the events and mechanisms underlying the pregnancy-induced suppression of EAE and MS.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CMS) which can present as a remitting/relapsing, primary progressive, secondary progressive or progressive relapsing form. Experimental autoimmune encephalomyelitis (EAE), a frequently studied animal model for MS, is inducible in the commonly used C57BL/6 mouse, which develops a progressive course of EAE when immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55. A major focus of research in EAE and MS has been what causes progression of disease and is there a signal which can be used to predict a relapse or worsening of disease. Migration inhibitory factor (MIF) has recently been re-discovered as an inducer of pro- inflammatory cytokines and an antagonist of glucocorticoid hormones. We examined the role of MIF in acute and chronic EAE using MIF knock-out mice. We found that MIF-/- mice showed signs of acute EAE identical to wild type (WT) controls but did not exhibit progression of disease. MIF-/- mice showed elevated levels of corticosterone and decreased levels of pro-inflammatory cytokines. In light of these findings and reports by others that colitis is suppressed in MIF-deficient animals, we pose the hypothesis that MIF derived from myeloid cells is necessary for progression of EAE. We will first determine the cellular source of MIF which is necessary for progressive EAE using a series of bone marrow chimeras in which various cell types are rendered MIF deficient. Second, we will gain a better understanding of the role played by MIF in disease progression by examining priming, CNS histopathologic changes, glucocorticoid hormone release, and cytokine changes. At the completion of these studies, we will have determined the source of the biologically relevant MIF and will have a clearer picture of the role played by MIF in causing progression of EAE. During the course of this project, we will repeatedly evaluate the possibility that MIF could serve as a target for new therapies in MS. RELEVANCE TO PUBLIC HEALTH: Multiple sclerosis is a disease affecting over 300,000 persons in the US which affects their ability to work and lead a productive life. It begins early in life and often progresses slowly leading to severe disability. This project studies a key factor involved in disease progression and will identify specific points in disease when therapeutic intervention is likely to be successful.

DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is an inflammatory disease of the central nervous system which involves autoreactive CD4+ T cells of a TH1 and TH17 phenotype. Identifying novel factors that control the inflammatory phenotype of T cells will enable a more complete understanding of T cell regulation and MS pathogenesis. MicroRNAs (miRs), which can negatively regulate multiple target genes, are positioned to act as regulatory nodes in gene networks such as those controlling T cell differentiation and function. MiRs are a class of small non-coding RNAs that negatively regulate post-transcriptional gene expression. MiRs have an established role in the immune system and profiling studies have shown that certain miRs are aberrantly expressed in autoimmune diseases, including MS. Mice deficient in small RNA processing machinery, Dicer, produce more cytokines when their immune cells are activated. Together, these data indicate that miR(s) are central to immune system homeostasis and may act as negative regulators of inflammation. However, the miRs responsible for modulating inflammatory cytokines have not been defined, and the function of specific miRs in CD4+ T cells is the subject of this proposal. Using target prediction algorithms, we found that the miR-29 family (miR-29a, miR-29b, miR-29c) is predicted to target both IFN3, the prototypical TH1 cytokine, as well as T-bet, a T-box transcription factor considered the master regulator of TH1 differentiation. MiR-29 is therefore positioned to modulate multiple developmental stages in TH1 cells, including effector cytokine production and transcriptional programming. We hypothesize that miR-29 modulates CD4+ T cell differentiation and propose to explore the biological consequences of altered miR-29 expression using in vitro TH1-polarized murine and human T cells in specific aim 1. The miR-29 promoters each contain 3 conserved IFN3-activated site (GAS) elements within 5kb of other demonstrated transcription factor binding sites, suggesting miR-29 may be transcriptionally regulated through interferon signaling. We have shown that IFN3 stimulation of naove murine T cells is sufficient to up-regulate miR-29 expression. We propose to extend our findings to IFN2 treatment of MS patients, since both IFN3 and IFN2 signal through the STAT pathway. We hypothesize that miR-29 expression is induced through IFN2 treatment of MS patients, and propose to use a unique collection of PBMC from MS patients pre- and post- IFN2 therapy to analyze changes in miR-29 expression in specific aim 2. These studies could result in the discovery of a novel regulatory network capable of influencing TH cell development and function, ultimately contributing to our understanding of the TH1/TH2 paradigm and IFN2 treatment in MS.

The pace of scientific discovery has been rapid in recent years owing to cyberinfrastructures that enable researchers to: (a) remotely access distributed computing resources and big data sets, and (b) effectively collaborate with remote peers, at a global-scale. However, wide-adoption of these advances has been a challenge to researchers mainly due to limitations in traditional cyberinfrastructure equipment, policies and engineering practices at campuses.

This project addresses the adoption challenges for researchers within The Ohio State University (OSU), and for their collaborators at the state, national and international levels. The project team is integrating advanced technologies (e.g., 100Gbps connectivity, perfSONAR, OpenFlow, RoCE/iWARP) relevant to the use cases of diverse OSU researchers in a "Science DMZ" environment. A 100Gbps border router at OSU will be setup to connect to the state-funded OARnet-Internet2 peered 100Gbps network in support of the use cases. The project will investigate the tradeoffs to be balanced between researcher flow performance and campus security practices. Project activities also involve wide-area network experimentation to seamlessly integrate OSU's cyberinfrastructure and Science DMZ with a remote campus (at University of Missouri) cyberinfrastructure. This project will create and document the role of a "Performance Engineer on campus", who will be the primary "keeper" and "help-desk" of the Science DMZ equipment, and will function as a liaison with researchers and their collaborators to configure wide-area cyberinfrastructures. Best-practices are to be published, and open-source software applications will be developed for handling researcher application flows in production networks across diverse science/engineering disciplines and multiple university campuses.