br METHODS br RESULTS br Discussion Numerous studies

METHODS
RESULTS
Discussion Numerous studies have proposed various hypotheses and models to explain how neuroinflammation could contribute to the chronic fatigue, postexertional fatigue, and cognitive deficits observed in patients with ME/CFS.12, 13, 14, 15 Although neuroimaging studies have found structural and functional alterations in the brains of patients with ME/CFS, only a single study has presented evidence of an increased activation of astrocytes and microglia in the brain of patients with ME/CFS, suggesting that widespread neuroinflammation was occurring. However, the underlying mechanisms that contribute to this neuroinflammatory reaction or reactions in patients with ME/CFS remain undefined. We have previously found that a subgroup of patients diagnosed with ME/CFS exhibited a statistically significant elevation in angiotensin 1 against the EBV dUTPase protein. In the present study we provide further evidence to support a mechanism by which abortive-lytic reactivation of a systemic latent infection of EBV and subsequent production of dUTPase protein, which occurs in a subgroup of patients with ME/CFS, could contribute to the development of a neuroinflammatory microenvironment in the brain by modulating BBB, microglia cell, and astrocyte gene expression/function, tryptophan, DA and serotonin metabolism, and synaptic plasticity, which in turn may contribute to the increased pain, postexertional fatigue, and cognitive impairments observed in some patients with ME/CFS. The BBB is composed of endothelial cells of the capillary wall, astrocytes end-feet ensheathing the capillary, and pericytes embedded in the capillary membrane. The function of the BBB is to prevent the free diffusion of substances and the movement of cells from the systemic circulation into the CNS (brain and spinal cord) and, thus, prevent unwanted activation of brain cells. We have previously reported that the EBV dUTPase protein induces the secretion of the proinflammatory cytokines TNF-α, IL-6, and IL-1β in hDCs and peripheral blood mononuclear cells.28, 29, 30 In this study we found that the EBV dUTPase protein strongly induces the expression of IL-6 and IL-1β in cerebral microvascular endothelial cells and microglia cells as well as TNF-α in both astrocytes and microglia cells. These proinflammatory cytokines and interferon-γ have been reported to disrupt BBB integrity. The EBV dUTPase also downregulated in vitro or in vivo the expression of genes that encode for the proteins which have critical and direct implications for both forming and maintaining tight junctures between endothelial cells in capillaries comprising the BBB as well as modulating cellular adhesion and the extracellular matrix. Altogether, these data suggest that the EBV dUTPase protein has the capacity to disrupt the BBB, which could result in neuroinflammation and/or neurodegeneration. In addition, the EBV dUTPase protein also induced a transient increase in the expression of PTGS2/COX-2 in astrocytes, whereas in microglia cells the dUTPase induced a strong and sustained PTGS2 expression, suggesting that microglia cells were the primary source of this proinflammatory enzyme. Cox-2 catalyzes the formation of prostaglandin E2, which is a key mediator of inflammatory responses. Although COX-2 is generally considered to be inducible, it is constitutively expressed in some glutamatergic neurons in the cortex and hippocampus, astrocytes, and microglia. The role of COX-2 as a contributor to neuroinflammatory toxicity in neurodegenerative disorders is well established. Our data also indicate that the EBV dUTPase altered the expression of genes involved with pain (GPR84 and GCH1) and fatigue (TBC1D1). Chronic fatigue and pain are characteristic symptoms in patients with ME/CFS.1, 2, 3 In addition to disrupting the integrity of the BBB and modulating genes involved with inflammatory processes, pain, and fatigue, our data suggest that the EBV dUTPase may alter synaptic plasticity in vivo, which is important in learning and memory processes, as indicated by the ability of the dUTPase protein to downregulate the expression of LIN7b, SYNPTO, and RAB33A and upregulate Egr-1 in mouse brain. These genes have critical functions in (1) ensuring proper localization of the GRIN2B subunit of the N-methyl-d-aspartate receptor (NMDAR), (2) long-term potentiation,55, 56 (3) mediating antegrade axonal transport of post-Golgi synaptophysin-positive vesicles and their fusion at growth cones, and (4) NMDAR-mediated downregulation of PSD95 and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPAR) trafficking, all of which are important for synaptic development, plasticity, and functions. AMPAR and NMDAR play critical roles in the plasticity of most excitatory synapses, as indicated by a number of neurologic disorders associated with synaptic dysfunction that have altered NMDAR and AMPAR expression, trafficking, and signaling. These data suggest that the EBV dUTPase is capable of altering synaptic structure and function as well as neuronal communication, which would affect cognitive processes.