MOLECULAR AND CELLULAR MECHANISMS UNDERLYING INFLAMMATORY PROCESSES PhD Programme in Molecular and Cellular Mechanisms underlying inflammatory processes

TABLE OF CONTENTS 7TH AUGUST 2014

TCD Oral Presentations IFN-γ production by Natural Killer Cells induces TCD M1 macrophage activation and upregulates VLA-4 The role of metabolism and hypoxia in the regulation of expression on CD4+ T cells in experimental autoimmune human T cell subsets encephalomyelitis D. Cluxton, K.H.G. Mills, J.M. Fletcher ------1 N.C. Mc Guinness, L.S. Dungan, M.A. Lynch, K.H.G. Mills - - - - 9 UCC NUIG IGF-1R signalling is essential for mitochondrial Modelling ATR disease-related mutations using chicken maintenance and function DT40 cells A. Lyons, R. O’ Connor ------2 M. Llorens Agost,- J.K. Eykelenboom, N.F. Lowndes - - - - -10 - UCD UCC CX3CL1 plays a role in memory-associated synaptic Altered regulation of adipogenesis in disease processes plasticity in the rat hippocampus S. Davies, T. McCarthy ------11- - A. Wdowicz, G.K. Sheridan, D.J. O’Connell, K.J. Murphy - - - -3 - TCD TCD Noradrenaline-mediated protection against Functional analysis of nucleic acid sensors in the TNF-α-induced neuronal atrophy CNS - Implications for infection, brain injury and E. O’Neill, A. Harkin, V.A. Campbell, T.J. Connor ------12 - neurodegeneration D. Cox, A.G. Bowie, M.A. Lynch, C. Cunningham, A. Dunne - - - 4 UCD Regulation of the human Prostacyclin receptor gene NUIG within the cardiovascular system: Influence of gender Regulatory T-cells of mouse display distinctive surface specific hormones glycosylation patterns that correlate with their S.B. Eivers, B.T. Kinsella ------13 - - suppressive function J. Cabral, S. Hanley, J. Gerlach, L. Joshi, T. Ritter, R. Ceredig, M.D. NUIG Griffin ------5 - Nucleosome - organisation of the vertebrate centromere UCC J.G.W. McCarter, K.F. Sullivan ------14 - - Cytotoxic effects of UPEC on human macrophage TCD I. O’Neill, D. Clarke ------6 Modulation of the endocannabinoid system and PanX1- mediated neuroinflammation in anin vitro model of TCD Alzheimer’s Disease Comparative bioenergetics in parental sensitive and Cisplatin resistant cancer cell subline S.G. Fagan, V.A. Campbell ------15 F. Geoghegan, E. O’Connor, K. Grankvist, T. Nilsson, P.B. Motlagh, R.K. Porter ------7- - - UCD Poster Presentations Serotonin transporter regulation in an animal model TCD of Rheumatoid Arthritis – Toward understanding S1P receptor activation attenuates psychosine-induced depression in Chronic Inflammatory Diseases astrocyte cell death E. Brown, J. Haase ------8 - - C. O’Sullivan, K.K. Dev ------16 PRTLI Programme PhD PHD PROGRAMME IN MOLECULAR CELL BIOLOGY MOLECULAR AND CELLULAR MECHANISMS UNDERLYING INFLAMMATORY PROCESSES Phd Programme in The role of metabolism and hypoxia in the Molecular and Cellular Mechanisms regulation of human T cell subsets underlying inflammatory processes TCD TABLE OF CONTENTS D. Cluxton, K.H.G. Mills, J.M. Fletcher cont. 7TH AUGUST 2014 ORAL

UCC UCD h17 cells are important pathogenic effector cells in Treg cells. Glucose uptake was evaluated via culture Use of Lacticin 3147 and essential oils against food- Quantitative analysis of IL-17 induced protein expression in autoimmune diseases such as rheumatoid with a fluorescent glucose analog (2 - NBDG). It was borne pathogens profiles of Psoriatic Arthritis B cell models arthritis (RA) and multiple sclerosis (MS). shown that Th17 -lineage cells expressed increased A. Campion, D. Field, P.D Cotter, C. Hill, R.P. Ross ------17 M. Doyle, S. Pennington ------27 TOn the other hand, regulatory T (Treg) cells play a 2 -NBDG compared to CD4+CD161 - T cells. This suggests UCD TCD crucial role in maintaining tolerance and preventing that Th17 cells utilise glycolysis while Treg cells employ The role of the bone morphogenetic antagonist Gremlin mTORC1 regulation of Dendritic cell metabolism through autoimmunity. Under healthy conditions there is a the aerobic process of oxidative phosphorylation for in the pathogenesis of Chronic Lung Disease HIF1α and iNOS balance between Treg and effector T cell responses energy supply and consumption. J. Cornwell, P. McLoughlin ------18- - S. Lawless, D. Finlay ------28 - - which may be perturbed in autoimmunity. Therefore, This switch towards anaerobic glycolysis could TCD it is crucial to understand the factors that regulate this promote Th17 cell survival within inflamed tissues, Cytokine release inhibitor drug, CRID3, inhibits the balance. Recently, a role for metabolism in murine T cell NLRP3 inflammasome in glia Abstracts (in absentia) where oxygen supply is limited. Hypoxia inducible factor differentiation has been identified. C.N. Dempsey, R.C. Coll, A.A.B. Robertson, M.A. Cooper, L.A.J. TCD 1 α (HIF -1α), which is a transcription factor best known O’Neill, M.A. Lynch ------19 - - The effect of inflammatory caspases in Colitis and CD4+ T cells were sorted from PBMC using magnetic for orchestrating the cellular response to hypoxia, now NUIG Colorectal Cancer beads and further sorted via the MoFlo sorter into the also appears to be an important factor in regulating B. Flood, K. Oficjalska, E. Creagh ------29 - Examination of the inner centromere compartment in a CD161+, CD161 - and Treg cell populations. The metabolic metabolism and T cell differentiation. The data showed mammal UCD profile of each T cell population was determined via the that Th17 cells express increased levels of HIF -1α via T. Masterson, K. Sullivan ------20 Structure-based drug design for NADPH Oxidases Seahorse XF Analyzer. CD4+ T cells expressing the human flow cytometric analysis. UCC S. O’Neill, U. Knaus ------30 - - Th17 cell marker CD161 were found to have increased Screening protein Kinase mutants to identify novel In conclusion, the data suggests that Th17 lineage glycolysis compared with CD4+CD161 - cells. We genes required for biofilm formation inCandida Albicans TCD cells employ glycolysis for their energy supply, Combretastatin (CA)-4 and its novel analogue CA-432 confirmed this by investigating the effect of inhibiting N. Konstantinidou, F. O’Gara, J. Morrissey ------21 - orchestrated by the increased expression of HIF -1α, and impair T-cell migration through the Rho/ROCK signalling glycolysis following treatment with 2 -deoxy -D -glucose pathway have the potential to persevere within inflamed tissues. TCD (2 -DG) and rapamycin on CD4+ T cell growth, revealing The PYHIN protein MNDA is involved in the regulation of J. Pollock, D. Zisterer ------31- - a decrease in Th17 cell expansion and a parallel increase interferon alpha expression NUIG D. Casserly, S. Carpenter, K.A. Fitzgerald, A.G. Bowie - - - - 22 - Identification of new sugar binding bacterial adhesins UCD encoded by the human gut metagenome Exploring the immune modulatory role of MPB70 and C. Agbavwe, C. O’Byrne, A. Boyd, Lo. Joshi ------32 MPB83 by Mycobacterium tuberculosis TCD L. Carr, C. Brereton, E. Lavelle, S. Gordon ------23- - The anti-inflammatory actions of noradrenergic agents UCC as a target to prevent neurodegeneration in Parkinson’s The effect of Isoellipticines on Leukaemia cells disease E.G. Russell, T.G. Cotter ------24 - - J.D.Yssel, T.J. Connor, A. Harkin ------33- - TCD TCD γβ T cells, a novel T cell subset with a pathogenic role in Double stranded RNAs of different lengths have Il-17-mediated CNS autoimmunity divergent effects on innate immune activation in the periphery and the brain Th17 cells have the ability to survive and S.C. Edwards, K.H.G. Mills ------25 - - expand within inflamed environments, N. McGarry, K. Mitchell, C. Cunningham ------34- - such as those found in rheumatic joints, NUIG due to their utilization of glycolysis Investigation into the regulation of p75NTR by NGF, and and increased expression of HIF-1α, its pro-survival signaling in breast cancer cells whereas Treg cells utilise oxidative R. Chakravarthy, J. Hillis, A. Gorman ------26- - phosphorylation for their metabolism. Hypoxia and metabolism may be targeted to maintain a balance between pro and

PRTLI Programme PhD anti-inflammatory T cells. PRTLI Programme PhD

1 PHD PROGRAMME IN MOLECULAR CELL BIOLOGY MOLECULAR AND CELLULAR MECHANISMS UNDERLYING INFLAMMATORY PROCESSES IGF-1R signalling is essential for mitochondrial CX3CL1 plays a role in memory-associated synaptic maintenance and function plasticity in the rat hippocampus UCC UCD

A. Lyons, R. O’ Connor A. Wdowicz, G.K. Sheridan, D.J. O’Connell, K.J. Murphy ORAL ORAL

ancer cells exhibit increased glycolytic rates and [1] Favre, C., et al. (2010). “Mitochondrial pyrimidine nucleotide carrier number of cytokines and chemokines act plasticity. Finally, we confirm that CX3CL1 activates glucose transport to facilitate adaptation to (PNC1) regulates mitochondrial biogenesis and the invasive as regulators of cognitive function under second messenger signalling molecules such as Akt in a phenotype of cancer cells.” Oncogene 29(27): 3964 -3976. the high energy demand imposed by increased immunologically unchallenged brain dose dependent manner, Erk at both doses and p -38 at a Cproliferation rates and the hypoxic environment. This is [2] Floyd, S., et al. (2007). “The Insulin -like Growth Factor -I mTOR Signaling Aconditions. Interestingly, they have been implicated in 500pM concentration in hippocampal neurons and glia. Pathway Induces the Mitochondrial Pyrimidine Nucleotide Carrier to often accompanied by reduced mitochondrial activity hippocampal memory consolidation processes such as Promote Cell Growth.” Mol. Biol. Cell 18(9): 3545 -3555. In conclusion, this study provides additional due to the accumulation of mitochondrial DNA (mtDNA) synaptic plasticity, neurogenesis and synaptic scaling [3] Ishikawa, K., et al. (2008). “ROS -generating mitochondrial DNA documentation for a role of fractalkine in normal brain [1]. The chemokine fractalkine (CX3CL1) functions mutations. However, evidence also exists which mutations can regulate tumor cell metastasis.” Science’s STKE function in protective process of synaptic scaling. as a modulator of synaptic transmission protecting indicate that mitochondria play an active role in cancer 320(5876): 661. Thus, targeting CX3CL1 and CX3CR1 may provide a from cellular over -excitation in the hippocampus [2]. progression, notably through their effect on glutamine novel therapeutic intervention for many chronic brain At basal levels fractalkine is abundantly expressed on and fatty acid metabolism. Moreover, mitochondrial inflammatory disorders such as Alzheimer’s disease. mature hippocampal neurons, which further suggests deficiency and mitochondrial ROS production has also IGF-1R Glucose a physiological role for this chemokine in control of [1] McAfoose, J., and Baune, B.T. (2009) ‘Evidence for a cytokine model of been implicated in the induction of metastasis [3]. cognitive function’ Neuroscience and Behavioural Reviews 33: 355 - Glut1 memory function. 366. doi: 10.1016/j.neubiorev.2008.10.005 PNC1 is an IGF -I inducible mitochondrial carrier protein which is essential for mitochondrial Adult rats were subjected to Morris water maze [2] Bertollini, C., Ragozzino, D., Gross, C., Limatola, C., and Eusebi, F. (2006) Glucose behavioural learning paradigm and samples were ‘Fractalkine/CX3CL1 depresses central synaptic transmission in mouse maintenance. Suppression of PNC1 causes reduced Hexose kinase hippocampal slices’. Neuropharmacology 51: 816–821. doi:10.1016/j. PI3K further tested by immunostaining and ELISA approaches oxidative phosphorylation and leakage of reactive ROS Glucose-6-Phosphate neuropharm.2006.05.027. to investigate fractalkine and expression of its oxygen species (ROS), which in turn causes a profound Phosphofructokinase receptor CX3CR1 following learning. A mixed primary ROS -dependent epithelial–mesenchymal transition AMPK AKT Fructose-1,6-BisPhosphate hippocampal cell model was established from G19 (EMT) in MCF -7 cells, whereas cells overexpressing Glycolysis Wistar rat embryos and calcium imaging was performed PNC1 are protected from the induction of EMT [1], [2]. mTOR PGC-1a using a custom built liquid infusion chamber to test for This suggests that PNC1s induction by IGF -I facilitates Pyruvate Lactate roles of basal fractalkine levels in synaptic plasticity. cell growth while also protecting cells from an ROS - Immunofluorescent analysis of primary cultures was promoted differentiation programme that arises Mitochondrial performed to investigate molecular mechanisms Biogenesis from mitochondrial dysfunction. Since PNC1 is an I II III IV V Mitochondrial ATP involved in the process. PNC1 IGF -I responsive gene we hypothesized that the IGF -1 Activity signalling pathway may directly influence mitochondrial In the current study we show CX3CL1 protein activity in cancer cells even in the presence of a switch The insulin-like growth factor (IGF-I) signalling pathway, through upregulation at 2h following water maze learning in PI3-kinase, Akt and mTOR, can directly enhance both glycolysis and the CA1, CA3 and dentate gyrus of a rat hippocampus. to the glycolytic phenotype. We have shown that mitochondrial activity. The IGF-1 responsive mitochondrial carrier IGF -I stimulation increases mitochondrial mass and protein, PNC1, is essential for maintenance of mtDNA and respiration, and Furthermore, we show CX3CR1 expression on mature suppression of PNC1 or inhibition of the IGF-1R signalling pathway gives hippocampal neurons in culture. At physiologically Glutamate-induced calcium dynamics in hippocampal neurons and glia. mitochondrial activity in several cancer cell lines, and rise to dysfunctional mitochondrial and leakage of ROS. relevant concentration, CX3CL1 reduced glutamate - Live cell imaging in primary hippocampal mixed neuron-glia cultures. At that inhibition of IGF -1R signalling gives rise to defective 19DIV cells were loaded with fluo-4 AM calcium indicator and exposed to mediated intracellular calcium rises in both neurons and mitochondria. We therefore conclude that mitochondrial 30 μM glutamate which causes an influx of intracellular calcium. Scale bar glia in a dose dependent manner in a model of synaptic 100μm. activity and biogenesis in cancer cells is enhanced by IGF -1 and that attenuation of this pathway, even if beneficial considering the effect of IGF -I on cell survival, may lead to deficient mitochondrial regulation, loss of mitochondrial activity, release of mitochondrial ROS and cancer progression. PRTLI Programme PhD PRTLI Programme PhD

2 3 PHD PROGRAMME IN MOLECULAR CELL BIOLOGY MOLECULAR AND CELLULAR MECHANISMS UNDERLYING INFLAMMATORY PROCESSES Functional analysis of nucleic acid sensors in the Regulatory T-cells display distinctive surface CNS — Implications for infection, brain injury and glycosylation patterns that correlate with their neurodegeneration suppressive function TCD NUIG

D. Cox, A.G. Bowie, M.A. Lynch, J. Cabral, S. Hanley, J. Gerlach, L. Joshi, C. Cunningham, A. Dunne T. Ritter, R. Ceredig, M.D. Griffin ORAL ORAL

he recognition of nucleic acids is essential for This study has implications for viral DNA sensing urface protein glycosylation status profoundly T -reg surface glycosylation is distinct from combating viral pathogens. The sensors and and also for sterile brain insults where cell free DNA is influences T -cell biology and functional that of T -conv in mouse. On -going experiments pathways involved in RNA/DNA detection have elevated and could therefore contribute to excessive characteristics [1], [2]. The main goals of this work are investigating the hypothesis that enhanced Tonly recently been discovered and have expanded neuroinflammation. Swere to identify surface glycosylation characteristics T -reg α -galactose surface expression contributes to + over the last number of years. TLR9 was the first DNA [1] Wu J, Chen ZJ. Innate immune sensing and signaling of cytosolic that distinguish T -reg from conventional CD4 T -cell suppressive function in vivo. sensor to be discovered followed by the cytosolic DNA nucleic acids. Annual review of immunology. 2014;32:461 -88. populations (T -conv) in mouse, to evaluate glycosylation [1] Grigorian, A., Torossian, S., Demetriou, M. T -cell growth, cell surface receptors including members of the PYHIN family, changes following activation and to correlate T -reg organization, and the galectin -glycoprotein lattice. Immunol. Rev., DAI and cGAS [1]. Activation of these proteins by DNA surface glycosylation with expression of suppression - 2009; 230(1): 232 -46. culminates in the production of anti viral- and pro - related proteins. [2] Daniels, M.A., Hogquist, K.A., Jameson, S.C. Sweet ‘n’ sour: the impact inflammatory cytokines. of differential glycosylation on T cell responses. Nat. Immunol., 2002; Cell suspensions of lymphoid organs and peripheral 3(10): 903 -10. Microglia and astrocytes can respond to cytosolic blood were obtained from C57BL/6 -FoxP3 -EGFP mice. RNA via RIG -like receptors, however the contribution Surface glycosylation and marker expression was then of DNA sensors has not been fully explored in evaluated by multi -colour flow cytometric analysis using neuroimmune cells. Furthermore, the contribution a panel of 17 biotinylated lectins (carbohydrate binding of individual cell types, in particular astrocytes, has proteins) and 9 fluorochrome -labelled antibodies with gating on T -reg (GFP+) and T -conv (GFP -). Naïve been confounded by difficulties in obtaining pure (CD4+CD62L+) splenocytes were purified, labelled with populations devoid of contaminating microglia. Finally, a fluorescent indicator of proliferation and subjected to most studies to date have used poly(dA:dT), however weak (ConA 1.5 μg/ml) and strong (plate -coated anti - this can be reverse transcribed into RNA by RNA pol III, CD3/anti -CD28) activation stimuli in culture for 5 days thus activating RNA sensors. followed by analysis of surface lectin binding. Mixed glia, microglia and purified astrocytes were Splenic T -reg demonstrated higher surface binding transfected with Vaccinia virus 70mer, poly(dA:dT) or of 8/17 lectins compared to T -conv. In particular, GSL -I, poly(I:C) for 24hrs. IFN -β, IL -6 and RANTES production which specifically recognizes α -galactose motifs, was quantified by ELISA. The expression of DNA sensors distinguished T -reg and T -conv at multiple anatomical was measured with and without IFN -β stimulation sites. GSL -I binding was lower in CD62Llo compared to in microglia and purified astrocytes using RT -PCR. CD62Lhi T -reg. Finally, expression of DNA sensors was examined in brains from mice exposed to poly(I:C) and a model of Differences in GSL -I binding between splenic T -reg neurodegeneration. and T conv were maintained in unstimulated culture or following a weak stimulus whereas strong stimulation We have found that p204, AIM2, cGAS and other resulted in higher GSL -I binding in both T -cell types. PYHIN family members are expressed in microglia and Using 6 -colour analysis, it was also demonstrated that Regulatory T-cells display distinctive surface glycosylation characteristics astrocytes. These cells produce high levels of IFN -β, IL -6 T -reg level of GSL -I surface binding correlated with than Conventional T‑cells. Surface glycosylation of freshly isolated resting Nucleic acid induced IL-6 is dependent on IFN-β in primary astrocytes. and RANTES in response to DNA and RNA. Furthermore CD4+ T cells from C57Bl/6 FoxP3.EGFP mice spleen was evaluated by lectin Primary astrocytes derived from wild type or IFNAR-/- mice were higher expression of multiple known mediators of profiling using flow cytometry. The histograms show GSL-I binding by the we have found that key sensors are upregulated in a transfected with poly(dA:dT) (1μg/ml) or poly(I:C) (5μg/ml) for 24hrs. T -reg suppressive function. Finally, in a preliminary GFP+(T-reg) and GFP- (T-conv) CD4+ T-cell populations. murine model of neurodegeneration and that this is Secretion of IFN-β(A), IL-6(B) and CCL5(C) was quantified by ELISA. experiment, purified CD62Lhigh/GSL -Ihigh T -reg exhibited Results shown are means ± SD for triplicate cultures. ***p≤0.001. dependent on the type I interferon, IFN -β. increased suppression of memory but not naïve T -conv when compared to their GSL -Ineg/low counterparts. PRTLI Programme PhD PRTLI Programme PhD

4 5 PHD PROGRAMME IN MOLECULAR CELL BIOLOGY MOLECULAR AND CELLULAR MECHANISMS UNDERLYING INFLAMMATORY PROCESSES Cytotoxic effects of UPEC on human macrophage Comparative bioenergetics in parental sensitive and Cisplatin resistant cancer cell subline UCC TCD

I. O’Neill, D. Clarke F. Geoghegan, E. O’Connor, K. Grankvist, T. Nilsson, P.B. Motlagh, R.K. Porter ORAL ORAL

scherichia coli has the ability to colonise the gut THP -1 human macrophage cell line and identified 25 isplatin is one of the most potent expression was assessed by flow cytometric analysis. of mammals either as a commensal, a pathobiont mutants that had significantly less cytotoxicity than chemotherapeutic drugs used in the clinic to It was found that the parental sensitive cell lines or as a highly virulent pathogen. This versatility wild type UTI89. Sequencing indicated that 22 of 25 treat solid cancers such as; sarcoma, small cell demonstrate 3 fold greater oxygen consumption rates, Eis reflected in the genetic and phenotypic diversity of the Tn5 insertions were in the hlyCABD operon, Clung cancer, germ cell tumours, lymphoma, and ovarian 3 fold greater mitochondrial abundance, greater growth observed across the species. Therefore we are interested encoding a well -characterised alpha -hemolysin (HlyA) cancer, however, resistance often develops. One way rates and 10 fold greater Gb3 expression than their in establishing how host cells interact with different and the proteins required for its secretion from the cell. resistance can occur is through multidrug resistance resistant sub -lines. strains of E. coli. The 3 other mutants had insertions in a transcriptional proteins -1 (MDR1). Overexpression of an enzyme activator rfaH, an outer membrane protein precursor, glucosylceramide synthase (GCS), the first enzyme in Currently work is being carried out to determine the During preliminary studies to look at the response tolC, and a putative ADP -heptose synthase respectively. the glycosphingolipids (GSL) synthesis is associated mechanism behind these mitochondrial/bioeneregtic of macrophages to a set of 11 strains of E. coli (carefully Interestingly, rfaH and tolC have previously been with multidrug resistance. Glycosphingolipids are differences. selected to represent commensal, pathobiont and implicated in hlyCABD transcription and HlyA transport components of all vertebrate cells and have a crucial pathogenic strains) we identified 3 strains (CFT073, [1] Kovbasnjuk, O., et al., The glycosphingolipid globotriaosylceramide respectively. The putative ADP -heptose synthase has role during development and cell differentiation. in the metastatic transformation of colon cancer. Proceedings of the UTI89 and A034/86) that caused rapid, catastrophic not previously been implicated in hemolysin activity Cells that express MDR1 have shown to also express National Academy of Sciences of the United States of America, 2005. cell death in the human macrophage cell line THP -1. 102(52): p. 19087 19092.- but may affect structure bacterial LPS structure and, glucosylceramide. MDR1 can function to translocate Interestingly 2 of these strains, the uropathogenic strain thus, would be expected to interfere with normal HlyA glucosylceramide into the Golgi apparatus for neutral [2] Behnam -Motlagh, P., et al., Verotoxin -1 Treatment or Manipulation of CFT073 and a commensal strain A034/86, also caused secretion. In conclusion, we have identified HlyA as the GSL synthesis, including Globotriasosylceramide (Gb3). its Receptor Globotriaosylceramide (Gb3) for Reversal of Multidrug similar levels of cell death in murine macrophage, whilst Resistance to Cancer Chemotherapy. Toxins, 2010. 2(10): p. 2467 -2477. primary virulence factor involved in the UTI89 -induced Functional interplay between Gb3 and MDR1 has been with UTI89 (another uropathogenic strain), cytotoxicity cytotoxicity of human macrophages. Our data also suggested and Gb3 expression in colorectal cancer was appeared to be specific for human macrophages. indicates that mouse macrophage are impervious to the correlated with invasiveness and metastatic potential To identify the UTI89 virulence factor responsible effects of UTI89 hemolysin suggesting that its cytotoxic [1]. Behnam -Motlagh, Tyler [2] were able to demonstrate for this cytotoxicity, we screened a library of >4000 effects on human macrophage may be host adapted. a correlation between increased expression of Gb3 and individual Tn5 mutants for reduced cytotoxicity in the Further studies to address this will be undertaken. acquired cisplatin resistant non -small lung cancer cells (H1299 cells). In this study it was decided to determine the metabolic changes behind acquired cisplatin resistance in the non small cell lung cancer cell line, H1299. Analysis of the bioenergetic differences between the parental sensitive cell line and resistant sub -line was determined by the Seahorse extracellular flux analyser, determination of mitochondrial abundance was assessed by the citrate synthase assay, growth rate Mechanism of action of Verotoxin-1 induced cell death. was measured by the alamar blue assay, Gb3 and MDR PRTLI Programme PhD PRTLI Programme PhD

6 7 PHD PROGRAMME IN MOLECULAR CELL BIOLOGY MOLECULAR AND CELLULAR MECHANISMS UNDERLYING INFLAMMATORY PROCESSES Serotonin transporter regulation in an animal IFN-γ production by Natural killer cells induces model of Rheumatoid Arthritis — Toward M1 macrophage activation and upregulates understanding depression in Chronic Inflammatory VLA-4 expression on CD4+ T cells in experimental Diseases UCD TCD autoimmune encephalomyelitis

E. Brown, J. Haase N.C. McGuinness, L.S. Dungan, M.A. Lynch, K.H.G. Mills ORAL ORAL

epression has long been associated with Future experiments will investigate; 1) signalling xperimental autoimmune encephalomyelitis at least 2 weeks after administration of the final dose chronic inflammatory disease and has pathways linking increased SERT activity to decreased (EAE) is a mouse model for multiple sclerosis (MS), of antibody. Neutralization of VLA -4 prior to the onset been observed as a side effect in patients BDNF mRNA, 2) if BNDF protein expression is where central nervous system (CNS) inflammation of symptoms also abrogated the development of Dreceiving cytokine therapy, as well as this patients downregulated in the hippocampus, 3) if this is Eis mediated by infiltrating autoantigen -specific IL -17 - actively -induced EAE. We also found that a failure to suffering from depression are known to display influencing hippocampal neurogenesis in CIA mice and producing Th17 cells and IFN -γ -producing Th1 cells. transfer EAE from IFN -γ knockout to wild -type mice was elevated levels of proinflammatory cytokines [1]. As 4) if CIA mice display depression like behaviour. However, cells of the innate immune system, including associated with reduced M1 macrophage activation

the serotonin transporter (SERT) is known to regulate [1] Dantzer, R., O’Connor, J. C., Freund, G. G., Johnson, R. W., & Kelley, K. δγT cells, natural killer (NK) cells and macrophages are and VLA -4 expression on T cells. Co -incubation mood and behaviour [2] and is the target of modern W. (2008). From inflammation to sickness and depression: when the also involved [1]. Here we found that NK cells infiltrate experiments revealed that NK cells could polarise naïve antidepressant drugs (SSRIs) the regulation of this immune system subjugates the brain. Nature Reviews Neuroscience, the CNS early in disease and provide an innate source of macrophages to the M1 phenotype, and these NK - protein under inflammatory conditions has emerged as 9, 46 -56 IFN -γ before the onset of clinical signs, whereas Th1 and activated macrophages promoted VLA -4 expression on a new area of study in depression research. [2] Torres, G. E., Gainetdinov, R. R., & Caron, M. G. (2003). Plasma Th17 cells are present at high numbers in the CNS at the MOG -specific CD4+ T cells. Our findings suggest that membrane monoamine transporters: structure, regulation and peak of disease. Depletion of NK cells with anti -asialo IFN -γ production by NK cells plays a pathogenic role Our research employs a mouse model of chronic function. Nature Reviews Neuroscience, 4, 13 -25 GM1 at the induction of EAE delayed the onset of clinical in the induction of EAE by promoting M1 macrophage inflammatory disease, collagen induced arthritis (CIA), in signs of EAE. This correlated with a reduction in the activation and upregulating VLA -4 expression on and combination with biochemical techniques such as 5 -HT infiltration of Th1 and Th17 cells into the CNS. encephalitogenic activity of CD4+ T cells. uptake assays, qPCR and Western blot to investigate pathways which may link peripheral inflammation to The migration of Th1 and Th17 cells into the [1] Mayo, L., Quintana, F.J. & Weiner, H.L. The innate immune system in altered SERT function in the CNS. CNS and their resulting encephalitogenic activity demyelinating disease. Immunological Reviews, 2012, 248 (1): 170 - 187. is mediated in part by VLA -4 which is a therapeutic Results thus have far have revealed a hippocampal target in MS2. We found that treatment of mice with [2] Clifford, D. B., Deluca, A., Simpson, D. M., Arendt, G., Giovannoni, specific upregulation of SERT activity in CIA mice relative G. & Nath, A. Natalizumab -associated progressive multifocal an anti VLA- -4 neutralizing antibody prevented EAE to controls (p < = 0.01, n = 11). Using qPCR a significant leukoencephalopathy in patients with multiple sclerosis: lessons from induced by T cell transfer, and this effect persisted for decrease in BDNF mRNA levels was found in the 28 cases. The Lancet Neurology, 2010 9, 438 -446. hippocampus of CIA mice (p < = 0.05, n = 6). Expression of glycoprotein M6a has also been found to also be downregulated region specific to the hippocampus of Western blot using fluorescent detection of SERT N- and C-terminus, CIA mice and experiments with M6a/M6b DKO mice β-actin and glycoprotein M6a in synaptosomal protein fraction isolated from wild type (WT), M6a knockout (M6a), M6b knockout (M6b) and double have demonstrated increased SERT activity in the knockout (DKO) mice. absence of M6a/M6b (p < = 0.05, n = 7). It remains unclear if M6a downregulation is the sole

factor influencing the upregulation of SERT activity IFN-γ produced by NK cells promotes observed in the hippocampus of CIA mice, currently in M1 macrophage activation, which in turn + vitro experiments are ongoing to investigate the nature enhances VLA-4 expression by CD4 T cells in EAE. of M6a regulation of SERT activity. PRTLI Programme PhD PRTLI Programme PhD

8 9 PHD PROGRAMME IN MOLECULAR CELL BIOLOGY MOLECULAR AND CELLULAR MECHANISMS UNDERLYING INFLAMMATORY PROCESSES Modelling ATR disease-related mutations using Altered regulation of adipogenesis in chicken DT40 cells disease processes NUIG UCC

M. Llorens-Agost, J.K. Eykelenboom, S. Davies, T. McCarthy N.F. Lowndes ORAL ORAL

he ATR kinase acts as a master regulator of the A new mutation in the ATR gene (6431A→G) ysregulation of adipose tissue metabolism is The promoters of a number of adipocyte DNA Damage Response. This protein responds has also been implicated for the first time in cancer associated with multiple metabolic disorders. differentiation markers including PPARγ, CEBPα and to different types of lesions that lead to the predisposition, in particular, oropharyngeal cancer. One such disease, known as Dunnigan -type AdipoQ, have been amplified and cloned upstream of Tgeneration of single stranded DNA. Upon activation, Tanaka and collaborators found a missense mutation Dfamilial partial lipodystrophy (FPLD2) is characterized the secreted luciferase reporter gene. These constructs ATR co -ordinates cell cycle checkpoints, replication fork in the ATR protein (Q2144R) that translates into an by defective fat metabolism and storage. FPLD2 is will be used to validate a novel secreted luciferase stability and restart, and origin firing [1]. autosomal dominant inherited disease [3]. In this project caused by specific mutations in the LMNA gene. The reporter assay system for the continuous assessment of we have generated a stable cell line in DT40 cells to gene promoter activity throughout the differentiation Mutations in the ATR gene have been implicated mechanisms by which LMNA mutations lead to the model this cancer -related mutation. programme. This in turn will enable early detection of in different human diseases. In 2003, a mutation in the adipose specific FPLD2 phenotype have yet to be cell differentiation and can be used as an alternative to ATR gene (2101A→G) was related to Seckel syndrome, a Our results show that a single point mutation determined. the standard 10 day differentiation protocol of 3T3 L1- rare recessive disorder [2]. This mutation affects splicing, (Q2163R in Gallus gallus) causes a proliferation defect. RNA -Seq analysis carried out in this laboratory to cells and subsequent Oil Red O staining of lipid droplets. resulting in reduced but residual levels of normal More interestingly, this mutation also interferes with assess the effects of wild -type and mutant LMNA on Preliminary experiments have begun to characterise the transcript and protein. Patients with this mutation ATR signaling: It produces a complete loss of Chk1 the differentiation of 3T3 -L1 mouse preadipocytes has activity of these promoters in 3T3 -L1 differentiation. suffer from microcephaly and developmental defects. phosphorylation. We are currently addressing the identified altered expression in a number of candidate Recently, two novel mutations in the ATR gene were hypothesis that the neighbouring residue, S2162, is a genes including IGFBP5 and WNT6. This research aims found in two unrelated Seckel sufferers (M1159I and novel phosphorylation site that regulates ATR activation. to examine the functional relationship between LMNA K1665N). In previous studies, we have modeled these Results of this research will greatly contribute to and these genes. mutations using chicken DT40 cells. Contrary to our understand how ATR function is affected by the disease - expectations, the missense mutations had no effect Secreted luciferase reporter vectors were related mutations. on the protein function in DT40 cells. Currently, we are constructed containing 2.7, 2.4 and 1.2 kb regions of planning to investigate if these mutations affect splicing [1] Cimprich, KA, Cortez, D ATR: an essential regulator of genome the IGFBP5 promoter to evaluate IGFBP5 expression integrity. Nat Rev Mol Cell Biol, 9(8), 616 -27, 2008. using patient -derived cells. throughout 3T3 -L1 differentiation and in the presence [2] O’Driscoll M, Ruiz -Perez VL, Woods CG, Jeggo PA, Goodship JA. A of LMNA. The larger IGFBP5 promoter regions (2.4kb and splicing mutation affecting expression of ataxia -telangiectasia and 2.8kb) do not direct luciferase activity which indicates Rad3 -related protein (ATR) results in Seckel syndrome. Nat Genet, 33(4), 497 -501, 2003. the presence of an inhibitory element in the IGFBP5 promoter. IGFBP5 promoter activity appears to be down - IGFBP5 promoter activity during 3T3-L1 cell differentiation in the absence [3] Tanaka A, Weinel S, Nagy N, O’Driscoll M, Lai -Cheong JE, Kulp -Shorten and presence of mutant and wild-type LMNA. Promoter activity was CL, Knable A, Carpenter G, Fisher SA, Hiragun M, Yanase Y, Hide regulated upon induction of 3T3 -L1 differentiation while reduced by LMNA both before and after induction of differentiation at day M, Callen J, McGrath JA. Germline mutation in ATR in autosomal - preliminary data suggests further down -regulated in 0. From Day 0 to day 2 IGFBP5 promoter activity is reduced in response to dominant oropharyngeal cancer syndrome. Am J Hum Genet, 90(3), response to both wild -type and mutant LMNA. dexamethasone, an ingredient of the differentiation cocktail. 511 -7, 2012.

Model for ATR activation: We hypothesize that the residue S2143 (in human) might be a new phosphorylation site that is crucial for ATR activation. This post-translational modification could affect ATR function by regulating its recruitment or PRTLI Programme PhD PRTLI Programme PhD signaling 10 11 PHD PROGRAMME IN MOLECULAR CELL BIOLOGY MOLECULAR AND CELLULAR MECHANISMS UNDERLYING INFLAMMATORY PROCESSES Noradrenaline-mediated protection against Regulation of the human Prostacyclin receptor TNF-α-induced neuronal atrophy gene within the cardiovascular system: Influence of gender specific hormones TCD UCD

E. O’Neill, A. Harkin, V.A. Campbell, S.B. Eivers, B.T. Kinsella T.J. Connor ORAL ORAL

he catecholaminergic neurotransmitter NA -induced increase in expression of growth factors he prostanoid prostacyclin is a key hIP expression in both megakaryocytic and vascular noradrenaline (NA) is depleted in several (nerve growth factor, glial -derived neurotrophic factor, cardioprotective agent within the vasculature, endothelial cells.

neurodegenerative disorders including vascular endothelial growth factor, interleukin -6), acting as a potent inhibitor of platelet [1] Turner, E.C. and B.T. Kinsella, Estrogen increases expression of the TAlzheimer’s disease, Parkinson’s disease and multiple an effect that was also blocked by propranolol. Taggregation, as a vasodilator and promotes vascular human prostacyclin receptor within the vasculature through an sclerosis, diseases which also include a significant Furthermore, conditioned media from glial cells repair in response to injury. Previous studies established ERalpha -dependent mechanism. J Mol Biol, 2010. 396(3): p. 473 -86. neuroinflammatory component. The inflammatory stimulated with the β2 -adrenoceptor agonist salmeterol that the female hormone estrogen increases expression processes observed are characterised by breakdown of (1, 10 μM) was shown to prevent the neuronal atrophy of the prostacyclin receptor, the IP, within the the blood -brain barrier, infiltration of peripheral immune induced by TNF -α. vasculature accounting for some of the cardioprotective cells, activation of microglia and increased release effects of estrogen and prostacyclin [1]. There is These results demonstrate the potential of of pro -inflammatory cytokines. Of the detrimental emerging evidence that the androgen testosterone/ harnessing the endogenous noradrenergic system outcomes following these events, neuronal atrophy is dihydrotestosterone may also have cardioprotective to rescue neurons from TNF -α -induced atrophy with known to lead to loss of functional synaptic connections effects in men, but through unknown mechanisms. wider implications for the treatment of inflammatory and neurodegeneration. NA binds to adrenoceptors neurodegenerative disorders. This study investigated whether androgens may on neuronal and glial cells and the β2 -adrenoceptor regulate expression of the human (h) IP gene, the PTGIR, subtype is thought to exert anti -inflammatory and within the vasculature. neurotrophic effects in the central nervous system (CNS). Using genetic reporter based -luciferase assays, QPCR, site directed mutagenesis, cAMP generation We hypothesised that activation of β2 - assays, chromatin immunopreciptitation (ChIP), and adrenoceptors on glial cells may protect neurons immunocytochemistry, the effect of dihydrotestosterone against inflammatory neuronal atrophy in vitro. Primary (DHT) on prostacyclin receptor promoter (PrmIP) - cortical neurons were treated (24, 48 h) with the directed luciferase gene expression, IP mRNA expression pro -inflammatory cytokine tumour necrosis factor α an IP protein expression was investigated in human (h) (TNF -α; 1, 10, 100 ng/ml), which was found to induce Expression of human prostacyclin receptor (IP) in 1°HUVECs. megakaryocytic and vascular endothelial cells. Immunofluorescence microscopy of 1°HUVECs preincubated for 24hr with neuronal atrophy by inhibition of neurite outgrowth. DHT (10 nM), and immunolabelled with anti-hIP sera and Alexa-Fluor Primary cortical mixed glial cells were treated (24 h) DHT increased hIP mRNA and protein expression, 488-conjugated anti-rabbit IgG (green), followed by counterstaining with DAPI (blue). with NA (0.1, 1, 10, 100 μM) and glial conditioned media effects that were abrogated by the androgen receptor was transferred to neurons, significantly increasing Representative image of neuron treated with conditioned media from glial (AR) antagonist hydroxyflutamide (HF). Furthermore, neurite outgrowth. This effect was blocked by the cells stimulated with noradrenaline (10 μM). Neuron is stained with neuron- DHT led to substantial increases in PrmIP -directed specific marker βIII-tubulin (white) and nuclear marker DAPI (blue). Scale β -adrenoceptor antagonist propranolol (10 μM). The bar = 50 μm. luciferase reporter gene expression. The DHT -responsive increase in outgrowth is proposed to be mediated by region was localized to the core promoter region of PrmIP in megakaryocytic HEL.92.1.7, vascular endothelial EA.hy926 cells and primary (1o) HUVECs. Mutational disruption of a putative Androgen Response Element (ARE) within PrmIP abolished DHT - induced reporter gene expression while ChIP analysis confirmed direct binding of the androgen receptor (AR) to the PrmIP in vivo. DHT increased hIP -induced signalling (cAMP second messenger generation) while immunocytochemistry confirmed DHT -induction of PRTLI Programme PhD PRTLI Programme PhD

12 13 PHD PROGRAMME IN MOLECULAR CELL BIOLOGY MOLECULAR AND CELLULAR MECHANISMS UNDERLYING INFLAMMATORY PROCESSES Nucleosome organisation of the vertebrate Modulation of the Endocannabinoid system and centromere PanX1-mediated neuroinflammation in anin vitro model of Alzheimer’s disease NUIG TCD

J.G.W. McCarter, K.F. Sullivan S.G. Fagan, V.A. Campbell

ORAL ORAL

entromeres provide the segregation function of individuals of the same species. Quantitative analysis lzheimer’s disease is an age -related and fatal This data demonstrates a role for PanX1 in mitotic chromosomes. Centromeres are specified has also revealed that even with the varying CENP A- neurodegenerative disease characterised by promoting neurodegeneration and microglial migration by a unique type of chromatin that contains distribution the amount of CENP A- associated DNA still the progressive deterioration of cognition and in response to mediators produced by neurons Cthe histone H3 variant - CENP A.- In addition to CENP A,- remains remarkably uniform. This suggests that even Amemory resulting from synaptic loss and neuronal following exposure to Aß. Furthermore, a downstream histone H3 and a novel nucleosome -like structure, though centromeres occupy different spans of DNA that death. The accumulation of toxic amyloid -beta (Aβ) interaction between the endocannabinoid system and CENP T/W/S/X,- are essential for centromere function. at a molecular level centromeres may have a very well oligomers results in chronic neuroinflammation leading PanX1 regulation may represent a novel mechanism These nucleate assembly of the kinetochore in G2 and defined structure in terms of CENP A- nucleosomes. to synaptic dysfunction and severe neurodegeneration for modulation of the neuroimmune response in M through recruitment of a large group of centromere [1]. Modulation of the endogenous cannabinoid system Alzheimer’s disease.

and kinetochore associated proteins. Despite the critical is known to confer neuroprotection against Aβ -induced [1] Salminen, A., Ojala, J., Kapuppinen, A., Kaarniranta, K. & Suuronen, T. importance of centromeric chromatin, little is known neurotoxicity through the inhibition of apoptotic 2009. Inflammation in Alzheimer’s disease: amyloid -beta oligomers about its detailed structure due to its association cascades and the immune response [2]. A recently trigger innate immunity defense via pattern recognition receptors. with repetitive satellite DNA. The equid species have identified and ubiquitously expressed membrane Prog Neurobiol, 87, 181 -94. numerous centromeric regions associated with unique channel, PanX1, has been implicated in the release of [2] Fagan, S.G. and Campbell, V.A. 2014. The influence of cannabinoids sequence. ATP from damaged cells as well as activation of the on generic traits of neurodegeneration. Brit Jour Pharmacol, 171(6), 1347 -1360. NLRP3 inflammasome [3]. The aim of this study was to This project is focused on dissecting the investigate the involvement of PanX1 in cannabinoid - [3] Wicki -Stordeur, L.E. and Swayne, L.A. 2014. The emerging Pannexin1 molecular architecture of this chromatin using ChIP - signalome: a new nexus revealed? Frontier Cell Neuro, 7. mediated neuroprotection against Aβ. seq methods uniquely suitable for equid unique sequence centromeres. Here we present CENP A- Cultured primary rat cortical neurons treated with immunoprecipitated DNA sequence data from various Aβ (10 µM, 72 hours) release chemoattractant signals equids and equid hybrid offspring revealing 29 unique that increased the migration of BV2 microglial cells by sequence centromeres. Our data show what we call 115% (p=0.0017; n=4; ANOVA) using a Boyden chamber “allelic positional variation” where centromeres on assay. Both the pharmacological inhibition of PanX1 two homologues of a diploid cell can occupy slightly ChIP-Seq of CENP-A reveals centromere position and behaviour in by mimetic peptide, 10panx (200 µM; p=0.0415; n=5; equine cells. Shown are ChIP-Seq read count profiles for donkey (top two different genomic positions. We also show that CENP A- panels) and mule (bottom three panels) CENP-A associated DNA of two ANOVA) and treatment with URB597 (5 µM; p=0.0028; distribution or its “genomic footprint” spans between chromosomes. Analysis of the location and transmission of centromeres n=5; ANOVA) an inhibitor of cannabinoid degradation, reveals plasticity and stability of these essential chromosomal loci. 50 -250kb. This varying footprint can be seen not only significantly reduced the microglial migration that is within one individual but also between two different stimulated by amyloid -beta -primed neuronal media. Furthermore, no additive effect from co -treating neurons with both 10panx and URB597 on microglial migration was found indicating a possible overlap in the TUNEL assay for DNA fragmentation (red arrows) carried out on two pathways. Using the TUNEL assay Aβ was shown to primary cortical neurons treated with [A] neurobasal medium control, [B] oligomerised Aβ, [C] URB597, an inhibitor of cannabinoid increase DNA fragmentation in primary cortical neurons, degradation, [D] URB597 and Aβ, [E] probenecid, a pharmacological an affect that was abrogated by both URB597 and inhibitor of PanX1, [F] probenecid and Aβ. treatment with probenicid, a pharmacological inhibitor of PanX1 (p=0.3043; n=3; ANOVA). The apoptotic effector molecule caspase -3 is one of the primary regulators of PanX1 activity. In primary cortical neurons URB597 significantly reduced the Aβ -induced rise in caspase -3 levels (p=0.038; n=5; ANOVA). PRTLI Programme PhD PRTLI Programme PhD

14 15 PHD PROGRAMME IN MOLECULAR CELL BIOLOGY MOLECULAR AND CELLULAR MECHANISMS UNDERLYING INFLAMMATORY PROCESSES S1P receptor activation attenuates Use of Lacticin 3147 and essential oils psychosine-induced astrocyte cell death against food-borne pathogens TCD UCC

C O’Sullivan, K.K. Dev A. Campion, D. Field, P.D Cotter, C. Hill, R.P. Ross POSTER POSTER

rabbe disease is a rare autosomal recessive effect psychosine on human and rat astrocyte antibiotics are ribosomally synthesised neurodegenerative disorder affecting 1:100,000 survival and demonstrate the protective effects of antimicrobial peptides that undergo extensive births. This illness is rapidly progressing, the phosphorylated version of FTY720 (pFTY720) post -translational modifications resulting in the Kappearing within the first three to six months of life and against cell -toxicity induced by psychosine. In brief, Lformation of unusual amino acid residues. Lacticin 3147 is usually fatal by the age of two years. Krabbe disease we find psychosine induces astrocyte cell death is a two -peptide lantibiotic with a broad spectrum of is caused by a deficiency in the lysosomal enzyme in a concentration - and time -dependent manner. activity. It is one of the most well studied lantibiotics galactocerebrosidase (GALC), which results in the Importantly, pre -treatment with pFTY720 attenuates and exerts its activity through a dual mode of action accumulation of a toxic metabolite in the brain termed psychosine induced cell death and significantly by binding lipid II, an intermediate involved in psychosine. The build -up of psychosine is believed prolongs astrocyte cell survival. Taken together, these peptidoglycan biosynthesis, followed by insertion into to be the main pathogenic agent in Krabbe disease results suggest that S1PRs could be potential drug the target cell membrane. Essential oils are secondary resulting in widespread oligiodendrocyte cell death and targets for Krabbe disease. metabolites extracted from plants. They often possess demyelination. antimicrobial activities and are thought to be important This work was supported in part by research grants in plant defense. Essential oils are now primarily used To date, most studies have focused on the toxic from Trinity College Dublin, The Health Research Board in the food industry as flavourings but there has been effects of psychosine on oligiodendrocytes, however Ireland, Science Foundation Ireland and The Higher increased interest in recent years in their application little is known about the effects of psychosine on Education Authority Ireland (Programme for Research in as natural food preservatives. Here, we investigate the astrocytes. Astrocytes, the most abundant cell Third Level Institutions [PRTLI]). COS is a PRTLI funded activities of purified and semi -purified lacticin 3147 and type in the brain, have many important functions PhD Scholar. the essential oils, thymol, carvacrol and cinnamaldehyde such as protection and support of neurons and against food -borne pathogens of interest. Lacticin oligodendrocytes. Astrocytes also play key roles in 3147 and the essential oils are both effective at limiting regulating metabolic and ion homeostasis in the CNS. bacterial growth however, a greater potency is seen Importantly, astrocytes express S1P receptors (S1PR) when the antimicrobials are used in conjunction. which are targets for the first oral therapy developed for multiple sclerosis, FTY720 (Fingolimod). S1PRs have various roles in the CNS, including astrocyte migration,

oligodendrocyte survival, and neurogenesis. Organotypic Slice cultures from P10 mice stained for NFH (Neurofilament H = red) and MBP (Mylein Basic Protein = green) More recently, S1PRs have been found to play a role in inhibiting the release of pro‐inflammatory cytokines from glial cells. Here we investigate the

Effect of semi-purified lacticin 3147 and the essential oil thymol on the growth of Bacillus cereus DPC6336. Lacticin 3147 and thymol are both capable of inhibiting bacterial growth of B. cereus DPC6336. Although, a greater antimicrobial potency is observed when thymol and lacticin are used in conjunction. PRTLI Programme PhD PRTLI Programme PhD

16 17 PHD PROGRAMME IN MOLECULAR CELL BIOLOGY MOLECULAR AND CELLULAR MECHANISMS UNDERLYING INFLAMMATORY PROCESSES The role of the Cytokine release inhibitor drug, CRID3, inhibits bone morphogenetic antagonist Gremlin the NLRP3 inflammasome in glia in the pathogenesis of Chronic Lung Disease UCD TCD

J. Cornwell, P. McLoughlin C.N. Dempsey, R.C. Coll, A.A.B. Robertson, M.A. Cooper, POSTER L.A.J. O’Neill, M.A. Lynch POSTER

hronic hypoxic lung disease is one of the world’s within HPMVEC and whole lung. Three ligands (Wnt3, ssembly of the cytosolic multiprotein complex, activated caspase 1 activity in LPS -treated microglia leading causes of death and disability. Hypoxic Wnt3a and Wnt5a) appeared to be hypoxia responsive the NLRP3 inflammasome, results in activation and astrocytes, represented by the CD11b+ and CD11b - pulmonary hypertension (HPH) is a common on end -point PCR. On real -time PCR Wnt3 was expressed of caspase 1 and processing of IL -1β to permit populations in mixed glial cultures; this effect was Ccomplication of chronic lung disease which frequently but not hypoxic responsive, Wnt3a was undetectable Arelease of the active form of the cytokine. We have significantly attenuated by CRID3. Consistent with a leads to heart failure and reduced life expectancy. and Wnt5a was increased in hypoxia. Therefore, the previously reported that amyloid beta (Aβ) induces specific effect on IL -1β processing, CRID3 exerted no Microvascular endothelial cells play an important role in interaction of grem1 with Wnt5a will be examined assembly of the inflammasome in microglia resulting significant effect on the Aβ -induced increase in mRNA the vascular changes associated with hypoxia. Gremlin, further. in release of IL -1β, but not IL -6 or TNFα. Recent expression of IL -1β, TNFα and IL -6 in LPS -pretreated a BMP antagonist, is upregulated during hypoxia [1] Cahill E, Costello CM, Rowan SC, Harkin S, Howell K, Leonard MO, findings indicate that a novel NLRP3 inhibitor, cytokine microglia or astrocytes. and contributes to the development of pulmonary et al. Gremlin plays a key role in the pathogenesis of pulmonary release inhibitor drug 3 (CRID3), effectively inhibits These data show that CRID3 selectively inhibits hypertension through its action within the BMP pathway hypertension. Circulation. 2012;125(7):920 -30. Epub 2012/01/17. inflammasome activation in bone marrow -derived inflammasome activation in both glial subtypes and [1]. More recently it has been shown that gremlin may [2] de Jesus Perez VA, Alastalo TP, Wu JC, Axelrod JD, Cooke JP, Amieva M, macrophages. The aim of this study was to investigate identify it as a potential therapy for neurodegenerative act on other non -BMP pathways including the Wnt et al. Bone morphogenetic protein 2 induces pulmonary angiogenesis the effects of CRID3 on NLRP3 inflammasome activation disorders which are characterized by neuroinflammatory pathway [2]. The aims of the project are to explore the via Wnt -beta -catenin and Wnt -RhoA -Rac1 pathways. The Journal of cell biology. 2009;184(1):83 -99. Epub 2009/01/14. in glia. changes. non -BMP signalling pathways by which gremlin acts in the lung and to examine the role of endothelial gremlin Primary cultures of purified microglia, purified in the development of HPH in vivo. astrocytes and mixed glia, generated from neonatal mice, were pre -treated with lipopolysaccharide (LPS; The expression of 12 Wnt ligands expressed in 1µg/ml) for 4 hours and thereafter incubated with endothelial cells of other organs, and their receptors, different concentrations of CRID3 for 30 minutes, after was examined in human pulmonary microvascular which time human Aβ (4.3µM) + Aβ (5µM) was endothelial cells (HPMVEC) exposed to 24 hour 1 -40 1 -42 added. Incubation continued for a further 24 hours. normoxia (21% O ) or hypoxia (5% O ) using end -point 2 2 Concentrations of IL -1β, IL -6 and TNFα were assessed in PCR (39 cycles). Real Time- PCR was used to investigate samples of the supernatant and cells were harvested for the hypoxia response of three selected Wnt ligands. Wild analysis of mRNA expression of these cytokines. Flow type mice were exposed to 48 hours normoxia (21% O ) 2 cytometry was used to assess expression of activated or hypoxia (10% O ) then anaesthetised and lung tissue 2 caspase 1 activity in CD11b+ and CD11b - cells in mixed harvested for RNA extraction. End -point PCR was used glial cultures. to examine the expression of Wnt ligands and receptors in these lungs. Aβ significantly increased release of IL -1β in LPS - Wnt5a Expression in HMVEC-L . Wnt5a expression in Human pretreated microglia and astrocytes and this was Seven Wnt ligands were found to be expressed Microvascular Endothelial cells from the lung. Real-Time qPCR showed Wnt5a expression to be increased in hypoxia (5%), a level which mimics significantly attenuated by CRID3 in a dose -dependent CRID3 and the NLRP3 inflammasome. CRID3 selectively inhibits the within HPMVEC exposed to either normoxia or hypoxia. oxygen levels in chronic lung disease. NLRP3 inflammasome by preventing the oligomerization of NLRP3 and the These seven were also expressed in the in vivo lung. manner; CRID3 did not affect release of TNFα or IL -6 adaptor protein ASC. This prevents the cleavage of procaspase 1 therefore preventing the cleavage of proIL-1β and release of the active cytokine. Cognate Wnt receptors were also found to be present in either cell type. In parallel with the increase in IL -1β release, Aβ also significantly increased expression of PRTLI Programme PhD PRTLI Programme PhD

18 19 PHD PROGRAMME IN MOLECULAR CELL BIOLOGY MOLECULAR AND CELLULAR MECHANISMS UNDERLYING INFLAMMATORY PROCESSES Examination of the inner centromere compartment Screening protein Kinase mutants to identify in a mammal novel genes required for biofilm formation in Candida Albicans NUIG UCC

T. Masterson, K. Sullivan N. Konstantinidou, F. O’Gara, J. Morrissey POSTER POSTER

ntil now, it was generally accepted that ChIPSeq experiments will be carried out on espite the extensive utilization of drugs, their ability to form biofilms on polystyrene microtitre centromeres were usually associated with paraformaldehyde crossed -linked cells, sheered to a size microbial infections remain a leading cause plates. Some of the strains that were unable to form repetitive α satellite DNA which prevents range of between 200 -300bp. Antibodies are validated of human mortality worldwide. In particular, biofilms were deleted in genes already known to be Uapplication of chromatin profiling methods to examine for use in ChIPSeq by carrying out ChIP on 5 x106 cells Dmedical devices can serve as the substrate for the required for biofilm formation, but several novel genes the biochemical structure of this locus. Our recent and examining the immunoprecipitated protein and growth of polymicrobial communities that form were also identified. Currently, these are being analysed work in collaboration with the Giulotto lab, established DNA by western blot and qPCR. RNA will be extracted drug -resistant biofilms. The fungusC. albicans and the to ascertain whether they are connected to known that equid species contain from one (Equus caballus- from horse and donkey fibroblasts and sent for whole bacterium Pseudomonas aeruginosa are two clinically biofilm formation pathways or represent new processes. horse) [1] to sixteen (Equus asinus-donkey) centromeres genome sequencing. A core set bioinformatics skills important biofilm formers that can sometimes be Filamentation studies showed that all the very poor associated with unique sequence DNA. Localisation and workflow analysis is also required for downstream co -isolated from infected sites of human body. Recent biofilm forming strains were defective in the switch from of inner centromere components such as the of analysis of the data generated and this has been studies established that molecular interactions yeast to hyphal growth, establishing this as the primary chromosomal passenger complex (CPC) in interphase established within the lab. defect. The effect ofP. aeruginosa supernatants (with between these two microbes can have important is dependent on heterochromatin, which is lacking in or without homoserine lactones) on the ability of the [1] Piras, F.M., Nergadze, S.G., Magnani, E., Bertoni, L., Attolini, C., Khoriauli, consequences for expression of virulence traits. For equid neocentromeres. Cohesin cleavage in prophase L., Raimondi, E., and Giulotto, E. Uncoupling of satellite DNA and mutants to form biofilms was assessed but none showed example, P. aeruginosa signaling molecules have is dependent on the CPC kinase Aurora B [2]. ChIPSeq centromeric function in the genus Equus. PLoS Genet 6, e1000845. specific defects indicating either that protein kinases are at least two independent effects onC. albicans: approaches will be applied to the equid system using (2010) not part of this signalling pathway or that redundancy impairment of the switch from yeast to hyphal growth, antibodies against specific CPC subunits as well as [2] Carmena, M., Wheelock, M., Funabiki, H., and Earnshaw, W.C. The may exist. Identification of new biofilm -related genes cohesin subunits to establish how protein localization is chromosomal passenger complex (CPC): from easy rider to the and inhibition of biofilm development. To better and understanding yeast -bacterial cross talk contributes godfather of mitosis. Nat Rev Mol Cell Biol 13, 789 -803 (2012). impacted by lack of alpha satellite repeats. understand biofilm formation inC. albicans and how to our knowledge of fungal signalling pathways and this might be affected byP. aeruginosa, we screened may ultimately lead to the identification of novel drug Centromeric regions are generally considered to a library of 80 independent C. albicans protein kinase targets. be transcriptionally inactive. This is not strictly the mutants in biofilm and morphology assays. Mutants case although centromere -derived transcripts do not were categorized into five different classes based on generally accumulate at significant levels. Functional centromeres possess histone marks that are typically associated with transcriptionally active chromatin. The equine system provides a platform to evaluate centromere -associated transcription by virtue of the availability of centromere -active versus centromere - inactive chromosomal domains present in very closely related species. The hypothesis is that centromeres possess a transcriptional “signature” and we propose to evaluate that hypothesis by RNASeq analysis of horse versus donkey cells, correlating RNA with centromere Investigating the molecular anatomy of the centromere with novel satellite- Biofilm development of 11 free centromeres in equine species. Mitotic centromere domain structure position in parallel samples prepared at different stages very poor biofilm formers. α| (left). Recombinant proteins (top right) are being used to make antibodies for first screen, β| second screen, of the cell cycle. equine cells (lower right). ChIPSeq will be used to map centromere domains grey notched boxplots| wild to the linear sequence of the DNA. type control Day286, red dots| mean, middle line of the boxplots and calculations| median, white circles| outliers, vertical dashed lines| threshold OD4900.5 (1/3) and horizontal dotted lines|

PRTLI Programme PhD separate mutants. Statistical PRTLI Programme PhD analysis were performed in R. 20 21 PHD PROGRAMME IN MOLECULAR CELL BIOLOGY MOLECULAR AND CELLULAR MECHANISMS UNDERLYING INFLAMMATORY PROCESSES The PYHIN protein, MNDA is involved Exploring the immune modulatory role of in the regulation of Interferon alpha expression MPB70 and MPB83 by Mycobacterium tuberculosis TCD UCD

D. Casserly, S. Carpenter, L. Carr, C. Brereton, E. Lavelle, K.A. Fitzgerald, A.G. Bowie S. Gordon POSTER POSTER

ype I interferons (IFNs) are secreted by cells in a direct transcriptional activator of IFNα induction. Thus ycobacterium tuberculosis and response to pattern recognition receptor (PRR) human PYHIN proteins have diverse and distinct roles in Mycobacterium bovis display distinct host stimulation by viral nucleic acids. Recently type I IFN induction. preference, even though they are over Tmammalian PYHIN proteins have been implicated as 99.95% identical at the nucleotide level. In spite of [1] Hornung V, et al. AIM2 recognizes cytosolic dsDNA and forms M PRRs for intracellular viral DNA. PYHIN proteins are a caspase -1 -activating the inflammasome with ASC. Nature this identity there are proteins that are differentially characterized by the presence of a Pyrin and a HIN200 2009;458:514–518. expressed by M. bovis and M. tuberculosis. We DNA binding domain, and many of the PYHIN family are [2] Unterholzner L, et al. IFI16 is an innate immune sensor for intracellular hypothesise that some of these proteins may play an IFN -inducible. AIM2 was the first family member shown important role in virulence by modulating the host to be a PRR, and both mouse and human AIM2 are immune response. intracellular sensors of dsDNA which activate caspase MPB83 is a glycosylated lipoprotein embedded 1 leading to IL -1β production [1]. Human IFI16 is also in the outer membrane of the bacterium. MPB70 is a a PRR and mediates type I IFN induction in response homolog of MPB83 but it is not translationally modified to dsDNA, via the STING TBK1- -IRF3 signaling axis [2]. and is secreted from the cell. Their function is largely Apart from AIM2 and IFI16, the human PYHIN family also unknown but they have been shown to be expressed at includes MNDA and PYHIN1. higher levels in vitro in M. bovis than in M. tuberculosis. In order to determine whether MNDA is also a DNA They are up -regulated by M. tuberculosis when the sensor we used lentiviral shRNA to generate human bacterium is engulfed by a macrophage. They have monocytic cells with reduced expression of MNDA. In been shown to induce a T -cell response in cattle causing contrast to cells expressing IFI16 shRNA, DNA -induced IFN -gamma production and T -cell proliferation. MPB83 IFNβ was normal in MNDA shRNA -treated cells. However, has also been reported that it binds to TLR -1/2 and can DNA -induced IFNα expression was significantly reduced induce pro -inflammatory cytokine production. when MNDA levels were reduced. RNA -induced IFNα , In order to explore the immunomodulatory function but not IFNβ, was also dependent on MNDA, suggesting of these proteins on innate cells DCs and macrophages that MNDA plays a role downstream in the PRR -type DNA. Nat Immunol 2010;11:997–1004. were stimulated with recombinant proteins. Cytokine I IFN induction signalling pathway, being selectively Potential role for MNDA as a regulator of the IFNα promoter. Cytosolic production was measured by ELISA. To examine the required for IFNα and not IFNβ induction. IFNα is DNA is recognised by cGAS or IFI16 and results in the upregulation of IFNβ effect on a mixed cell population T -cell proliferation and secondarily induced by PRR -induced IFNβ signalling via through the STING-TBK1-IRF3 axis. IFNβ signals through the IFNAR and initiates JAK-STAT signalling and the formation of the ISGF3 transcription cytokine production by splenocytes was measured by STAT1, and STAT1 activation was normal in cells with factor, which induces the expression of many ISGs, including IRF7. Activated flow cytometry. reduced expression of MNDA. IRF7 acts as a transcription factor and can upregulate IFNα. As signalling events leading up to the induction of IFNα, it suggests that MNDA functions Our results demonstrate that MPB70 and MPB83 Given that MNDA is primarily localised within the by regulating the IFNα promoter. don’t induce pro -inflammatory cytokine production by nucleus, and has the ability to bind DNA, MNDA may be innate, antigen presenting cells. However, T cells, in a mixed cell population, are activated when stimulated with both proteins. PRTLI Programme PhD PRTLI Programme PhD

22 23 PHD PROGRAMME IN MOLECULAR CELL BIOLOGY MOLECULAR AND CELLULAR MECHANISMS UNDERLYING INFLAMMATORY PROCESSES The effect of Isoellipticines on leukaemia cells γβ T Cells, a novel T Cell subset with a pathogenic role in IL-17-mediated CNS autoimmunity UCC TCD

E.G. Russell, T.G. Cotter S.C. Edwards, K.H.G. Mills

POSTER POSTER

llipticine (5,11 -dimethyl -6H -pyrido[4,3 -b] therapy and we aim to explore this concept in future γ4+ T cells have been identified as the main IL - significantly impair their ability to induce EAE, with a carbazole) was first isolated from the leaf of studies. 17 -producing γδ T cell in the CNS of mice with reduction in inflammatory T cells infiltrating the CNS. Ochrosia elliptica Labill by Goodwin et al. in 1959 experimental autoimmune encephalomyelitis [1] Miller CM, O’Sullivan EC, Devine KJ, McCarthy FO (2012) Synthesis and Our study has identified a novel γβ T cell subset, Eand was found to display potent anticancer activity. biological evaluation of novel isoellipticine derivatives and salts. Org V(EAE), an animal model of multiple sclerosis. Vγ4β T cells, which together with conventional Vγ4δ Despite this, its use in a clinical setting has been limited Biomol Chem. 10(39):7912 -21 The findings of this study demonstrate that Vγ4+ T cells, play a critical role in the pathogenesis of EAE due to low solubility and bioavailability. To overcome [2] Ellipticine derivative induces potent cytostatic effect in acute myeloid T cells are present in T cell receptor (TCR)δ -/ - mice, and through innate IL -17 production, necessary to enhance these limitations, a range of novel ellipticine derivatives leukaemia cells (2014) Russell EG, O’Sullivan EC, Miller CM, Stanicka J, furthermore these Vγ4+ T cells co -express TCRβ. The Th17 and Th1 activation and migration into the CNS to have been synthesised and evaluated for potential McCarthy FO, Cotter TG. Investigational New Drugs (under revision) data reveals that Vγ4β T cells respond to IL -1β and mediate inflammation and autoimmunity. improvement in cytotoxicity [1]. Certain isoellipticine IL -23 stimulation in the absence of TCR engagement analogues displayed promising anti -tumour activity to produce IL -17A and IL -17F and express the master across a number of different cell lines, particularly transcription factor, RORγt and the integrin, MCAM. leukaemia cell lines. Furthermore, Vγ4β+ T cells, together with conventional The current study examines the effect of these Vγ4δ+ T cells were found in the brains of mice with EAE. derivatives in detail on the Acute Myeloid Leukaemia Vγ4β+ T cells were also found in the brains TCRδ -/ - mice (AML) cell line, MV4 -11. Cell cycle analyses revealed with EAE. Although, the course of EAE is somewhat that the compounds had a range of distinctive cell reduced in TCRδ -/ - compared with wild type (WT) mice, cycle effects. 7 -Hydroxyisoellipticine showed the depletion of Vγ4+ T cells from TCRδ -/ - as well as WT mice most promise with respect to cytostatic activity. significantly attenuated clinical disease and weight loss We demonstrated that this compound inhibited in mice with EAE. proliferation of leukaemia cells by preventing cells Anti Vγ4- treated mice with EAE had a significantly from progressing from G2 phase into mitosis over a reduced frequency of infiltrating IL -17+, IFN -γ+ and period of 24 hours at a concentration of 5 μM. Our Co-localised surface expression of Vγ4 and TCRβ on T cells. Overlay of IFN -γ+IL -17+ CD4+ T cells into CNS. Furthermore, in the research suggests that this is mediated by an induction DAPI, anti Vγ4 (AlexaFluor488) and anti TCR β (AlexaFluor647) on Moflo adoptive transfer model of EAE, depletion of Vγ4+ cells FACS sorted Vγ4+ CD3+ T cells. Fluorescent microscopy was performed of reactive oxygen species (ROS), as measured by flow from lymph node and spleen cells from TCRδ -/ - mice using 40X/60X zoom on an Olympus FV1000© confocal microscope cytometry, which in turn activates the DNA damage response pathway. As a result of the activation of p53, cyclin B1 is inhibited. These protein levels were measured by western blotting. The induction of this damage response pathway leads to apoptosis which is seen at 48 hours by microscopy using the same dose of 7 hydroxyisoellipticine- [2]. The project now focuses on establishing the mechanism of action of 7 -formyl -10 - methylisoellipticine, a more potent isoellipticine. This study provides for the first time detailed cellular

information on the potential use of isoellipticines as Schematic of 7-hydroxyisoellipticine mechanism of action. chemotherapeutic agents. By probing the mechanism of 7-hydroxyisoellipticine mechanism of action resulting in G2/M cell cycle action of this novel compound class we have uncovered arrest. A representative profile is shown of cell cycle of MV4-11 cells incubated with 5 μM of 7-hydroxyisoellipticine for 24 hours analyzed by flow a potential clinical application in the field of adjuvant cytometry with propidium iodide staining. Control = 0.5 % DMSO. PRTLI Programme PhD PRTLI Programme PhD

24 25 PHD PROGRAMME IN MOLECULAR CELL BIOLOGY MOLECULAR AND CELLULAR MECHANISMS UNDERLYING INFLAMMATORY PROCESSES Investigation into the regulation of p75NTR by NGF, Quantitative analysis of IL-17 induced and its pro-survival signaling in protein expression profiles of breast cancer cells Psoriatic Arthritis B cell models NUIG UCD

R. Chakravarthy, J. Hillis, A. Gorman M. Doyle, S. Pennington

POSTER POSTER

ro -survival nerve growth factor (NGF) signalling, We hypothesize that NGF increases the expression n 2010 multiple genome wide association studies expressed in response to various stimuli in numerous mediated by p75NTR, may contribute to the of p75NTR thus increasing the availability of the full linked two single nucleotide polymorphisms (SNPs) different cell lines. These proteins could be used to plan resistance of breast tumours to chemotherapy [1]. length receptor and facilitating the pro -survival effect of D10N/R74W in the TRAF3IP2 gene (Act1 protein) future experiments or as potential targets for inhibition PThus anti -NGF therapy could increase the effectiveness p75NTR in TNBC cells. Iwith psoriatic arthritis(1). My current project is directed in the treatment of PsA. of chemotherapeutic drugs used in breast cancer [1] Adriaenssens E, Vanhecke E, Saule P, Mougel A, Page A, Romon R, towards using immortalised B cells from psoriatic [1] Hüffmeier U, Uebe S, Ekici AB, Bowes J, Giardina E, Korendowych E, et treatment. NGF is produced by over 80% of primary Nurcombe V, Le Bourhis X, Hondermarck H: Nerve growth factor is a arthritis (PsA) patients with these SNPs to identify how al. Common variants at TRAF3IP2 are associated with susceptibility to breast tumours, in contrast to normal breast cells which potential therapeutic target in breast cancer. Cancer Research 2008, they could affect B cell responses in PsA. psoriatic arthritis and psoriasis. Nature Genetics. 2010;42(11):996 -9. do not secrete NGF [2]. Both express the NGF receptors 68:346 -351. Wild type (WT) and double mutant (DM) (n=3) B TrkA and p75NTR. Hence, NGF -targeted treatment [2] Dolle L, El Yazidi -Belkoura I, Adriaenssens E, Nurcombe V, Hondermarck cells (2x106 cells per ml) were induced with IL -17 (50ng/ would be more specific to breast tumour cells. H: Nerve growth factor overexpression and autocrine loop in breast cancer cells. Oncogene 2003, 22:5592 -5601. ml) for 4 hours. The IL -6 concentrations within the B cell MDA -MB -231, a triple negative breast cancer cell supernatants were measured by ELISA to quantify the line expressing p75NTR, was treated as desired and the cells response to IL -17. The lysate protein concentrations whole cell extracts were harvested for western blot -anti were quantified using a BCA assay. The lysates (100ug) p75NTR or to measure loss of mitochondrial membrane were digested with Trypsin and Lysine -C using a potential using TMRE assay. modified FASP protocol. The stage tipped digests (2ug) We have shown that by inhibiting the downstream were run on the Q -Exactive (LC -MS/MS) over a 90 minute activation of p75NTR or by interfering with the gradient. The protein and peptides were searched by interaction of NGF/p75NTR using Ro 08 -2750 (inhibits Andromeda (maxquant) on a uniprot database and NGF binding to its receptors), we can sensitise cells to the statistical analysis was performed using perseus. death in MDA -MB -231 cells (n=3; One way ANNOVA, Pathway analysis was done using ingenuity pathway Tukey’s post -hoc test p<0.05). This confirms an NGF/ analysis (IPA). p75NTR mediated pro -survival effect. We show here that Increased levels of IL -6 indicated an overactive the expression of p75NTR is regulated by exogenous/ response to IL -17 in the DM cells. Over 3000 proteins endogenous NGF. The secretion of NGF into the medium were identified in each of the digests (n=12). Analysis was quantified by ELISA. This regulation was diminished of the LC -MS/MS results showed consistent expression in the presence of NGF neutralizing antibody. NGF (0.91 -0.99 Pearson correlation) of a large portion of the mediated increase in expression of p75NTR is not due proteins from sample to sample. 32/92 unique proteins to inhibition of processing of p75NTR. Furthermore, that were only expression in the IL -17 induced WT/ LC-MS/MS analysis of Psoriatic Arthritis patient B cell lysates can identify significant proteins whose expression level is changed in response to IL17. inhibiting the processing of FLp75NTR (Full length) DM protein expression profiles were shown to interact These proteins can be linked using ingenuity pathway analysis software and sensitised cells to drug induced cell death (n=3, One with Ubiquitin (UBC). A couple of these proteins (AKT1, networks of the most significant proteins can be generated way ANNOVA, Tukey’s post -hoc test p<0.05). Regulation MAPK14) were involved in IPA’s IL -17 canonical pathway of p75NTR by NGF is dependent on transcription/ Regulation of expression of p75NTR by exogenous NGF; inhibition by (Act1 independent) and they are only expressed in the translation. This regulation of p75NTR by NGF may neutralizing antibody to NGF; regulation through NGF dependent IL -17 induced DM protein expression profile. contribute to resistance to chemotherapeutic drugs. transcription/translation These data are representative of n=3. The results indicate that post translational modifications specifically ubiquitination (UBC) may be playing a key role in the activation of various IL -17 signalling pathways. LC -MS/MS could be used to identify different key proteins and pathways that are over PRTLI Programme PhD PRTLI Programme PhD

26 27 PHD PROGRAMME IN MOLECULAR CELL BIOLOGY MOLECULAR AND CELLULAR MECHANISMS UNDERLYING INFLAMMATORY PROCESSES mTORC1 regulation of Dendritic cell metabolism The effect of inflammatory Caspases in through HIF1α and iNOS Colitis and Colorectal cancer TCD TCD

S. Lawless, D. Finlay B. Flood, K. Oficjalska, E. Creagh

POSTER

vidence is emerging that crucial metabolic [1] Finlay, D.K., Rosenzweig, E., Sinclair, L.V., Feijoo -Carnero, C., nflammatory Bowel Diseases (IBDs) affect a CASP11 -/ - mice, also indicating enhanced inflammation. changes occur in pro -inflammatory immune Hukelmann, J.L., Rolf, L., Panteleyev, A.A., Okkenhaug, K., Cantrell, D.A. significant and increasing proportion of Ireland’s Histological analysis revealed increased inflammatory PDK1 regulation of mTOR and hypoxia -inducible factor 1 integrate cells that are required for the normal effector metabolism and migration of CD8+ T cells. J. Exp. Med., 2012; 209(13): population. Persistent and sustained intestinal cell infiltration and mucosal damage in DSS -treated - - Efunctions of these cells. Thus, pro -inflammatory immune 2441 -53. Iinflammation can predispose patients to colorectal CASP11 / mice, with inflammation resulting in severe + cells, including CD8 cytotoxic T cells [1] and activated [2] Krawczyk, C.M., Holowka, T., Sun, J., Blagih, J., Amiel, E., DeBerardinis, cancer (CRC). The inflammatory caspases are a group loss of crypt architecture (see Figure). dendritic cells (DC) [2], increase glucose uptake which of enzymes involved in triggering pro -inflammatory R.J., Cross, J.R., Jung, E., Thompson, C.B., Jones, R.G., Pearce, E.J. Toll -like Our findings suggest a role for caspase -11 in colon is primarily metabolized to lactate rather than through receptor -induced changes in glycolytic metabolism regulate dendritic immune responses. Caspase -1, which is responsible repair and proliferation in a mouse model of colitis. oxidative phosphorylation (OxPhos). Understanding cell activation. Blood., 2010; 115(23): 4742 -9. for the activation and secretion of two major pro - Longer term DSS and azoxymethane (AOM)/DSS studies how cell metabolism relates to immune cell function is inflammatory cytokines, IL -1β and IL -18, has been are underway to determine whether CASP11 -/ - mice crucial to the design of improved immunotherapeutic implicated in IBD -associated inflammation [1]. The role are also more susceptible to chronic colitis and colitis strategies. of inflammatory caspase -11 during inflammation is less associated cancer (CAC). well understood, however, recent studies suggest that it The mammalian target of rapamycin complex 1 is required for the activation of caspase -1 during Gram [1] Dupaul -Chicoine J, Yeretssian G, Doiron K, Bergstrom KSB, McIntire (mTORC1) is required for the metabolic changes in LPS CR, LeBlanc PM, Meunier C, Turbide C, Gros P, Beauchemin N, Vallance negative bacterial infection [2]. stimulated bone marrow derived DC (BMDC). Both BA, Saleh M. Control of Intestinal Homeostasis, Colitis, and Colitis - HIF1α and iNOS, acting downstream of mTORC1, are To determine the role of caspase -11 during a model Associated Colorectal Cancer by the Inflammatory Caspases. Immunity 2010;32:367 -78. involved in controlling BMDC glucose metabolism. While of acute colitis, CASP11 -/ - mice and their wild type inhibition of mTORC1 shifts metabolism from glycolysis (WT) littermates were given 2% (w/v) dextran sodium [2] Kayagaki N, Warming S, Lamkanfi M, Vande Walle L, Louie S, Dong J, Newton K, Qu Y, Liu J, Heldens S, Zhang J, Lee WP, et al. Non -canonical to OxPhos, deletion of HIF1α inhibits the shift to sulphate (DSS) in their drinking water for 3 -7 days, and inflammasome activation targets caspase -11. Nature 2011;479:117 -21. glycolysis but has no effect on the decrease in OxPhos. disease activity was monitored. Colon sections were Interestingly, deleting HIF1α effects the expression of analyzed using hematoxylin and eosin (H&E) staining. inflammatory cytokines, thereby directly implicating Colitis severity was histologically assessed by scoring for cellular metabolism in the control of DC function for the cellular infiltration and crypt damage. first time. Colon homogenates from WT C57BL6/J mice The mTORC1/HIF1α/iNOS signaling axis is central to In the absence of HIF-1α the shift to glycolysis is blocked in LPS stimulated DC’s. 10 day BMDC’s from HIF-1αflox/flox (WT) or HIF-1αflox/ revealed that caspase -11 is robustly activated during the control of DC metabolism and seems to be directly flox Vav-Cre (knockout) were left unstimulated or stimulated for 20 hours the inflammatory phase of experimental colitis.CASP11 - related to DC effector function. with LPS (100ng/ml). Cells were then analysed on a Seahorse Extracellular / - mice had increased susceptibility to DSS -induced Flux Analyser to measure a glycolytic rate. inflammation compared with WT littermate control Protective role for caspase-11 during 2% DSS-induced colitis. (A) H&E staining of distal colon sections (magnification 200X); arrows indicating mice, exhibiting more pronounced weight loss, more loss of crypt architecture. (B) Colonic tissues were assessed by a combined severe and earlier incidences of diarrhea and rectal histological score of colon cellular infiltration (Inflammation) and tissue disruption (crypt damage). Data represent mean ±SEM of n=5 bleeding over the disease course. Assessment of mice and n=2 for the control group (*p<0.05, **p<0.01, ***p<0.001). colon length revealed more significant shortening in PRTLI Programme PhD PRTLI Programme PhD

28 29 PHD PROGRAMME IN MOLECULAR CELL BIOLOGY MOLECULAR AND CELLULAR MECHANISMS UNDERLYING INFLAMMATORY PROCESSES Structure-based drug design for NADPH Oxidases Combretastatin (CA)-4 and its novel analogue CA-432 impair T-Cell migration through the Rho/ROCK signalling pathway UCD TCD

S. O’Neill, U. Knaus J. Pollock, D. Zisterer

ADPH oxidases are a family of enzymes whose overexpressing Cos7 cells, revealing optimal efficacy by he capacity of T -cell lymphocytes to migrate and [1] Smith, A., et al., LFA -1 -induced T -cell migration on ICAM -1 involves sole cellular function is to generate reactive exposure to 5uM pepducin for 30min at 37°C. The HVA localise in tissues is important in their protective regulation of MLCK -mediated attachment and ROCK -dependent detachment. J Cell Sci, 2003. 116(Pt 15): p. 3123 -33. oxygen species (ROS). The seven members, assay determined the inhibitory effect of pepducins function against infectious agents, however, [2] Greene, L.M., et al., The vascular targeting agent combretastatin A4- NNox1 5,- Duox1 and Duox2, differ from each other in on H2O2 production, while superoxide production in Tthe ability of these cells to migrate and infiltrate the and a novel cis -Restricted {beta} -Lactam Analogue, CA -432, induce their distribution and function, the type of ROS they neutrophils obtained from mouse bone marrow was tumour microenvironment is also a major contributing apoptosis in human chronic myeloid leukemia cells and ex vivo generate, and their mode of activation. Currently, very determined using a chemiluminescence assay. factor in the development of cancer. T -cell migration patient samples including those displaying multidrug resistance. J few NADPH oxidase inhibitors are available, all of them requires ligand (ICAM -1)/integrin(LFA -1) interaction, Pharmacol Exp Ther, 2010. 335(2): p. 302 -13. Treatment of Cos Nox2 cells with Nox2 pepducins lacking specificity for individual Nox/Duox isoforms. activating intracellular signalling pathways which result targeting the B -loop resulted in ~25% reduction of This is primarily due to our limited knowledge of in a distinct polarised morphology, with an actin - H O production and was comparable to treatment unique features that distinguish oxidase isoforms. The 2 2 rich lamellipodium and microtubule -rich uropod1. with gp91ds tat peptide. Superoxide generation was only well -characterized specific Nox inhibitor is the Combretastatin (CA) A -4 is a microtubule -destabilising markedly reduced (~50%) in mouse neutrophils treated moderately cell -permeable gp91ds Tat peptide, which agent that possesses potent anti -tumour properties2. with Nox2 pepducin while the gp91ds Tat peptide targets a region in the Nox2 B -loop [1]. The current study had no effect, presumably due to poor penetration or In this study, the effect of CA -4 and a novel analogue uses proteolytically stable, N -terminal palmitylated proteolysis. To identify further structural regions for CA -432 on human T -cell morphology and migration oligopeptides (pepducins) to allow for entry of peptides the design of inhibitory pepducins, key sites for the was assessed. CA -treated cells displayed altered T -cell and anchoring into lipid membranes of cells [2]. Using activation mode of Nox2 and Nox4 will be elucidated. migratory polarity of HuT -78 and peripheral blood this approach we hope to find specific inhibitors for T -lymphocyte (PBTL) cells, shown by high content Nox2 and Nox4. [1] Csanyi G, Cifuentes -Pagano E, Al Ghouleh I, et al. 2011. Nox2 B -loop peptide, Nox2ds, specifically inhibits the NADPH oxidase Nox2. Free analysis, and inhibited active migration of PBTLs in a To date, seven pepducins were designed against Radic. Biol. Med. 51(6): 1116 -1125 transwell assay. Both compounds induced activation phox regions in Nox2, Nox4 and p22 that were previously [2] Covic L, Gresser AL, Talavera J, et al. 2002. Activation and inhibition of of the RhoA/RhoA associated kinase (ROCK) signalling described as being important for functional activity. G protein -coupled receptors by cell -penetrating membrane -tethered pathway, leading to the phosphorylation of myosin peptides. Proc. Natl. Acad. Sci. USA 99(2): 643 -648 A time/dose response curve for H2O2 production after light chain (MLC), acto -myosin contractility and pepducin treatment was established in Nox2 and Nox4 impaired migration. Disruption of the MT network of T -cells through CA -induced MT depolymerisation was associated with reduced acetylated tubulin expression and decreased MT stability. GEF -H1 is a MT -associated nucleotide exchange factor that activates RhoA upon release from MTs. We have demonstrated for the first HuT-78 T cells display polarised morphology. Cells were stimulated to migrate on LFA-1 coated plates for 4 hours at 37°C. DNA was stained with time that siRNA -mediated depletion of GEF -H1 in HuT - DAPI 388 (blue) and microtubules with anti-α-tubulin amplified with Alexa 78 cells prevented CA -induced phosphorylation of MLC fluor 488 (green) antibodies. Slides were imaged on Olympus FV1000© confocal microscope (60X) and analysed using FV10-ASW 2.0 viewer and attenuated the formation of actin -rich membrane software©. protrusions and cell contractility. These results suggest an important role for a GEF -H1/RhoA/ROCK/MLC signalling pathway in mediating CA -induced contractility of T cells. Therapeutic agents that target cytoskeletal proteins and are effective in inhibiting cell migration may open new avenues in the treatment of cancer and metastasis. PRTLI Programme PhD PRTLI Programme PhD

30 31 PHD PROGRAMME IN MOLECULAR CELL BIOLOGY MOLECULAR AND CELLULAR MECHANISMS UNDERLYING INFLAMMATORY PROCESSES Identification of new sugar binding bacterial The anti-inflammatory actions of noradrenergic adhesins encoded by the human gut metagenome agents as a target to prevent neurodegeneration in Parkinson’s disease NUIG TCD

C. Agbavwe, C. O’Byrne, A. Boyd, J.D.Yssel, T.J. Connor, A. Harkin Lo. Joshi

o persist in the gut, bacteria must colonize their identify. To complement the functional metagenomic arkinson’s disease (PD) is a common partially rescued following treatment. Furthermore host. Gut bacteria express various molecules approach, bioinformatic analysis was performed to neurodegenerative disorder characterised by there was a complete restoration of nigral DA levels able to promote attachment to host cells. These identify novel proteoglycan binding elements encoded progressive degeneration of dopaminergic and a trend towards reduced striatal DA depletion. In Tadhesins rely on interactions with host cell surface in the human gut microbial metagenome. In silico Pneurons of the nigrostriatal tract. Much of our addition, following LPS administration in the nigra, receptors acting as a bridge between bacteria and analysis of the human gut metagenome yielded several understanding of the pathologies underlying PD have IBA -1 immunohistochemical staining of microglia their host [1]. Proteoglycans and sugars are amongst putative adhesins of which four have been generated been obtained from neurotoxin -based animal models displayed a greater number of ramified processes the types of structures that can act as host receptors. by PCR amplification. Two of the four PCR products such as the 6 -hydroxydopamine (6 -OHDA) model. In after treatment with atomoxetine/idazoxan. Microglia Adhesion is a critical first step prior to attachment, are homologs of a known adhesin, mucus adhesion addition, neuroinflammation is now recognised as displayed an amoeboid morphology indicating their colonization and persistence; and therefore a key event promoting protein (MapA). MapA is 263 amino acids a key player in the progression of PD and interest is activation in rodents which were administered a saline to be studied [2]. long with a bacterial extracellular solute -binding increasing in inflammatory models of PD including control treatment following LPS, indicating the anti - domain. The other two PCR products are homologs of a lipopolysaccharide (LPS) -induced degeneration of inflammatory actions of NA leading or contributing Despite increasing number of scientific reports starch binding outer membrane protein (551aa). dopaminergic neurons in the substantia nigra (SN). ultimately to neuroprotection. describing adhesion of bacteria to components of the human intestinal mucosa, information on the [1] Miyoshi, Y., Okada, S., Uchimura, T. and Satoh, E. 2006. A mucus We have developed an animal model using both surface molecules mediating this adhesion and their adhesion promoting protein, MapA, mediates the adhesion of 6 -OHDA and LPS in an effort to combine these two Lactobacillus reuteri to Caco2 human intestinal. corresponding receptors are poorly understood [3]. contributory elements believed to be of significance to In this project, a functional metagenomic approach [2] Van Pijkeren, J -P., C. Canchaya, K. A. Ryan, Y. Li, M. J. Claesson, B. the pathogenesis and progression of PD. These rodents Sheil, L. Steidler, L. O’Mahony, G. F. Fitzgerald, D. van Sinderen and was undertaken to construct metagenomic libraries P. W. O’Toole. 2006. Comparative and functional analysis of sortase - display functional motor deficits, reduced striatal for screening of novel proteoglycan binding elements dependent proteins in the predicted secretome of Lactobacillus dopamine (DA) concentrations and activated microglia encoded in the human gut microbial metagenome. Two salivarius UCC118. App. Env. Microbiol. 72: 4143 -4153. following LPS injection in the SN. In recent years there types of libraries were generated; small -insert libraries [3] Rojas, M., Ascencio, F. and Conway, P.L. 2002. Purification and has been a focus on the degeneration of noradrenergic in plasmid vectors (less than 10kb) and large -insert characterization of a surface protein from Lactobacillus fermentum neurons in addition to dopamine loss in PD research. libraries in fosmid vectors (up to 40kb). Both libraries 104R that binds to porcine small intestinal mucus and gastric mucin. Degeneration within the locus coeruleus, the main Appl Environ Microbiol 68:23302336 were obtain from healthy adult female faeces and noradrenergic cell body region within the midbrain, is confirmed to be genetically diverse through sequencing. evident in PD, and a loss of noradrenaline (NA) appears to exacerbate both the demise of DA neurons and An in vitro assay of bacterial adhesion onto Caco - the motor symptoms of PD. NA is potentially involved 2 epithelial cells (model for intestinal epithelium) in an array of compensatory, anti -inflammatory and was used to select for adherent clones. Due to high possibly neuroprotective mechanisms in PD. Moreover background levels, potential clones were difficult to as a number of drugs that can increase noradrenergic function are already in clinical use for various conditions, the potential for targeting NA for treating the symptoms of PD is high. In the current investigation we have observed that a combination of treatment with the noradrenaline

reuptake inhibitor atomoxetine and the α2 -adrenoceptor antagonist idazoxan, a combination which serves to Following LPS administration in the nigra, IBA-1 immunohistochemical enhance the extrasynaptic availability of noradrenline, staining of microglia displayed a greater number of ramified processes after treatment with atomoxetine/idazoxan (A). Microglia displayed an amoeboid exerts neuroprotective effects in the LPS/6 -OHDA morphology indicating their activation, in rodents which were administered model of PD. Functional deficits in motor function were a saline control treatment following LPS (B). PRTLI Programme PhD PRTLI Programme PhD

32 33 PHD PROGRAMME IN MOLECULAR CELL BIOLOGY Double stranded RNAs of different lengths have divergent effects on innate immune activation in the periphery and the brain TCD

N. McGarry, K. Mitchell, C. Cunningham

oly -Inosinic: Poly -Cytidylic Acid (Poly I:C) is a This demonstrates that length of poly I:C is a key synthetic double stranded analogue of a double determinant of the activating alternate pathways in stranded RNA (dsRNA). This viral mimetic is the periphery. This has a very significant impact on Pused to study the role of Maternal Immune Activation current studies in neuroscience since similar studies are (MIA) in schizophrenia, type I interferon effects on apparently unknowingly examining different pathways neurophysiology, and the impact of anti viral- acute of innate immune activation using poly I:C. phase responses during neurodegenerative disease. We observed highly divergent outcomes in an MIA model using preparations of poly I:C from 2 different suppliers and it is clear that preparations from different suppliers may differ fundamentally. In the current study we examined the peripheral and central nervous system inflammatory responses to poly I:C (20 mg/kg) from different sources (Amersham, Sigma, Invivogen). We found very significantly different responses at 3 hours post poly I:C stimulation, with Sigma producing undetectable IL -1b, TNF -a and IFNb responses and a much reduced IL -6 response compared to the very robust response with Amersham poly I:C. These data suggested fundamentally different pathways and we thus compared their molecular weights and HMW and LMW poly I:C induce differential dsRNA receptor transcription innate immune responses to those of invivogen poly in peripheral and central tissues. Toll like receptor 3 (TLR3) is strongly induced in the periphery, while in brain tissue we see a greater reduction I:C high molecular weight (HMW) Poly I:C (800 -10000 of Retinoic acid-inducible gene 1 (RIG-1) and Melanoma Differentiation- bp) and low MW (200 -1000 bp). HMW induces a strong Associated protein 5 (MDA-5). These data stress that different lengths of dsRNA interact differently with pattern recognition receptors, activating IFNβ, IL -6 and TNFα response in serum, while LMW does different signalling pathways and producing alternate responses. not and DNA gels confirm that Amersham and Sigma provide HMW and LMW poly I:C respectively. Even at a higher dose of LMW poly I:C, which induces a similar IL -6 response to HMW, very low IFNβ and TNFα induction was observed. PRTLI Programme PhD

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