Amy Lovett-Racke - US grants

Abstract Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) that causes progressive neurological deficits, which affects 400,000 people in the USA. This incurable, life-long disease is quite costly to the health care system and society with a conservative estimate of a national annual cost of $20 billion in 2007. The prevalence and incidence of MS are increasing worldwide. Thus, the need for strategies to prevent MS is greater than ever. The cause of MS remains unknown; it is generally accepted that the combination of genetic and environmental factors determines the disease susceptibility. Vitamin D deficiency is emerging as an important environmental risk of MS. Epidemiological studies have shown that MS frequency increases with higher latitude, which is inversely correlated with sunlight and vitamin D levels. Furthermore, studies of migration and sun exposure indicate susceptibility to develop MS is determined in childhood, although disease onset is later in life. Providing sufficient vitamin D in early life may be beneficial to prevent MS. What is not known is how early life vitamin D deficiency influences the susceptibility to adult-onset MS. No studies have investigated whether low vitamin D during early life enhances the risk of developing autoimmune demyelinating disease in adulthood. Vitamin D receptors are prevalent in both the immune system and central nervous system (CNS), making both systems potentially altered due to low vitamin D during early life. MS is the autoimmune disease most closely linked to sunlight/vitamin D levels suggesting that there is a CNS-specific component related to vitamin D signals that may contribute to the risk of developing MS. The longterm goal of this study is to understand the mechanism by which sufficient vitamin D in early life imprints the protection against MS development later in life. The overall objective of the proposed project is to compare the susceptibility of mice to EAE in which there is impaired vitamin D signaling in the immune system or CNS during early life. Our main hypothesis is that reduced vitamin D signaling during early life makes the CNS more vulnerable to inflammation, enhancing the susceptibility to autoimmune demyelinating disease. The rationale that underlies this study is that, once the correlation of early life vitamin D deficiency and MS susceptibility is fully defined, this will be the foundation for making a public health policy to manipulate this easily modifiable risk factor for MS. The specific aims are as follows: 1) determine if vitamin D insufficiency in the immune system in juvenile mice enhances susceptibility to EAE, and 2) determine if vitamin D insufficiency in the CNS in juvenile mice enhances susceptibility to EAE. The relative contribution of vitamin D signaling in the immune system versus the CNS is necessary to understand the role that vitamin D plays in MS risk, and how the data may be applicable to other autoimmune diseases and neurodegenerative diseases. Understanding how this environmental factor influence MS risks will be a significant step towards the ultimate goal - preventing MS.

Abstract Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) that causes progressive neurological deficits, which affects 400,000 people in the USA. In 2007, total MS costs in the USA exceeded $20 billion, and today it would cost $18 billion per year if every MS patient in the USA was treated with just one disease modifying drug, not to mention the additional costs of lost productivity and other medical care. The cause of MS remains unknown; it is generally accepted that the combination of genetic and environmental factors determines the disease susceptibility. Vitamin D deficiency is emerging as an important environmental risk of MS. What is not known is how early life vitamin D deficiency influences the susceptibility to MS. Previous studies have shown that higher serum vitamin D levels in MS patients are associated with lower risk of relapse, in agreement with its immune-modulatory functions. However, this immune-regulatory activity of vitamin D does not explain the onset of a CNS-specific autoimmune disease. A high vitamin D diet attenuates microglia activation and reduces demyelination, suggesting a neuroprotective role of vitamin D. Interleukin-34 (IL-34) has recently been shown to be essential for the homeostasis of microglia. IL-34 is produced by neurons in the CNS, and its expression reaches its peak during postnatal development then declines in adulthood and vitamin D can positively regulate IL-34 expression. Together, the expression timing/pattern and functions of IL-34 make it a prime candidate as a vitamin D-mediated neuroprotective molecule, which may ultimately contribute to decreased MS susceptibility. The longterm goal of this study is to understand the mechanism by which sufficient vitamin D in early life imprints the protection against MS development later in life. The overall objective of the proposed project is to use a transgenic mouse - neuron-specific inducible vitamin D receptor knockout mice to elucidate how impaired vitamin D signaling on neurons during early life alters microglia phenotypes. Our main hypothesis is that vitamin D primes microglia into the neuroprotective phenotype through enhancing the production of IL-34 and/or other factors in neurons in a developing CNS. The rationale that underlies this study is that, once the correlation of early life vitamin D deficiency and MS susceptibility is fully defined, this will be the foundation for making a public health policy to manipulate this easily modifiable risk factor for MS. The aims are: 1) Elucidate the functional role of IL-34 in vitamin D-mediated neuroprotection, 2) determine if vitamin D modulates IL-34 and inflammatory cytokine responses in the CNS during early life infection, and 3) determine if vitamin D insufficiency in early life enhances susceptibility to EAE, an animal model of MS. These outcomes will have an important positive impact because they will define the cellular mechanism of vitamin D-mediated neuroprotection in early life. Understanding how this environmental factor influence MS risks will be a significant step towards the ultimate goal - prevent MS.

Specific Aims Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system (CNS) that affects an estimated 1 million Americans1. The cause of MS is unknown, there is no cure, and the current therapies have limited efficacy. My laboratory focuses on identifying molecules critical to the pathogenicity of encephalitogenic T cells. Since the CNS is an immune-privileged tissue and immunological surveillance is limited, we hypothesize that encephalitogenic T cells express unique molecules that enhance their encephalitogenic capacity, which are distinct from the CD4 T cells that protect us from infection, and that these molecules may be therapeutic targets for MS. The goal of this study is to identify molecular targets in encephalitogenic effector CD4 T cells that could be therapeutically manipulated to minimize the differentiation of encephalitogenic T cells, as well as extinguish or anergize established encephalitogenic T cells, while sparing pathogen-specific CD4 T cells. We hypothesize that encephalitogenic T cells, regardless of whether they are Th1 or Th17, share specific molecular pathways that can be therapeutically targeted to halt progression of CNS autoimmunity. Aim 1: Determine the role of genes differentially expressed in both encephalitogenic Th1 and Th17 cells. Aim 2: Analyze the validity of encephalitogenic molecules as MS-specific therapeutic targets with minimal immune compromise. The role that these encephalitogenic-associated molecules play in the generation and/or function of encephalitogenic T cells will be determined in both mouse and human CD4 T cells, as well as whether there is a differential role of these molecules in pathogenic versus protective T cells. Many studies have focused on the differentiation of encephalitogenic T cells, but far fewer studies have analyzed the unique characteristics of encephalitogenic effector/memory T cells in MS. This data will help identify therapeutic targets in newly differentiating encephalitogenic T cells to limit the generation of potentially pathogenic T cells in MS. Importantly, this study will also identify molecules that are critical to the function of encephalitogenic effector/memory CD4 T cells that may be contributing to disease progression and may be less vulnerable to therapies that target T cell differentiation or specific T cell subsets. This study is novel in that the focus is on T cell encephalitogenicity, irrespective of whether the T cells have a Th1 or Th17 phenotype. Furthermore, this study will compare protective versus pathogenic CD4 T cell responses in MS patients to identify therapeutic targets that would not compromise protection to infections.

Abstract Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system (CNS) which can result in severe neurological deficits. The cause of the disease is unknown, the immune and neurodegenerative mechanisms underlying the pathophysiology of the disease are poorly understood, and current therapies are only partially effective at slowing disease progression. There is a tremendous need to investigate novel mechanisms that may be contributing to disease susceptibility and the pathophysiology of this disease. To this end, my laboratory has performed a large miRNA profiling study on naïve and effector/memory CD4 T cell in untreated MS patients to determine if miRNA may contribute to disease susceptibility or progression. MiRNA negatively regulate gene expression via the RNA interference pathway, and thus play a key role modulating the level of specific proteins in cells. We have identified at least two pathways that are altered in MS patients that may contribute to their susceptibility to develop MS. First, miRNAs targeting components of the Th2 cell differentiation pathway were elevated in MS patients T cells, skewing differentiation into pro-inflammatory Th1 cells. Second, miRNAs targeting the TGF? signaling pathway limited the differentiation of regulatory T cells. Thus, the defects observed in MS patients CD4 T cells may be at least partially mediated by miRNA dysregulation. In this proposal, we will address the following questions. Aim 1: Are MS patients? CD4 T cells defective in their ability to differentiate into Tregs in a miRNA-dependent manner? Preliminary data indicates that naïve CD4 T cells from MS patients fail to efficiently differentiate into Tregs. Using miRNA inhibitors, we will determine if this failure to differentiate into Tregs is dependent on specific miRNAs over-expressed in MS patients and whether Treg differentiation can be normalized MS patients? naïve CD4 T cells. Aim 2: How does over-expression of MS-associated miRNAs affect the development and progression of CNS autoimmunity in a mouse model? Using EAE, the role of miRNAs that target CD4 T cell differentiation into effector and regulatory T cells will be analyzed in vivo to complement the human in vitro experiments. Aim 3: Can expression level of miR-128, which targets both effector and regulatory CD4+ T cells, modulate the risk of CNS autoimmunity? We found that miR-128 targets proteins in the Th2 and TGF? signaling pathways, promoting the differentiation of Th1 cells and preventing the development of Tregs. Using CD4-specific miR-128-/- mice and mice overexpressing miR-128 in CD4 T cells, we will determine if loss of miR-128 in CD4 T cells minimizes susceptibility to CNS autoimmunity, normalizes CD4 T cell differentiation into effector and regulatory cells, and thus, is a potential therapeutic target to correct both effector and regulatory T cell defects in MS. In contrast, we will use mice overexpressing miR-128 in CD4 T cells to determine if miR-128 is sufficient to skew CD4 T cell differentiation and enhance the risk of developing CNS autoimmunity. This study will test the hypothesis that miRNA dysregulation in naïve CD4+ T cells is an underlying risk factor in CNS autoimmunity that can be therapeutically targeted to normalize effector and regulatory CD4 T cell function. If our hypothesis is correct, miRNA-based therapies may not only prevent CNS autoimmunity in susceptible individuals, but ameliorate autoreactive T cells in patients with MS.