Acanthamoeba culbertsoni is an opportunistic free-living amoeba that is causative of granulomatous amoebic encephalitis (GAE), a chronic and often fatal central nervous system (CNS) disease that is most prevalent in immune compromised individuals. One hallmark of this disease is the formation of granulomas within the CNS, which are commonly absent in immune compromised individuals. Granulomas are usually composed of amoebae, microglia (CNS macrophages), macrophages, T cells, B cells, and neutrophils. Previous studies have demonstrated that microglia respond to Acanthamoeba by producing pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF)-α, interleukin (IL)-1α, and IL-1β. In addition, activated microglia and macrophages have been demonstrated to be cytolytic (i.e., amoebicidal) to Acanthamoeba. Furthermore, previous studies also indicated that Acanthamoeba secrete a myriad of factors including proteases. The role of these proteases during GAE has not been fully elucidated; however, it is thought that these factors may aid in the chronic persistence of Acanthamoeba within the CNS by modulating the host immune response. Using two-dimensional (iso-dalt) gel electrophoresis, we demonstrated that A. culbertsoni secrete factors that degrade culture medium proteins. Initial gelatin zymography studies demonstrated that propagation of A. culbertsoni in medium with high iron content leads to augmentation of protease activity. Gelatin zymography in concert with protease inhibitors demonstrated that A. culbertsoni secrete proteases predominantly of the serine protease class. Using an in vitro co-culture model, we demonstrated that co-culture of A. culbertsoni with mouse microglial-like cells (BV-2 cells) results in the augmentation of A. culbertsoni serine protease activity and stimulation of pro-inflammatory cytokine and chemokine protein expression by microglial-like cells. However, the A. culbertsoni-elicited proteases were shown to degrade microglial-like cell elicited cytokines and chemokines. Collectively, our results suggest that A. culbertsoni- secreted serine proteases may play a role in A. culbertsoni CNS immune evasion by increasing A. culbertsoni CNS dissemination via the diminution of granuloma formation and by dampening microglial-dependent cytokine response.

Neuroinflammation is considered to be an important underlying process in the pathology of major depressive disorder (MDD) within a subpopulation of patients. MDD is associated with increased levels of proinflammatory cytokines in the blood, and cytokine-based treatments can induce depression. In mice, the induction of systemic inflammation with lipopolysaccharide (LPS) can induce depressive-like behaviours that are associated with symptoms of MDD. Microglia mediate the neuroinflammatory response within the brain and have a critical role in inflammation-induced depressive- like behaviours. Microglia within the brain exist in low O2 conditions (~5 %), though experimentation in vitro is typically carried out in high O2 conditions (20 %). The NLRP3 inflammasome is a molecular complex central to the production of the proinflammatory cytokine IL-1β and the propagation of the inflammatory response. NLRP3 inflammasome activity has been implicated in chronic stress and inflammation-based models of depressive-like behaviours in mice. The aims of this thesis were to study LPS-induced depressive-like behaviour in C57BL/6J mice, the role of NLRP3 in the behavioural output and the influence of oxygen (O2) availability on NLRP3 inflammasome activity in microglia cell cultures. Acute LPS induced depressive-like behaviours were observed in hedonia-based tasks but not in the forced swim test (FST). However, acute LPS induces a brief period of inflammation that does not address the sustained nature of depression. A FST depressive-like behaviour was observed in a novel 3-day increasing dose LPS model of sustained inflammation, whilst circumventing the development of LPS tolerance. The LPS-induced sickness was partially dependent upon NLRP3, though the resulting depressive-like behaviour was not. NLRP3 inflammasome signalling in microglia was studied in 5 % O2 conditions to replicate the hypoxic environment within the brain. Primary microglia isolated from mixed glial cultures by mild trypsinisation exhibited functional NLRP3 inflammasome expression and activity. When exposed to 5 % O2 (24 hours), NLRP3 inflammasome activity and adenosine triphosphate (ATP)-induced cell death was attenuated, whilst the production of other proinflammatory cytokines were unaffected. These data demonstrate the O2 sensitivity of NLRP3 inflammasome signalling in microglia. This thesis demonstrates a novel model of sustained inflammation and that inhibiting NLRP3 signalling may provide a target for attenuating neuroinflammation and the resulting behavioural changes. The importance of understanding the influence of O2 in microglia function and neuroinflammation was highlighted by the sensitivity of NLRP3 inflammasome activity to low O2.

No description available.

Inhibitors of apoptosis (IAPs) modulate cell death and play critical role in signal transduction that promotes inflammation. Recently, Smac mimetics, which are IAP antagonists, have attracted attention as novel cancer therapeutics. Cellular Inhibitor of Apoptosis 2 (cIAP2), a member of IAP family, positively affects both NF-κB and MAPK activation in response to many inflammatory stimuli. We observed that the lack of cIAP2 ablates LPS-induced neuroinflammation. Also, cIAP2-/- macrophages demonstrated diminished antigen presentation potential that could contribute to ablated immunity. In addition to these functions, we have previously reported that cIAP2 also regulates the activation of Interferon Regulatory Factor 1 (IRF1). Since IRF1-/- mice are resistant to experimental autoimmune encephalomyelitis (EAE), we hypothesized that cIAP2-/- mice will be protected from the disease. Surprisingly, induction of EAE in cIAP2-/- mice resulted in exaggerated infiltration of immune cells increased expression of proinflammatory cytokines and demyelination within CNS. We found that the lack of cIAP2 induces caspase-8 expression in microglia derived macrophages, contributing to their activation and polarization towards M1 phenotype, and exacerbates the symptoms of EAE. These findings suggest that cIAP2 limits neuroinflammation in the CNS and thus the use of Smac mimetics as chemotherapeutics needs to be reevaluated.

Alzheimer’s disease (AD) is a progressive, neurodegenerative disorder and the most common form of dementia. The hallmark pathologies of AD are extracellular aggregates of amyloid-beta, intracellular aggregates of microtubule associated protein tau and increased neuroinflammation. Current therapeutics offer only symptomatic relief and clinical trials investigating therapeutic benefits of non-steroidal anti-inflammatory drugs have yielded no positive results. Therefore, recent work has focused on immunomodulators, such as CD200 and fractalkine, as potential therapeutic targets for AD. Fractalkine (CX3CL1; FKN) is expressed as a transmembrane protein with an N-terminal chemokine domain followed by a long, mucin-like stalk. FKN can signal as a membrane-bound protein or, upon cleavage, as a soluble ligand (sFKN). Upon binding its receptor, FKN reduces expression of pro-inflammatory genes in activated microglia. Disrupting FKN signaling has been shown to exacerbate neurodegeneration in a number or neurodegenerative diseases. Relevant to this study, there have been conflicting reports on how FKN signaling affects AD pathology and whether a soluble FKN is beneficial or not. Here, we examine the ability of soluble FKN over expression to impact tauopathy and the resulting cognitive deficits in the rTg4510 mouse model of tauopathy, focusing on cognitive improvement after the onset of tau deposition. Furthermore, we explore the functional activity of proteolytic fragments of FKN on activated microglia in vitro to rectify the contradictory findings in the literature. We observed that sFKN over expression can significantly reduce both soluble and insoluble phospho-tau in both a preventative and an early interventional study design. However, in animals with significant pathology and neurodegeneration we did not observe an impact of sFKN over expression on tau pathology. Interestingly, in these late stage animals we did observe an improvement in spatial learning and memory as well as a reduction in hyperactivity. This suggests that earlier intervention would likely be most beneficial in reducing tau pathology but in late stage AD FKN signaling can still have benefits on cognition, likely due to reductions in the inflammatory milieu. Current publications suggest that different proteolytic fragments of FKN may have different functional signaling. Here we demonstrate that the this may be due to differences in receptor binding. sFKN (which includes the mucin-like stalk) exhibited a lower EC50 than the ckFKN (soluble chemokine domain), which leads to reduced functional efficacy of ckFKN at low concentrations. More interestingly, we also observed that high concentrations of FKN, regardless of cleavage variant, is ineffective at reducing pro-inflammatory activation of microglial and may in fact elicit a proinflammatory response. We hypothesize that FKN may signal through an alternative receptor at high concentrations, suggesting an as yet unidentified signaling pathway for FKN. Furthermore, we show that the ckFKN does not rescue pathology in the rTg4510 mouse, as sFKN does. These data may clarify conflicts in the literature and demonstrate that care must be taken with respect to in vitro and in vivo studies using FKN.

Abstract not available

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 127-155).

Inflammation can be up-regulated by microglia and macrophages through the release of pro-inflammatory cytokines such as tumour necrosis factor-alpha (TNF-α). Excess production of TNF-α can lead to a variety of diseases and even tissue necrosis. Recently, the expression of alpha seven acetylcholine receptors (α7AChR) by microglia have been shown to decrease the amount of TNF-α released. This anti-inflammatory pathway has been studied extensively where researchers are able to reduce TNF-α concentration through α7AChR expression and increases in the concentration of its ligand. I have shown that Neuregulin is able to increase the expression of α7AChR in microglia and macrophages. Using three immortalized cell lines, BV-2, EOC-20 and RAW 264.7, and primary microglial cells harvest from mice I investigated the role that neuregulin plays in the anti-inflammatory process. Neuregulin signals through the ErbB receptors, a family of tyrosine kinase receptors, to facilitate the effects on ACh expression. My results show that ErbB4 is expressed in BV-2, EOC-20 and RAW 264.7 cell lines while ErbB2-4 receptors are expressed in primary microglia. As well, I was able to show that ErbB4 became phosphorylated upon binding to NRG in immortalized cell lines. Using an Enzyme Linked Immunsorbent Assay to analyze TNF- α concentration in microglia and macrophages, I was able to demonstrate that increased levels of α7AChRs did not result in a reduction in TNF-α concentration. These results showed that NRG is able to increase α7AChRs in microglia and macrophages after the phosphorylation of the ErbB4 receptors. As well, this increase in α7AChR does not relate to a reduction in TNF-α, thus under these experimental conditions does not have an effect on the anti-inflammatory pathway.

Inflammation can be up-regulated by microglia and macrophages through the release of pro-inflammatory cytokines such as tumour necrosis factor-alpha (TNF-α). Excess production of TNF-α can lead to a variety of diseases and even tissue necrosis. Recently, the expression of alpha seven acetylcholine receptors (α7AChR) by microglia have been shown to decrease the amount of TNF-α released. This anti-inflammatory pathway has been studied extensively where researchers are able to reduce TNF-α concentration through α7AChR expression and increases in the concentration of its ligand. I have shown that Neuregulin is able to increase the expression of α7AChR in microglia and macrophages. Using three immortalized cell lines, BV-2, EOC-20 and RAW 264.7, and primary microglial cells harvest from mice I investigated the role that neuregulin plays in the anti-inflammatory process. Neuregulin signals through the ErbB receptors, a family of tyrosine kinase receptors, to facilitate the effects on ACh expression. My results show that ErbB4 is expressed in BV-2, EOC-20 and RAW 264.7 cell lines while ErbB2-4 receptors are expressed in primary microglia. As well, I was able to show that ErbB4 became phosphorylated upon binding to NRG in immortalized cell lines. Using an Enzyme Linked Immunsorbent Assay to analyze TNF- α concentration in microglia and macrophages, I was able to demonstrate that increased levels of α7AChRs did not result in a reduction in TNF-α concentration. These results showed that NRG is able to increase α7AChRs in microglia and macrophages after the phosphorylation of the ErbB4 receptors. As well, this increase in α7AChR does not relate to a reduction in TNF-α, thus under these experimental conditions does not have an effect on the anti-inflammatory pathway.

Microglia have the potential to produce specific neurotrophic molecules in response to injury and brain diseases. Activated microglia are seen after brain injury or in neurological disease, such as Parkinson¡¦s disease (PD). PD is a progressive neurodegenerative disorder. Although its cause remains unknown, it is believed that enhanced oxidative stress is a major component in the pathogenesis of nigral cell death in PD. Previous results have shown that DA induces apoptosis of dopaminergic neurons in a time- and concentration- dependent manner. In addition, a number of studies have shown that Zn2+ may enter the cell to reach toxic concentrations and that Zn2+ concentration is higher in the striatum of the postmortem brains of PD patients than that of the control brains. We have previously shown that Zn2+ synergistically enhanced the dopamine- and H2O2- induced PC12 cell death. To study the role of microglia in the cell death, I have examined the effect of conditioned medium from a human microglia cell line on the PC12 cell death induced by dopamine and Zn2+. The result shows that conditioned medium inhibits the PC12 cell death and the phosphorylation of JNK induced by dopamine and Zn2+ is diminished by the conditioned medium. It appears that the factor(s) that are responsible for the protection is heat-stable because the conditioned medium heated in 70¢Jfor 30 minutes still has the ability to protect the cell death. Cell death induced by A23187 and C2-ceramide, but not by staurosporine can be protected by the conditioned medium. Results from this study suggest that the microglia secrete some factors which can protect neuron.