Leptin modifies the prosecretory and prokinetic effects of the inflammatory cytokine interleukin-6 on colonic function in Sprague–Dawley rats

Leptin ameliorates the prosecretory and prokinetic effects of the pro-inflammatory cytokine interleukin-6 on rat colon. Leptin also suppresses the neurostimulatory effects of irritable bowel syndrome plasma, which has elevated concentrations of interleukin-6, on enteric neurons. This may indicate a regulatory role for leptin in immune-mediated bowel dysfunction. In addition to its role in regulating energy homeostasis, the adipokine leptin modifies gastrointestinal (GI) function. Indeed, leptin-resistant obese humans and leptin-deficient obese mice exhibit altered GI motility. In the functional GI disorder irritable bowel syndrome (IBS), circulating leptin concentrations are reported to differ from those of healthy control subjects. Additionally, IBS patients display altered cytokine profiles, including elevated circulating concentrations of the pro-inflammatory cytokine interleukin-6 (IL-6), which bears structural homology and similarities in intracellular signalling to leptin. This study aimed to investigate interactions between leptin and IL-6 in colonic neurons and their possible contribution to IBS pathophysiology. The functional effects of leptin and IL-6 on colonic contractility and absorptosecretory function were assessed in organ baths and Ussing chambers in Sprague–Dawley rat colon. Calcium imaging and immunohistochemical techniques were used to investigate the neural regulation of GI function by these signalling molecules. Our findings provide a neuromodulatory role for leptin in submucosal neurons, where it inhibited the stimulatory effects of IL-6. Functionally, this translated to suppression of IL-6-evoked potentiation of veratridine-induced secretory currents. Leptin also attenuated IL-6-induced colonic contractions, although it had little direct effect on myenteric neurons. Calcium responses evoked by IBS plasma in both myenteric and submucosal neurons were also suppressed by leptin, possibly through interactions with IL-6, which is elevated in IBS plasma. As leptin has the capacity to ameliorate the neurostimulatory effects of soluble mediators in IBS plasma and modulated IL-6-evoked changes in bowel function, leptin may have a role in immune-mediated bowel dysfunction in IBS patients.

Paracetamol metabolism in postoperative patients

Introduction: Despite being available for more than 50 years, there is still much to learn about paracetamol. Postoperative analgesic regimens that maintain good pain control while minimising exposure to opiates are beneficial and paracetamol has had a resurgence in this role since an IV formulation came to market. However there is evidence to suggest currently licensed doses are sub-therapeutic, especially when administered orally or rectally. Higher, unlicensed doses are now being advocated but, prior to this study, there was little evidence of their safety in surgical patients. When assessing drug safety in surgical patients a number of surgery and patient related factors influence results, and these must be considered. Methods: Major and intermediate surgical patients were recruited from two hospitals in Ireland. They were administered IV paracetamol at either 9g or 4g daily doses. In addition they received daily sub therapeutic doses of four other medicines to indicate the activity of their CYP450 enzymes that are involved in paracetamol metabolism. Urine and blood samples were collected to determine paracetamol pharmacokinetics, CYP450 activity, inflammatory cytokine concentration and for evidence of hepatotoxicity. Results: There were 33 patients that participated in the study. There was no evidence of clinically significant hepatotoxicity occurring in any patient during the study period, but there could have been changes following this time. Paracetamol disposition was shown to change, however half-life remained relatively constant. There were a number of changes to the way paracetamol was metabolised following surgery that maintained this rate of elimination. Conclusion: Doses of up to 9g per day given to major surgical patients for up to five days postoperatively produced no evidence of hepatotoxicity. Further research is warranted to determine the clinical utility of these higher doses

The role of metabolism in the pathogenesis of adherent invasive Escherichia coli (AIEC)

Crohn’s disease (CD) is a chronic, relapsing inflammatory condition affecting the gastrointestinal tract of humans, of which there is currently no cure. The precise etiology of CD is unknown, although it has become widely accepted that it is a multifactorial disease which occurs as a result of an abnormal immune response to commensal enteric bacteria in a genetically susceptible host. Recent studies have shown that a new group of Escherichia coli, called Adherent Invasive Escherichia coli (AIEC) are present in the guts of CD patients at a higher frequency than in healthy subjects, suggesting that they may play a role in the initiation and/or maintenance of the inflammation associated with CD. Two phenotypes define an AIEC and differentiate them from other groups of E. coli. Firstly, AIEC can adhere to and invade epithelial cells; and secondly, they can replicate in macrophages. In this study, we use a strain of AIEC which has been isolated from the colonic mucosa of a CD patient, called HM605, to examine the relationship between AIEC and the macrophage. We show, using a systematic mutational approach, that while the Tricarboxylic acid (TCA) cycle, the glyoxylate pathway, the Entner-Doudoroff (ED) pathway, the Pentose Phosphate (PP) pathway and gluconeogenesis are dispensable for the intramacrophagic growth of HM605, glycolysis is an absolute requirement for the ability of this organism to replicate intracellularly. We also show that HM605 activates the inflammasome, a major driver of inflammation in mammals. Specifically, we show that macrophages infected with HM605 produce significantly higher levels of the pro-inflammatory cytokine IL-1β than macrophages infected with a non-AIEC strain, and we show by immunoblotting that this is due to cleavage of caspase-1. We also show that macrophages infected with HM605 exhibit significantly higher levels of pyroptosis, a form of inflammatory cell death, than macrophages infected with a non-AIEC strain. Therefore, AIEC strains are more pro-inflammatory than non-AIEC strains and this may have important consequences in terms of CD pathology. Moreover, we show that while inflammasome activation by HM605 is independent of intracellular bacterial replication, it is dependent on an active bacterial metabolism. Through the establishment of a genetic screen aimed at identifying mutants which activate the inflammasome to lower levels than the wild-type, we confirm our observation that bacterial metabolism is essential for successful inflammasome activation by HM605 and we also uncover new systems/structures that may be important for inflammasome activation, such as the BasS/BasR two-component system, a type VI secretion system and a K1 capsule. Finally, in this study, we also identify a putative small RNA in AIEC strain LF82, which may be involved in modulating the motility of this strain. Overall this works shows that, in the absence of specialised virulence factors, the ability of AIEC to metabolise within the host cell may be a key determinant of its pathogenesis.

Optimisation of immune effector function within the tumour microenvironment

Cancer represents a leading of cause of death in the developed world, inflicting tremendous suffering and plundering billions from health budgets. The traditional treatment approaches of surgery, radiotherapy and chemotherapy have achieved little in terms of cure for this deadly disease. Instead, life is prolonged for many, with dubious quality of life, only for disease to reappear with the inevitable fatal outcome. “Blue sky” thinking is required to tackle this disease and improve outcomes. The realisation and acceptance of the intrinsic role of the immune system in cancer pathogenesis, pathophysiology and treatment represented such a “blue sky” thought. Moreover, the embracement of immunotherapy, the concept of targeting immune cells rather than the tumour cells themselves, represents a paradigm shift in the approach to cancer therapy. The harnessing of immunotherapy demands radical and innovative therapeutic endeavours – endeavours such as gene and cell therapies and RNA interference, which two decades ago existed as mere concepts. This thesis straddles the frontiers of fundamental tumour immunobiology and novel therapeutic discovery, design and delivery. The work undertaken focused on two distinct immune cell populations known to undermine the immune response to cancer – suppressive T cells and macrophages. Novel RNAi mediators were designed, validated and incorporated into clinically relevant gene therapy vectors – involving a traditional lentiviral vector approach, and a novel bacterial vector strategy. Chapter 2 deals with the design of novel RNAi mediators against FOXP3 – a crucial regulator of the immunosuppressive regulatory T cell population. Two mediators were tested and validated. The superior mediator was taken forward as part of work in chapter 3. Chapter 3 deals with transposing the RNA sequence from chapter 2 into a DNA-based construct and subsequent incorporation into a lentiviral-based vector system. The lentiviral vector was shown to mediate gene delivery in vitro and functional RNAi was achieved against FOXP3. Proof of gene delivery was further confirmed in vivo in tumour-bearing animals. Chapter 4 focuses on a different immune cell population – tumour-associated macrophages. Non-invasive bacteria were explored as a specific means of delivering gene therapy to this phagocytic cell type. Proof of delivery was shown in vitro and in vivo. Moreover, in vivo delivery of a gene by this method achieved the desired immune response in terms of cytokine profile. Overall, the data presented here advance exploration within the field of cancer immunotherapy, introduce novel delivery and therapeutic strategies, and demonstrate pre-clinically the potential for such novel anti-cancer therapies.

The role of the IL-1 type 1 receptor in the life, death and differentiation of adult hippocampal neural precursor cells

Neurogenesis occurs in two distinct regions of the adult brain; the subgranular zone (SGZ) of the dentate gyrus (DG) in the hippocampus, and the subventricular zone (SVZ) lining the lateral ventricles. It is now well-known that adult hippocampal neurogenesis can be modulated by a number of intrinsic and extrinsic factors e.g. local signalling molecules, exercise, environmental enrichment and learning. Moreover, levels of adult hippocampal neurogenesis decrease with age, at least in rodents, and alterations in hippocampal neurogenesis have been reported in animal models and human studies of neuropsychiatric and neurodegenerative conditions. Neuroinflammation is a common pathological feature of these conditions and is also a potent modulator of adult hippocampal neurogenesis. Recently, the orphan nuclear receptor TLX has been identified as an important regulator of adult hippocampal neurogenesis as its expression is necessary to maintain the neural precursor cell (NPC) pool in the adult DG. Likewise, exposure of animals to voluntary exercise has been consistently demonstrated to promote adult hippocampal neurogenesis. Lentivirus (LV)- mediated gene transfer is a useful tool to elucidate gene function and to explore potential therapeutic candidates across an array of conditions as it facilitates sustained gene expression in both dividing and post-mitotic cell populations. Both intrinsic and extrinsic factors are important regulators of adult hippocampal neurogenesis. Examining how these factors are affected by an inflammatory stimulus, and the subsequent effects on adult hippocampal neurogenesis provides important information for the development of novel treatment strategies for neuropsychiatric and neurodegenerative conditions in which adult hippocampal neurogenesis is impaired. The aims of the series of experiments presented in this thesis were to examine the effect of the pro-inflammatory cytokine interleukin-1β (IL-1β) on adult hippocampal NPCs both in vitro and in vivo. In vitro, we have shown that IL-1β reduces proliferation of adult hippocampal NPCs in a dose and time-dependent manner. In addition, we have demonstrated that TLX expression is reduced by IL-1β. Blockade of IL-1β signalling prevented both the IL-1β-induced reduction in cell proliferation and TLX expression. In vivo, we examined the effect of short term and long term exposure to LV-IL-1β in sedentary mice and in mice exposed to voluntary running. We demonstrated that impaired hippocampal neurogenesis is only evident after long term exposure to IL-1β. In mice exposed to voluntary running, hippocampal neurogenesis is significantly increased following short-term but not long-term exposure to running. Moreover, short-term running effectively prevents any IL-1β-induced effects on hippocampal neurogenesis; however, no such effects are seen following long-term exposure to running.

Investigation of toll-like receptor tolerance in the regulation of inflammation

Inflammation is a complex and highly organised immune response to microbes and tissue injury. Recognition of noxious stimuli by pathogen recognition receptor families including Toll-like receptors results in the expression of hundreds of genes that encode cytokines, chemokines, antimicrobials and regulators of inflammation. Regulation of TLR activation responses is controlled by TLR tolerance which induces a global change in the cellular transcriptional expression profile resulting in gene specific suppression and induction of transcription. In this thesis the plasticity of TLR receptor tolerance is investigated using an in vivo, transcriptomics and functional approach to determine the plasticity of TLR tolerance in the regulation of inflammation. Firstly, using mice deficient in the negative regulator of TLR gene transcription, Bcl-3 (Bcl-3-/-) in a model of intestinal inflammation, we investigated the role of Bcl-3 in the regulation of intestinal inflammatory responses. Our data revealed a novel role for Bcl-3 in the regulation of epithelial cell proliferation and regeneration during intestinal inflammation. Furthermore this data revealed that increased Bcl-3 expression contributes to the development of inflammatory bowel disease (IBD). Secondly, we demonstrate that lipopolysaccharide tolerance is transient and recovery from LPS tolerance results in polarisation of macrophages to a previously un-described hybrid state (RM). In addition, we identified that RM cells have a unique transcriptional profile with suppression and induction of genes specific to this polarisation state. Furthermore, using a functional approach to characterise the outcomes of TLR tolerance plasticity, we demonstrate that cytokine transcription is uncoupled from cytokine secretion in macrophages following recovery from LPS tolerance. Here we demonstrate a novel mechanism of regulation of TLR tolerance through suppression of cytokine secretion in macrophages. We show that TNF-α is alternatively trafficked towards a degradative intracellular compartment. These studies demonstrate that TLR tolerance is a complex immunological response with the plasticity of this state playing an important role in the regulation of inflammation.

Assessment of the immunomodulatory effects of raw/farm milk and probiotic bacteria

The overall aims of this study were to investigate the differences between raw/farm milk and pasteurised milk with respect to potential immune modifying effects following consumption and investigate the bacterial composition of raw milk compared to pasteurised milk. Furthermore, in this thesis, panels of potential probiotic bacteria from the Bifidobacterium and Lactobacillus genera were investigated. The overall bacterial composition of raw milk was compared with pasteurised milk using samples obtained from commercial milk producers around Ireland using next generation sequencing technology (454 pyrosequencing). Here the presence of previously unrecognised and diverse bacterial populations in unpasteurised cow’s milk was identified. Futhermore the bacterial content of pasteurised milk was found to be more diverse than previously thought. The global response of the adenocarcinoma cell line HT-29 to raw milk and pasteurised milk exposures were also characterised using whole genome microarray technology. Over one thousand differentially expressed genes were identified which were found to be involved in a plethora of cellular functions. Interestingly a reduction in immune related activity (e.g. Major histocompatability complex class II signalling and T and B cell proliferation) was identified in cells exposed to pasteurised milk compared with raw milk exposures. Further studies comparing human cell response to raw versus pasteurised milk was performed using peripheral blood mononuclear cells (PBMC) from healthy donors. A reduction in CD14 was identified following raw milk exposures compared with pasteurised milk and the pattern of cytokine production may indicate that gram positive bacteria in the raw milk were contributing to the differences in the cellular response to raw versus pasteurised milk. Panels of potentially probiotic bacteria (comprising of lactobacilli and bifidobacteria) were further assessed for immunomodulatory capabilities using cell culture based models. Gene expression and cytokine production were used to evaluate stimulated and unstimulated (LPS) cellular responses as well as interaction mechanisms

Identification and characterization of innate immune receptor substrates of γ-secretase enzyme complex

The γ-secretase protease complexes and associated regulated intramembrane proteolysis play an important role in controlling receptor-mediated intracellular signalling events, which have a central role in Alzheimer’s disease, cancer progression and immune surveillance. It has previously been reported that the Interleukin-1 receptor, type 1, (IL-1R1) is a substrate for regulated intramembrane proteolysis, mediated by presenilin (PS)-dependent γ-secretase activity. The aims of this project were twofold. Firstly, to determine the conservation of regulated intramembrane proteolysis as a physiological occurrence amongst other cytokine receptors. In this regard, similar to IL-1R1, we identified the Tumour necrosis factor receptor type 1 (TNFR1) and the Toll like receptor 4 (TLR4) as novel γ-secretase substrates. Secondly, given that the diversity of signalling events mediated by the IL-1R1, TLR4 and TNFR1 are spatially segregated, we investigated the spatial distribution, subcellular trafficking and subcellular occurrence of regulated intramembrane proteolysis of IL-1R1, TLR4 and TNFR1. Using dynasore an inhibitor of clathrin-dependent receptor endocytosis, both ectodomain shedding and γ-secretase-mediated cleavage of IL-1R1 were observed post-internalization. In contrast, TNFR-1 underwent ectodomain shedding at the cell surface followed by endosomal γ-secretase-mediated cleavage. Furthermore, immortalised fibroblasts from PS1-deficient mice showed impaired γ-secretasemediated cleavage of IL-1R1 and TNFR1, indicating that both are cleaved by PS1-and not PS2-containing γ-secretase complexes. Subcellular fractionation and immunofluorescence studies revealed that the γ-secretase generated IL-1R1 ICD translocates to the nucleus on IL-1β stimulation. These observations further demonstrate the novel PS-dependent means of modulating IL-1β, LPS and TNFα- mediated immune responses by regulating IL-1R1/TLR4/TNFR1 protein levels within the cells.

Pregnancy-specific glycoprotein function, conservation and receptor investigation

Pregnancy-specific glycoproteins (PSGs) are highly glycosylated secreted proteins encoded by multi-gene families in some placental mammals. They are carcinoembryonic antigen (CEA) family and immunoglobulin (Ig) superfamily members. PSGs are immunomodulatory, and have been demonstrated to possess antiplatelet and pro-angiogenic properties. Low serum levels of these proteins have been correlated with adverse pregnancy outcomes. Objectives: Main research goals of this thesis were: 1). To attempt to replicate previously reported cytokine responses to PSG-treatment of immune cells and subsequently to investigate functionally important amino acids within PSG1. 2). To determine whether candidate receptor, integrin αvβ3, was a binding partner for PSG1 and to investigate whether PSG1 possessed functionality in a leukocyte-endothelial interaction assay. 3). To determine whether proteins generated from recently identified putative PSG genes in the horse shared functional properties with PSGs from other species. Outcomes: 1). Sequential domain deletion of PSG1 as well as mutation of conserved residues within the PSG1 Ndomain did not affect PSG1-induced TGF-β1. The investigated response was subsequently found to be the result of latent TGF-β1 contaminating the recombinant protein. Protein further purified by SEC to remove this showed no induction of TGF-β1. The most N-terminal glycosylation site was demonstrated to have an important role in PSG N domain secretion. PSG1 attenuated LPS-induced IL-6 and TNF-α. Investigations into signalling underpinning this proved inconclusive. 2). Integrin αvβ3 was identified as a novel PSG1 receptor mediating an as yet unknown function. Preliminary investigations into a role for PSGs as inhibitors of leukocyte endothelial interactions showed no effect by PSG1. 3). Horse PSG protein, CEACAM49, was shown to be similarly contaminated by latent TGF-β1 particle and once removed did not demonstrate TGF-β1 release. Interestingly horse PSG did show anti-platelet properties through inhibition of the plateletfibrinogen interaction as previously published for mouse and human PSGs.

Generation and characterisation of biologically active milk-derived protein and peptide fractions

In recent years, extensive research has been carried out on the health benefits of milk proteins and peptides. Biologically active peptides are defined as specific protein fragments which have a positive impact on the physiological functions of the body; such peptides are produced naturally in vivo, but can also be generated by physical and/or chemical processes, enzymatic hydrolysis and/or microbial fermentation. The aims of this thesis were to investigate not only the traditional methods used for the generation of bioactive peptides, but also novel processes such as heat treatment, and the role of indigenous milk proteases, e.g., in mastitic milk, in the production of such peptides. In addition, colostrum was characterised as a source of bioactive proteins and peptides. Firstly, a comprehensive study was carried out on the composition and physical properties of colostrum throughout the early-lactation period. Marked differences in the physico-chemical properties of colostrum compared with milk were observed. Various fractions of colostrum were also tested for their effect on the secretion of pro- and anti-inflammatory cytokines from a macrophage cell line and bone marrow dendritic cells, as well as insulin secretion from a pancreatic beta cell line. A significant reduction in the secretion of the pro-inflammatory cytokines, TNF-α, IL-6, IL-1β and IL-12, a significant increase in the secretion of the anti-inflammatory cytokine, IL-10, as well as a significant increase in insulin secretion were observed for various colostrum fractions. Another study examined the early proteomic changes in the milk of 8 cows in response to infusion with the endotoxin lipopolysaccharide (LPS) at quarter level in a model mastitic system; marked differences in the protein and peptide profile of milk from LPS challenged cows were observed, and a pH 4.6-soluble fraction of this milk was found to cause a substantial induction in the secretion of IL-10 from a murine macrophage cell line. Heat-induced hydrolysis of sodium caseinate was investigated from the dual viewpoints of protein breakdown and peptide formation, and, a peptide fraction produced in this manner was found to cause a significant increase in the secretion of the anti-inflammatory cytokine, IL-10, from a murine macrophage cell line. The effects of sodium caseinate hydrolysed by chymosin on the gut-derived satiety hormone glucagon-like peptide-1 (GLP-1) were investigated; the resulting casein-derived peptides displayed good in vitro and in vivo secretion of GLP-1. Overall, the studies described in this thesis expand on current knowledge and provide good evidence for the use of novel methods for the isolation, generation and characterisation of bioactive proteins and/or peptides.

Molecular mechanisms underpinning IFN-gamma and TNF-alpha synergy in intestinal epithelial cells

Cytokine-driven signalling shapes immune homeostasis and guides inflammatory responses mainly through induction of specific gene expression programmes both within and outside the immune cell compartment. These transcriptional outputs are often amplified via cytokine synergy, which sets a stimulatory threshold that safeguards from exacerbated inflammation and immunopathology. In this study, we investigated the molecular mechanisms underpinning synergy between two pivotal Th1 cytokines, IFN-γ and TNF-α, in human intestinal epithelial cells. These two proinflammatory mediators induce a unique state of signalling and transcriptional synergy implicated in processes such as antiviral and antitumour immunity, intestinal barrier and pancreatic β-cell dysfunction. Since its discovery more than 30 years ago, this biological phenomenon remains, however, only partially defined. Here, using a functional genomics approach including RNAi perturbation screens and small-molecule inhibitors, we identified two new regulators of IFN-γ/TNF-α-induced chemokine and antiviral gene and protein expression, a Bcl-2 protein BCL-G and a histone demethylase UTX. We also discovered that IFN-γ/TNF-α synergise to trigger a coordinated shutdown of major receptor tyrosine kinases expression in colon cancer cells. Together, these findings extend our current understanding of how IFN-γ/TNF-α synergy elicits qualitatively and quantitatively distinct outputs in the intestinal epithelium. Given the well-documented role of this synergistic state in immunopathology of various disorders, our results may help to inform the identification of high quality and biologically relevant druggable targets for diseases characterised by an IFN-γ/TNF-α high immune signature

Investigations into the cellular and molecular changes in the amygdala in models of temporal lobe epilepsy and maternal immune activation

The amygdala is a limbic structure that is involved in many of our emotions and processing of these emotions such as fear, anger and pleasure. Conditions such as anxiety, autism, and also epilepsy, have been linked to abnormal functioning of the amygdala, owing to improper neurodevelopment or damage. This thesis investigated the cellular and molecular changes in the amygdala in models of temporal lobe epilepsy (TLE) and maternal immune activation (MIA). The kainic acid (KA) model of temporal lobe epilepsy (TLE) was used to induce Ammon’s-horn sclerosis (AHS) and to investigate behavioural and cytoarchitectural changes that occur in the amygdala related to Neuropeptide Y1 receptor expression. Results showed that KA-injected animals showed increased anxiety-like behaviours and displayed histopathological hallmarks of AHS including CA1 ablation, granule cell dispersion, volume reduction and astrogliosis. Amygdalar volume and neuronal loss was observed in the ipsilateral nuclei which was accompanied by astrogliosis. In addition, a decrease in Y1 receptor expressing cells in the ipsilateral CA1 and CA3 sectors of the hippocampus, ipsi- and contralateral granule cell layer of the dentate gyrus and ipsilateral central nucleus of the amygdala was found, consistent with a reduction in Y1 receptor protein levels. The results suggest that plastic changes in hippocampal and/or amygdalar Y1 receptor expression may negatively impact anxiety levels. Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the brain and tight regulation and appropriate control of GABA is vital for neurochemical homeostasis. GABA transporter-1 (GAT-1) is abundantly expressed by neurones and astrocytes and plays a key role in GABA reuptake and regulation. Imbalance in GABA homeostasis has been implicated in epilepsy with GAT-1 being an attractive pharmacological target. Electron microscopy was used to examine the distribution, expression and morphology of GAT-1 expressing structures in the amygdala of the TLE model. Results suggest that GAT-1 was preferentially expressed on putative axon terminals over astrocytic processes in this TLE model. Myelin integrity was examined and results suggested that in the TLE model myelinated fibres were damaged in comparison to controls. Synaptic morphology was studied and results suggested that asymmetric (excitatory) synapses occurred more frequently than symmetric (inhibitory) synapses in the TLE model in comparison to controls. This study illustrated that the amygdala undergoes ultrastructural alterations in this TLE model. Maternal immune activation (MIA) is a risk factor for neurodevelopmental disorders such as autism, schizophrenia and also epilepsy. MIA was induced at a critical window of amygdalar development at E12 using bacterial mimetic lipopolysaccharide (LPS). Results showed that MIA activates cytokine, toll-like receptor and chemokine expression in the fetal brain that is prolonged in the postnatal amygdala. Inflammation elicited by MIA may prime the fetal brain for alterations seen in the glial environment and this in turn have deleterious effects on neuronal populations as seen in the amygdala at P14. These findings may suggest that MIA induced during amygdalar development may predispose offspring to amygdalar related disorders such as heightened anxiety, fear impairment and also neurodevelopmental disorders.

Investigating the role of Fas (CD95) signalling in the modification of innate immune induced inflammation

Background/Aim: It has been demonstrated that a number of pathologies occur as a result of dysregulation of the immune system. Whilst classically associated with apoptosis, the Fas (CD95) signalling pathway plays a role in inflammation. Studies have demonstrated that Fas activation augments TLR4-mediated MyD88-dependent cytokine production. Studies have also shown that the Fas adapter protein FADD is required for RIG-I-induced IFNβ production. As a similar signalling pathway exists between RIG-I, TLR3 and the MyD88- independent of TLR4, we hypothesised that Fas activation may modulate both TLR3- and TLR4-induced cytokine production. Results: Fas activation reduced poly I:C-induced IFNβ, IL-8, IL-10 and TNFα production whilst augmenting poly I:C-, poly A:U- and Sendai virus-induced IP-10 production. TLR3-, RIG-I- and MDA5-induced IP-10 luciferase activation were inhibited by the Fas adapter protein FADD using overexpression studies. Poly I:C-induced phosphorylation of p-38 and JNK MAPK were reduced by Fas activation. Overexpression of FADD induced AP-1 luciferase activation. Point mutations in the AP-1 binding site enhanced poly I:C-induced IP- 10 production. LPS-induced IL-10, IL-12, IL-8 and TNFα production were enhanced by Fas activation, whilst reducing LPS-induced IFNβ production. Absence of FADD using FADD-/- MEFs resulted in impaired IFNβ production. Overexpression studies using FADD augmented TLR4-, MyD88- and TRIF-induced IFNβ luciferase activation. Overexpression studies also suggested that enhanced TLR4-induced IFNβ production was independent of NFκB activation. Conclusion: Viral-induced IP-10 production is augmented by Fas activation by reducing the phosphorylation of p-38 and JNK MAPKs, modulating AP-1 activation. The Fas adapterprotein FADD is required for TLR4-induced IFNβ production. Studies presented here demonstrate that the Fas signalling pathway can therefore modulate the immune response. Our data demonstrates that this modulatory effect is mediated by its adapter protein FADD, tailoring the immune response by acting as a molecular switch. This ensures the appropriate immune response is mounted, thus preventing an exacerbated immune response.