Ning Quan - US grants

Two neuroimmune communication pathways, the ascending vagus nerve and cells of the blood-brain barrier, have recently been identified to relay signals of peripheral infection to the brain by inducing the expression of IL-1 and TNF-alpha in the central nervous system (CNS). Chronic expression of IL-1 and TNF-alpha in the CNS, however, has been shown to contribute to the pathogenesis of many CNS diseases including chronic fatigue syndrome, AIDS dementia, and various neurodegenerative diseases. Whether chronic peripheral infection can cause CNS diseases by driving chronic production of IL-1 and TNF-alpha in the brain has not been studied. In a recently created infectious disease model, striking patterns of neuropathological changes were found in the brain without infiltration of either the pathogen or peripheral inflammatory cells into the brain parenchyma. The neuropathological changes were closely associated with the chronic expression of IL-1 and TNF-alpha in the brain. These pathological changes was enhanced by blocking the inhibitory mechanisms for IL-1 and TNF-alpha expression and reduced by intracerebral administration of specific antagonists of IL-1 and TNF-alpha. Therefore, it is hypothesized that induction of IL-1 and TNF-alpha in the brain by chronic peripheral infection is a mechanism for the pathogenesis of CNS diseases. Using this infectious disease model, the following specific aims are proposed to test this hypothesis: 1) Determine and characterize the neurotoxic effects mediated by chronic CNS production of IL-1 and/or TNF-alpha; and 2) Determine the role of glucocorticoids and prostaglandins in regulating the chronic expression of IL-1 and TNF-alpha in the brain. Specific Aim 1 is designed to characterize the neurotoxic effects attributable to the chronic expression of IL-1 and/or TNF-alpha in the brain induced by neuroimmune activation. In Aim 2, whether glucocorticoids and prostaglandins importantly controls the levels of chronic expression IL-1 and TNF-alpha and the manifestation of related neurotoxic effects in the brain will be determined. Glucocorticoids and prostaglandins are the two major feedback inhibitory regulators for IL-1 and TNF-alpha expression. Finally, the use of anti-inflammatory drugs in modulating the neurotoxic effects of chronic CNS production of IL-1 and TNF- alpha will also be evaluated in Specific Aim 2. This study will attempt to elucidate the mechanisms of neurotoxicity caused by chronic activation of the pathways for neuroimmune communication. The results will also provide critical information regarding the use of anti-inflammatory drugs for the treatment of CNS diseases.

The overall goal of these studies is to elucidate the mechanisms mediating the recent finding that under certain conditions stress can enhance skin immunity. We initially reported that acute of short-duration stress induces a redistribution of immune cells from the blood to organs such as the skin. Since the skin is the body's first line of defense, we examined the functional consequences of this leukocyte trafficking using the delayed type hypersensitivity (DTH) response as an in vivo assay for skin cell mediated immunity. Studies showed that acute stress experienced immediately before primary ( sensitization phase) or secondary (challenge phase) antigen exposure significantly enhanced skin DTH. In contrast, chronic stress suppressed skin DTH. In agreement with these studies, several investigators have reported stress-induced enhancement of DTH to different antigens administered to different sites of sensitization and challenge. The long- term objective of our research program is to elucidate the neuroendocrine and immune mediators and health consequences of the bi-directional effects of acute versus chronic stress on immune function. The overall goal of the proposed studies is to elucidate the mechanisms mediating the effects of acute stress on leukocyte trafficking and skin immunity. These studies will use wild type and gene knockout mice, immunoneutralization, flow cytometry, immunohistochemistry, in situ hybridization, RT-PCR, and ELISA to conduct analyses at the level of the organism, cell, protein, and gene expression. Three specific aims will be addressed: 1)Identify cell adhesion molecules that mediate the stress-induced redistribution of leukocytes. 2) Identify leukocyte subpopulations that mediate a stress-induced enhancement of the sensitization and challenge phases of DTH. 3) Identify chemokines and cytokines that mediate a stress-induced enhancement of both phases of DTH. These studies are important because stress is suspected to play a role in the etiology of many diseases and we propose to study the effects of stress on two important immune parameters: Leukocyte trafficking, which is crucial for the surveillance and effector functions of the immune system. And DTH, which mediates aspects of immunoprotection (e.g. resistance to infections and cancer and post-vaccination immunity) and immunopathology (e.g. autoimmune, dermatitis, and granulomatous disorders). It is hoped that the elucidation of mechanisms such as those proposed here will facilitate the development of biomedical treatments designed to harness and individual's physiology to selectively enhance (during surgery, wound healing, infections, or cancer) or suppress (during autoimmune or inflammatory disorders) an immune response depending on the clinical needs to the patient.

DESCRIPTION (provided by applicant): Neuroimmune communication has been implicated in both physiological defense against infection and pathogenic events contributing to many disorders of the central nervous system (CNS). A key mediator for this communication is interleukin-1. The following evidences indicate IL-1 acts on cells of the blood brain barrier to affect the CNS: 1) The functional 1L-1 receptor (type I IL-1 receptor, IL-1r1) is mostly expressed on CNS endothelial cells, 2) IL-1-responsive immediate early gene IkappaBalpha is induced primarily in CNS endothelium after central IL-1 injection;3) inflammatory mediators such as the prostaglandins are induced in brain endothelial cells after both peripheral and central immune challenges;and 4) IL-1r1 is essential for mediating the recruitment of leukocytes across the BBB during the development of CNS immune responses. The central hypothesis of this proposal, therefore, is that IL-1 affects the CNS through the activation of CNS endothelial cells. This hypothesis can be tested directly in our recently created transgenic mice with which we can specifically inhibit the expression of IL-1r1 on endothelial cells. The long range goal of this application is to elucidate the role of BBB in mediating neuroimmune crosstalk. Using these transgenic animals, the following specific aims will be tested: 1) Determine the distribution of IL-1r1 protein expression in the CNS in normal and immunologically challenged mice. 2) Determine the role of endothelial IL-1r1 in mediating the activation of the neural circuits and the functional consequences induced by peripheral and central immune challenges. 3) Determine the role of endothelial IL-1r1 in the recruitment of leukocyte across the BBB. 4) Determine the role of endothelial IL-1r1 in mediating the development and progression of experimental autoimmune encephalomyelitis (EAE). The results of this project should provide a definitive analysis to determine the role of endothelial IL-1r1 in several aspects of neuroimmune interaction.

DESCRIPTION (provided by applicant): Type I interleukin-1 receptor (IL-1R1) is the functional receptor for IL-1. IL-1 mediates numerous activities in the neuroendocrine, nervous, and immune systems. The transcriptional regulation of IL-1R1 is poorly understood. Current work in our lab has identified three promoters that control the transcription of IL-1R1 in mouse. The activities of these IL-1R1 promoters are tissue-specific, cell type-specific and dependent on developmental stages. In addition, these promoters are found to be regulated by exogenous as well as endogenous genetic regulatory elements. In vivo, the activities of the three murine IL-1R1 promoters showed distinct distribution patterns. Further, these IL-1R1 promoters are differentially regulated by signaling molecules, such as glucocorticoids. A species comparison study between murine and human IL-1R1 structures has been conducted. Aligning the resulting human and murine IL-1R1 promoter regions revealed a conserved framework by which IL-1R1 gene in both species are likely to be regulated by conserved regulatory sequences. These preliminary discoveries lead us to define a genetic region termed IL-1R1 promoter complex. The central hypotheses of this application are: 1) the level and type of IL-1R1 mRNAs expressed in a given cell are regulated by the IL-1R1 promoter complex; 2) the transcriptional control mechanisms in the IL-1R1 promoter complex determine the tissue- and cell type-specific expression of IL-1R1; and 3) IL-1R1 promoter complex contributes to the precise response characteristics of IL-1R1-bearing cells when they are stimulated by signaling molecules. We will pursue the following specific aims: 1) Determine the pattern of distribution of the promoter-specific IL-1R1 expression; 2) Elucidate transcriptional control mechanisms of the IL-1R1 promoter complex; and 3) Investigate promoter activity of IL-1R1 gene in selected cell types after the cells are stimulated by different signaling molecules. The results will reveal the transcriptional mechanisms that allow tissue-specific and cell type-specific expression and regulation of IL-1R1 that may be critical for the vast diversity of IL-1R1 function in multiple systems. This study will contribute significantly to IL-1R1 biology and provide a new foundation for our understanding of how IL-1R1 transcription might be involved in pathogenesis. for IL-1R1 promoter complex in the neuroendocrine, nervous, and immune system This study investigates the transcriptional mechanisms that control the expression of the type 1 interleukin-1 receptor. This receptor is important for many functions in the nervous, neuroendocrine, and immune systems. The results will provide a foundation for the understanding of how dysregulation of the expression of type 1 interleukin-1 receptor may be involved in the pathogenesis of diseases in multiple systems.

DESCRIPTION (provided by applicant): Inflammatory cytokine interleukin-1 (IL-1) performs multiple functions in the central nervous system (CNS). Two receptors have been identified for IL-1: the type I IL-1 receptor (IL-1R1) which is generally accepted as the receptor through which IL-1 activates cellular signaling;and the type II IL-1 receptor (IL-1R2) which serves as a decoy receptor. IL-1R1 is thought to mediate most, if not all, IL-1-induced effects. Several recent studies, however, show that IL-1- induced effects including its influence on brain tissue damage after cerebral ischemia, activation of JNK signaling pathway, and repression of synaptrophin mRNA expression persist in IL-1R1 knockout animals, suggesting the existence of a heretofore unidentified receptor for IL-1. Recently, we found that IL-1R1 gene contains an internal promoter which drives the transcription of a shortened IL-1R1 mRNA. This mRNA is the template for the production of an IL-1 receptor protein that is identical with IL-1R1 at the C-terminus, but with a shorter extracellular domain at the N-terminus. This receptor retains all the intracellular signaling machinery of IL-1R1. We have termed this molecule IL-1R3. Our preliminary results show that the mRNA and protein for IL-1R3 are expressed in normal and the two strains of commercially available IL-1R1 knockout animals. In addition, IL-1[unreadable] binds specifically to IL-1R3 and causes an increase in voltage-gated potassium current in neurons. On the other hand, IL-1[unreadable] stimulation of IL-1R3 does not activate NF-kB, the classical IL-1 signaling pathway. We propose that IL- 1R3 is the IL-1 receptor that accounts for the currently unexplained effects of IL-1 in the brain. In this application, we will investigate: 1) interaction between IL-1 and IL-1R3, 2) the role of IL- 1R3 in IL-1-induced activation of signal transduction pathways, and 3) the influence of IL-1R3 on NMDA-induced acute neurotoxicity. The results of this application should fill, at least in part, the apparent gap in our current knowledge on IL-1/IL-1 receptor interaction and potentially lead to discoveries of specific agonists or antagonists for IL-1R3, which could have significant clinical applications. PUBLIC HEALTH RELEVANCE: This study investigates a new IL-1 receptor that we recently discovered. IL-1 mediates many immunological, neuroimmunological, and neurophysiological activities in the brain. Currently, the type I IL-1 receptor (IL-1R1) is the only receptor known that mediates IL-1 signaling. We found that the new IL-1 receptor (IL-1R3) has the potential to account for many currently unexplained IL-1 effects. The results of this study will fill an apparent gap in IL-1 biology and potentially generate clinically useful molecules for IL-1 related brain disorders.

DESCRIPTION (provided by applicant): It is well known actions of interleukin-1 (IL-1) in the brain contributes to numerous behavior abnormalities, including illness behavior, heightened anxiety, major depression, and impairment in learning and memory. However, cellular mechanisms by which IL-1 affects neurons involved in these behaviors are unclear. In this exploratory grant, we will create a novel genetic mouse model that selectively expresses IL-1R1 on a defined cell type. In this mouse model, we can restrict cell type specific expression of IL-1R1 under its native promoters, thus permitting analysis of IL-1-mediated effects in mice which express IL-1R1 at physiological levels only in the cell type(s) of interest. Additional advantages of this model are IL-1R1 gene expression at mRNA level is able to be monitored by a knockin red fluorescence and detection of IL-1R1 protein will be facilitated by a 3HA tag added to the C-terminus of the IL-1R1 protein. We will use this model to determine the role cell type-specific IL- 1R1 expression plays in both illness behavior and spatial learning/memory following immune stimulation.

? DESCRIPTION (provided by applicant): Increasing evidence points to inflammation as contributing factor for the pathogenesis of psychopathologies, including depression and anxiety. Work from our collaborative group has established that the inflammatory cytokine interleukin-1 (IL-1) is a crucial mediator of stress induced anxiety. The pathways by which IL-1 induces inflammatory anxiogenesis, however, remain unknown. In this R01 application, we will investigate the novel hypothesis that IL-1 acts on brain endothelial cells to trigger inflammatory microglial activation, which is required for the induction of anxiety behaviors. We will use our newly created IL-1R1 restore mouse lines to study cell type specific IL-1R1 mediated microglial activation and isolate the pathogenic pathways by which IL-1 contributes to anxiogenesis.

Project Summary Neuroinflammation is a significant contributor to most CNS disorders including neurodegenerative diseases and psychological disorders. A hallmark of neuroinflammation is the increased expression of the proinflammatory cytokine interleukin-1 (IL-1) in the brain. How IL-1 causes neuro- and psycho- pathologies is poorly understood. We have recently discovered that the receptor for IL-1, IL-1R1, is expressed in specific sets of neurons in the brain. These neuronal IL-1R1s, nIL-1R1, modulate neuronal activity via non-canonical signaling pathways to alter neuronal function and circuit connectivity. This application is designed to: 1) map nIL-1R1 distribution in the brain, 2)demonstrate the involvement of nIL-1R1 in neuroinflammation induced behavioral deficits and neuropathology, and 3) elucidate the mechanisms of nIL-1R1 mediated neuromodulation.

Abstract Inflammatory and anti-inflammatory cytokines contribute to neuronal hyperexcitability in pain transmission pathways. One of the inflammatory cytokines, Interleukin-1 (IL-1), is involved in many neuroimmune responses. In particular, it has been demonstrated that IL-1 induces acute pain, reduces morphine analgesic activity, is involved in tolerance development, and is necessary for chronic neuropathic and inflammatory pain. This has been studied in many ways, including use of an endogenous IL-1 receptor antagonist, and the use of IL-1 and IL-1R knockout mice. The problem with these experiments from a mechanistic standpoint is that IL-1 receptors are found endogenously on a variety of cell types in the brain, including astrocytes, microglia, endothelial cells, and neurons, and global knockout experiments can't define the cell types that mediate the actions of IL-1. The development of novel genetic models, in which the IL-1 receptor, IL-1R1, can be reciprocally knocked out and restored (IL-1R1r/r) from a global knockout, into individual cell types in the brain, spinal cord, and dorsal root ganglion permits a much more specific identification of the actions of IL-1 with respect to pain. Specific Aim 1 will study morphine analgesia and tolerance development in 5 mouse genotypes, wild type, global IL-1R1 KO, and mice in which the IL-1R1 receptor is restored selectively into neurons, endothelial cells, and astrocytes. We expect to find that the global KO animals and two of the restored mouse genotypes will have more potent and prolonged morphine analgesia and reduced tolerance development, as described in the literature. Furthermore, one of the restored mouse lines will act like the wild type animals with reduced morphine activity and will exhibit tolerance development. Aim 2 will examine two chronic pain models, spinal nerve ligation and Complete Freund's Adjuvant, using the same mouse genotypes, to determine which cell type mediates the development of chronic pain. Aim 3 will develop a new mouse model by crossing the IL-1R1r/r mice with c-Fos-Cre/ERT2 (TRAP2) mice. With this new genetic model, IL-1R1 will be restored only in mice that have been subjected to some pain stimulus. The use of these novel genetic models will pinpoint the actions of IL-1 with respect to opioid analgesia and pain to specific cell types and develop a collaboration that will be able to determine the mechanisms of IL-1 actions, in future R01 applications.