Thesis (Ph. D.)--West Virginia University, 2010. / Title from document title page. Document formatted into pages; contains x, 182 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Master of Science / Department of Anatomy and Physiology / Lei Wang / Jishu Shi / Interleukin-1 beta (IL-1β) is an important mediator of inflammatory response, and the elevated expression of IL-1β is correlated with tumor growth and metastasis. Epithelial-mesenchymal transition (EMT) is a reversible transition between epithelial phenotype and mesenchymal phenotype. Usually, EMT can be identified by its unique morphology change and expression of EMT markers. In our study, we have found after treated HCT-116, a colon cancer cell line, and human primary colon cancer cells with IL-1β, cells began to display mesenchymal phenotype with highly down-regulated E-cadherin expression and up-regulated ZEB factors expression. For colon cancer cells, sphere formation assay in serum free medium (SFM) with the presence of growth factors is used to identify cancer stem cell population. We have shown that IL-1β can induce colon cancer stem cell proliferation and express stem cell markers (Bmi1, Nanog, and Nestin). In addition, besides the stem cell markers, we also found ZEB factors were highly up-regulated in spheroid cells as well. We silenced Zeb1 expression and investigated the effect of IL-1β on shZeb1 HCT-116 cells. The results indicated Zeb1 knockdown not only inhibited IL-1β-induced EMT but also reduced proliferation of spheroid cells and inhibited Bmi1 expression. Therefore, ZEB factors must play an important role in both EMT process and cancer stem cell development. From our data, we conclude that IL-1β promotes epithelial-mesenchymal transition and a stem cell phenotype in colon cancer via ZEB factors.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg for the Degree of Master of Science / The antiinflammatory effects of lnterleukin-4 (IL-4) and the synthetic glucocorticoid dexamethasone were studied in adhered monocytes and the leukaemic cells HL-60 and THP-1, with respect to the expression of interleukin-lB (IL-lLB), the (signalling) IL-1 receptor type I (IL..1RtI), and the (inhibitory) IL-1 receptor type I (IL-lRtI). (Abbreviation abstract) / AC2018

Neuroinflammation is a major driver of secondary brain cell death after ischaemic stroke, seizure activity and traumatic brain injury. In a model of excitotoxic neuroinflammation, striatal injection of a toxic dose of AMPA causes cell death in the striatum after 24 hours. Co-injection of AMPA with the pro-inflammatory cytokine interleukin-1β (IL-1β) leads to additional cortical cell death. Injected alone, IL-1β leads to little or no cell death. It is hypothesised that IL-1β may exacerbate cell death by interfering with blood flow coupling. In the first study, two-dimensional optical imaging spectroscopy was used to measure early changes in the haemodynamic response in the anaesthetised rat barrel cortex before and for 6 hours after injection of vehicle, AMPA, IL-1β, or AMPA+IL-1β. After injection of IL-1β, with or without AMPA, the oxygenated blood flow response to mechanical whisker stimulation approximately halved over the course of 6h. In the second study, to determine whether the IL-1β-dependent changes in blood flow response are reflected by altered cellular activity, local field potentials, multi-unit activity and local tissue oxygenation responses to whisker stimulation were recorded simultaneously from the active barrel before and up to 6h after injection. A similar reduction in the size of the oxygenation response was seen again in the IL-1β- and AMPA+IL-1β-treated groups. Importantly, the level of gamma frequency oscillations at stimulus onset decreased within the first hours after injection of AMPA+IL-1β or IL-1β, suggesting a disruption of the fast-spiking interneuron network in the barrel cortex. These findings, along with histological observations of IL-1β-dependent markers of neuroinflammation, suggest that IL-1β may exacerbate AMPA-induced excitotoxicity by potentiating seizure activity and decoupling the neurovascular response in the cortex.

Interleukin-1-beta (IL-1β) is a potent proinflammatory cytokine. A primary outcome of IL-1β signaling is the activation of NFκB, a transcription factor that induces a large number of immune molecules, apoptotic factors, anti-apoptotic factors, and other transcription factors. Recent work has demonstrated that the activation of NFκB involves a multistep redox-signaling cascade that requires endocytosis of the interleukin receptor (IL-1R1)/ligand pair and superoxide production by NADPH oxidase 2 (Nox2) within the resulting newly formed early endosome. Hydrogen peroxide produced by the rapid dismutation of superoxide is necessary for the subsequent downstream recruitment of IL-1R1 effectors (TRAF6, IKK kinases) and ultimately the activation of NFκB. In this thesis, I have further dissected the spatial and temporal events that coordinate signaling processes of the IL-1β pathway. Using a combination of biophotonic imaging, immunofluorescence imaging, and lipid raft density gradient isolation, I demonstrate that both Nox2 and IL-1R1 are constitutively present in lipid raft microdomains on the plasma membrane. Stimulation by IL-1β induces endocytosis of Nox2 and IL-1R1 from the plasma membrane into caveolin-1, lipid raft positive early endosomes. Further, inhibition of lipid raft mediated endocytosis or deletion of caveolin-1 inhibits activation of NFκB, by IL-1β. We have also identified Vav1 as the Rac1 guanine exchange factor that is recruited to caveolin-1 positive lipid rafts following IL-1β stimulation, and demonstrated that dominant negative Vav1 inhibits NFκB activation by IL-1β. Following this work, I utilized assays for redox sensitivity and mass spectrometry to demonstrate that C70, C73, and C105 are hydrogen peroxide sensitive cysteines within the RING domain of TRAF6. I further demonstrate that hydrogen peroxide does not alter the E3 ubiquitin ligase activity associated with the TRAF6 RING domain. My findings suggest that the redox sensitivity of the RING domain mediates TRAF6 recruitment to the receptor complex. This is supported by the observation that hydrogen peroxide treatment of TRAF6, but not early signaling effectors (IL-1R1, IRAK1, IRAK4, MyD88) mediates TRAF6 recruitment to the IL-1 receptor complex. Further, mutation of the identified redox sensitive cysteines inhibits IL-1β signaling and NFκB activation. This research has helped to refine the understanding of the IL-1β signaling pathway, and may ultimately lead to new therapeutic targets for controlling inflammation.

<p>Innate immunity has received impressive attention in the past decade owing to the discovery of the Toll like receptors (TLRs) and the NOD-like receptors (NLRs). While the TLRs specialize in fighting microbes at the cell surface, the NLRs complement by detecting and responding to intracellular microbes. Recently, the non-microbe sensing NLR called inflammasomes, have been identified, which senses metabolic stress as well as certain pathogenic microbes and elicits host’s inflammatory response. <strong></strong></p><p>The NLR, NALP3 (formerly known as cryopyrin) forms a large cytoplasmic complex called the ‘inflammasome’ when NALP3, activated by a stimuli, associates with the adaptor proteins ASC and CARD-8. This interaction leads to the activation of pro-inflammatory caspase-1 which subsequently results in the formation of Interleukin (IL)-1β and IL-18. Mutations in the gene encoding NALP3, termed <em>NLRP3</em> can lead to its constitutive activation resulting in an uncontrolled production of IL-1β. These mutations have been implicated in hereditary inflammatory diseases, often grouped under cryopyrin associated periodic syndromes (CAPS).</p><p>This thesis describes a patient with a long history of arthritis and antibiotic resistant fever, but without the typical symptoms of CAPS. The patient was found to be a heterozygous carrier of two common polymorphisms Q705K in <em>NLRP3 </em>and C10X in the <em>CARD-8</em>. Experimental studies showed elevated levels of caspase-1 and IL-1β in the patient, and a total clinical remission was achieved by IL-1β blockade. These two polymorphisms combined, were found to occur in approximately 4% of the control population, suggesting the possibility of a genetic predisposition for inflammation in these individuals. Therefore, a cohort of rheumatoid arthritis (RA) patients, where elevated IL-1β could be one of the reasons behind chronic inflammation, was investigated. We found that carrying the combined polymorphisms resulted in increased RA susceptibility and a more severe disease course. Hypothetically, this subgroup of patients might benefit from IL-1β blockade. Additional studies are warranted to elucidate the functional effects of the two polymorphisms and to determine whether they identify a subgroup of patients that could benefit from IL-1 targeted therapy. Given the structural similarity of NALP3 to other NALPs, the possibility of involvement of the alternative, homologous genes cannot be eliminated.</p>

IL-1 is an essential pro-inflammatory factor in inflammation response. The effect of IL-1 is through binding to the IL-1 receptor that triggers the following signal transduction pathway. To study the role of IL-1 receptor-mediated signal pathway in inflammatory response, injecting P. aeruginosa into trachea of wild-type (WT) and type-1 IL-1 receptor knock-out (IL-1R1-/-) mice was used as the experimental model. Injecting bacterium into trachea of mice will induce pneumonia which increases accumulation of neutrophils, production of nitric oxide, expression of intercellular adhesion molecule-1 as well as many kinds of cytokines and causes the lung damage. The pneumonia-induced lung damage and inflammation at 24 hr after injecting P. aeruginosa into trachea were more severe in knock-out than in WT mice, as demonstrated by increases in extravasations of Evans blue dye (EBD), myeloperoxidase (MPO) activity, expression of iNOS, IL-1 beta and ICAM-1, and higher mortality of knock-out mice. The cause of the high mortality in knock-out mice was further investigated by culturing the lung and blood samples for bacterial counts. The bacterial counts of lung and blood of IL-1R1-/- mice were all higher than that of WT mice in 8 to 24 hr after injection of bacterium. Finally, chimeric mice (WT ¡÷ WT, IL1R1-/- ¡÷IL1R1-/-, WT ¡÷ IL1R1-/-, IL1R1-/- ¡÷ WT) were generated and used to determine the role of PMN cells of blood. Suggesting that increased amounts of bacteria in lung and blood is related to the higher mortality in knock-out mice and the type-1 IL-1 receptor is essential for mice to against pneumonia in this model.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / "May 2001." Typescript. Vita. Includes bibliographical references (leaves 180-198). Also available on the Internet.

The autonomic nervous system serves to maintain a constant inner environment, a process termed homeostasis. Thus, in response to the homeostatic challenge posed by infectious agents, the autonomic nervous system answers to signals from the immune system and elicits adaptive physiological and behavioral reactions. These so called sickness responses include fever, anorexia, hyperalgesia, social avoidance, and the release of stress hormones. Neuropeptides, used in the communication between neurons, are because of their release properties and sustained actions likely mediators of homeostatic responses. The enkephalinergic system constitutes one of the largest neuropeptidergic systems in the brain, but its involvement in inflammatory conditions has been little studied. We first examined the immune-induced activation of the parabrachial nucleus (paper I), an enkephalinergic autonomic relay center in the brain stem. We found that intravenous injection of bacterial endotoxin, lipopolysaccharide (LPS), activated the external lateral parabrachial subnucleus, as measured in terms of Fos expression, but that the enkephalinergic cell population in this subnucleus was largely separated from the LPS-activated neurons. Because Fos may not always be a reliable activity marker, we next examined by in situ hybridization the immune-induced expression of newly transcribed preproenkephalin (ppENK) heteronuclear RNA (hnRNA), which gives a direct indication of the utilization of enkephalin in a particular neuron (paper II). We detected induced expression of ppENK hnRNA in several autonomic structures in the brain, including the paraventricular hypothalamic nucleus (PVH) but not the parabrachial nucleus, indicating increased enkephalinergic signaling activity in the positively labeled structures during inflammatory condition. We then examined the projections of the immune-induced ppENK transcribing PVH neurons by injecting rats intraperitoneally with the retrograde tracer substance Fluoro-Gold, hence labeling neurons with axonal projections outside the blood-brain barrier, followed by systemic injection of LPS (paper III). Dual-labeling histochemical and hybridization techniques showed that the vast majority of the ppENK hnRNA expressing cells were hypophysiotropic cells, hence being involved in neuroendocrine regulation. These findings suggest that centrally produced enkephalin is involved in the coordination of the sickness responses during systemic immune challenge, including the modulation of the release of stress hormones or other hypothalamic hormones during inflammatory conditions. We next turned to the role of prostaglandins in the hypothalamic-pituitary-adrenal (HPA) axis response to inflammation. We injected mice deficient for the terminal prostaglandin (PG) E2 synthesizing enzyme mPGES-1 with LPS and studied their stress hormone release (paper IV). The genetically modified mice displayed attenuated plasma levels of adrenocorticotropic hormone (ACTH) and corticosterone during the later phases of the HPA-axis response compared with wild type mice, and this impairment did not depend on a changed activation pattern in the brain, but instead correlated to an early decrease in corticotropin-releasing hormone mRNA expression in the PVH, hence being the likely cause of the blunted ACTH and corticosterone responses at later time-points. Based on these findings we suggest that a neural, mPGES-1-independent pathway, and a humoral, mPGES-1-dependent pathway act in concert but in distinct temporal patterns to initiate and maintain the HPA-axis response during immune challenge. In addition to activating the central limb of the HPA-axis, inflammatory mediators have been suggested to act directly on the adrenal gland to induce the release of corticosterone, but little is known about the underlying mechanisms. We examined adrenal tissue isolated from rats injected with LPS or interleukin-1β (IL-1β) (paper V), and found that immune stimulation resulted in dynamic changes in the adrenal immune cell population, implying a rapid depletion of dendritic cells in the inner cortical layer and the recruitment of immature cells to the outer layers. These changes were accompanied by an induced production of IL-1β and IL-1 receptor type 1, as well as of cyclo-oxygenase-2 and mPGES-1 in these cells, implying local cytokine-mediated PGE2 production in the adrenals, which also displayed EP1 and EP3 receptors in the cortex and medulla. Additional mechanistic studies using an IL-1 receptor antagonist showed that IL-1β acts locally to affect its own synthesis, as well as that of cyclooxygenase-2. Taken together these data demonstrate a mechanism by which systemic inflammatory agents activate an intrinsically regulated local signaling circuit that may influence the adrenals’ response to immune stress and may help explain the dissociation between plasma levels of ACTH and corticosteroids during chronic immune perturbations.

Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 1998? / Bibliography: leaves 267-345.