School of Biomedical Sciences - Research Publications

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    A method for quantifying pulmonary Legionella pneumophila infection in mouse lungs by flow cytometry.
    Ang, DKY ; Ong, SY ; Brown, AS ; Hartland, EL ; van Driel, IR (Springer Science and Business Media LLC, 2012-08-20)
    BACKGROUND: Pulmonary load of Legionella pneumophila in mice is normally determined by counting serial dilutions of bacterial colony forming units (CFU) on agar plates. This process is often tedious and time consuming. We describe a novel, rapid and versatile flow cytometric method that detects bacteria phagocytosed by neutrophils. FINDINGS: Mice were infected with L. pneumophila via intratracheal or intranasal administration. At various times after bacteria inoculation, mouse lungs were harvested and analysed concurrently for bacterial load by colony counting and flow cytometry analysis. The number of L. pneumophila-containing neutrophils correlated strongly with CFU obtained by bacteriological culture. CONCLUSIONS: This technique can be utilised to determine pulmonary bacterial load and may be used in conjunction with other flow cytometric based analyses of the resulting immune response.
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    A Convenient Model of Severe, High Incidence Autoimmune Gastritis Caused by Polyclonal Effector T Cells and without Perturbation of Regulatory T Cells
    Tu, E ; Ang, DKY ; Hogan, TV ; Read, S ; Chia, CPZ ; Gleeson, PA ; van Driel, IR ; Piccirillo, CA (PUBLIC LIBRARY SCIENCE, 2011-11-09)
    Autoimmune gastritis results from the breakdown of T cell tolerance to the gastric H(+)/K(+) ATPase. The gastric H(+)/K(+) ATPase is responsible for the acidification of gastric juice and consists of an α subunit (H/Kα) and a β subunit (H/Kβ). Here we show that CD4(+) T cells from H/Kα-deficient mice (H/Kα(-/-)) are highly pathogenic and autoimmune gastritis can be induced in sublethally irradiated wildtype mice by adoptive transfer of unfractionated CD4(+) T cells from H/Kα(-/-) mice. All recipient mice consistently developed the most severe form of autoimmune gastritis 8 weeks after the transfer, featuring hypertrophy of the gastric mucosa, complete depletion of the parietal and zymogenic cells, and presence of autoantibodies to H(+)/K(+) ATPase in the serum. Furthermore, we demonstrated that the disease significantly affected stomach weight and stomach pH of recipient mice. Depletion of parietal cells in this disease model required the presence of both H/Kα and H/Kβ since transfer of H/Kα(-/-) CD4(+) T cells did not result in depletion of parietal cells in H/Kα(-/-) or H/Kβ(-/-) recipient mice. The consistency of disease severity, the use of polyclonal T cells and a specific T cell response to the gastric autoantigen make this an ideal disease model for the study of many aspects of organ-specific autoimmunity including prevention and treatment of the disease.
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    The Golgi apparatus in the endomembrane-rich gastric parietal cells exist as functional stable mini-stacks dispersed throughout the cytoplasm
    Gunn, PA ; Gliddon, BL ; Londrigan, SL ; Lew, AM ; van Driel, IR ; Gleeson, PA (PORTLAND PRESS LTD, 2011-12)
    BACKGROUND INFORMATION: Acid-secreting gastric parietal cells are polarized epithelial cells that harbour highly abundant and specialized, H+,K+ ATPase-containing, tubulovesicular membranes in the apical cytoplasm. The Golgi apparatus has been implicated in the biogenesis of the tubulovesicular membranes; however, an unanswered question is how a typical Golgi organization could regulate normal membrane transport within the membrane-dense cytoplasm of parietal cells. RESULTS: Here, we demonstrate that the Golgi apparatus of parietal cells is not the typical juxta-nuclear ribbon of stacks, but rather individual Golgi units are scattered throughout the cytoplasm. The Golgi membrane structures labelled with markers of both cis- and trans-Golgi membrane, indicating the presence of intact Golgi stacks. The parietal cell Golgi stacks were closely aligned with the microtubule network and were shown to participate in both anterograde and retrograde transport pathways. Dispersed Golgi stacks were also observed in parietal cells from H+,K+ ATPase-deficient mice that lack tubulovesicular membranes. CONCLUSIONS: These results indicate that the unusual organization of individual Golgi stacks dispersed throughout the cytoplasm of these terminally differentiated cells is likely to be a developmentally regulated event.
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    Inhibition of Breast Cancer Resistance Protein (ABCG2) in Human Myeloid Dendritic Cells Induces Potent Tolerogenic Functions during LPS Stimulation
    Jin, J-O ; Zhang, W ; Wong, K-W ; Kwak, M ; van Driel, IR ; Yu, Q ; Wu, Y (PUBLIC LIBRARY SCIENCE, 2014-08-11)
    Breast cancer resistance protein (ABCG2), a member of the ATP-binding cassette transporters has been identified as a major determinant of multidrug resistance (MDR) in cancer cells, but ABC transporter inhibition has limited therapeutic value in vivo. In this research, we demonstrated that inhibition of efflux transporters ABCG2 induced the generation of tolerogenic DCs from human peripheral blood myeloid DCs (mDCs). ABCG2 expression was present in mDCs and was further increased by LPS stimulation. Treatment of CD1c+ mDCs with an ABCG2 inhibitor, Ko143, during LPS stimulation caused increased production of IL-10 and decreased production of pro-inflammatory cytokines and decreased expression of CD83 and CD86. Moreover, inhibition of ABCG2 in monocyte-derived DCs (MDDCs) abrogated the up-regulation of co-stimulatory molecules and production of pro-inflammatory cytokines in these cells in response to LPS. Furthermore, CD1c+ mDCs stimulated with LPS plus Ko143 inhibited the proliferation of allogeneic and superantigen-specific syngenic CD4+ T cells and promoted expansion of CD25+FOXP3+ regulatory T (Treg) cells in an IL-10-dependent fashion. These tolerogenic effects of ABCG2 inhibition could be abolished by ERK inhibition. Thus, we demonstrated that inhibition of ABCG2 in LPS-stimulated mDCs can potently induce tolerogenic potentials in these cells, providing crucial new information that could lead to development of better strategies to combat MDR cancer.
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    AN AUTOIMMUNE-DISEASE WITH MULTIPLE MOLECULAR TARGETS ABROGATED BY THE TRANSGENIC EXPRESSION OF A SINGLE AUTOANTIGEN IN THE THYMUS
    ALDERUCCIO, F ; TOH, BH ; TAN, SS ; GLEESON, PA ; VANDRIEL, IR (ROCKEFELLER UNIV PRESS, 1993-08-01)
    Many autoimmune diseases are characterized by autoantibody reactivities to multiple cellular antigens. Autoantigens are commonly defined as targets of the autoimmune B cell response, but the role, if any, of these autoantigens in T cell-mediated autoimmune diseases is generally unknown. Murine experimental autoimmune gastritis is a CD4+ T cell-mediated organ-specific autoimmune disease induced by neonatal thymectomy of BALB/c mice. The murine disease is similar to human autoimmune gastritis and pernicious anemia, and is characterized by parietal and chief cell loss, submucosal mononuclear cell infiltrates, and autoantibodies to the alpha and beta subunits of the gastric H/K ATPase. However, the specificity of T cells that cause the disease is not known. To examine the role of the H/K ATPase in this T cell-mediated disease, transgenic mice were generated that express the beta subunit of the H/K ATPase under the control of the major histocompatibility complex class II I-Ek alpha promoter. We show that transgenic expression of the gastric H/K ATPase beta subunit specifically prevents the onset of autoimmune gastritis after neonatal thymectomy. In addition, thymocyte transfer experiments suggest that tolerance of pathogenic autoreactive T cells is induced within the thymus of the transgenic mice. We conclude that the beta subunit of the gastric H/K ATPase is a major T cell target in autoimmune gastritis and that thymic expression of a single autoantigen can abrogate an autoimmune response to multiple autoantigens.
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    Cooperation between Monocyte-Derived Cells and Lymphoid Cells in the Acute Response to a Bacterial Lung Pathogen
    Brown, AS ; Yang, C ; Fung, KY ; Bachem, A ; Bourges, D ; Bedoui, S ; Hartland, EL ; van Driel, IR ; Roy, CR (PUBLIC LIBRARY SCIENCE, 2016-06)
    Legionella pneumophila is the causative agent of Legionnaires' disease, a potentially fatal lung infection. Alveolar macrophages support intracellular replication of L. pneumophila, however the contributions of other immune cell types to bacterial killing during infection are unclear. Here, we used recently described methods to characterise the major inflammatory cells in lung after acute respiratory infection of mice with L. pneumophila. We observed that the numbers of alveolar macrophages rapidly decreased after infection coincident with a rapid infiltration of the lung by monocyte-derived cells (MC), which, together with neutrophils, became the dominant inflammatory cells associated with the bacteria. Using mice in which the ability of MC to infiltrate tissues is impaired it was found that MC were required for bacterial clearance and were the major source of IL12. IL12 was needed to induce IFNγ production by lymphoid cells including NK cells, memory T cells, NKT cells and γδ T cells. Memory T cells that produced IFNγ appeared to be circulating effector/memory T cells that infiltrated the lung after infection. IFNγ production by memory T cells was stimulated in an antigen-independent fashion and could effectively clear bacteria from the lung indicating that memory T cells are an important contributor to innate bacterial defence. We also determined that a major function of IFNγ was to stimulate bactericidal activity of MC. On the other hand, neutrophils did not require IFNγ to kill bacteria and alveolar macrophages remained poorly bactericidal even in the presence of IFNγ. This work has revealed a cooperative innate immune circuit between lymphoid cells and MC that combats acute L. pneumophila infection and defines a specific role for IFNγ in anti-bacterial immunity.
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    IFNs Modify the Proteome of Legionella-Containing Vacuoles and Restrict Infection Via IRG1-Derived Itaconic Acid
    Naujoks, J ; Tabeling, C ; Dill, BD ; Hoffmann, C ; Brown, AS ; Kunze, M ; Kempa, S ; Peter, A ; Mollenkopf, H-J ; Dorhoi, A ; Kershaw, O ; Gruber, AD ; Sander, LE ; Witzenrath, M ; Herold, S ; Nerlich, A ; Hocke, AC ; van Driel, I ; Suttorp, N ; Bedoui, S ; Hilbi, H ; Trost, M ; Opitz, B ; Zamboni, DS (PUBLIC LIBRARY SCIENCE, 2016-02)
    Macrophages can be niches for bacterial pathogens or antibacterial effector cells depending on the pathogen and signals from the immune system. Here we show that type I and II IFNs are master regulators of gene expression during Legionella pneumophila infection, and activators of an alveolar macrophage-intrinsic immune response that restricts bacterial growth during pneumonia. Quantitative mass spectrometry revealed that both IFNs substantially modify Legionella-containing vacuoles, and comparative analyses reveal distinct subsets of transcriptionally and spatially IFN-regulated proteins. Immune-responsive gene (IRG)1 is induced by IFNs in mitochondria that closely associate with Legionella-containing vacuoles, and mediates production of itaconic acid. This metabolite is bactericidal against intravacuolar L. pneumophila as well as extracellular multidrug-resistant Gram-positive and -negative bacteria. Our study explores the overall role IFNs play in inducing substantial remodeling of bacterial vacuoles and in stimulating production of IRG1-derived itaconic acid which targets intravacuolar pathogens. IRG1 or its product itaconic acid might be therapeutically targetable to fight intracellular and drug-resistant bacteria.
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    Plasmacytoid Dendritic Cells Provide Protection Against Bacterial-Induced Colitis
    Rahman, T ; Brown, AS ; Hartland, EL ; van Driel, IR ; Fung, KY (FRONTIERS MEDIA SA, 2019-04-09)
    We have examined the influence of depleting plasmacytoid dendritic cells (pDC) in mice on the immune response to the gut pathogen Citrobacter rodentium, an organism that is a model for human attaching effacing pathogens such as enterohaemorraghic E. coli. A significantly higher number of C. rodentium were found in mice depleted of pDC from 7 days after infection and pDC depleted mice showed increased gut pathology and higher levels of mRNA encoding inflammatory cytokines in the colon upon infection. pDC-depletion led to a compromising of the gut mucosal barrier that may have contributed to increased numbers of C. rodentium in systemic organs. pDC-depleted mice infected with C. rodentium suffered substantial weight loss necessitating euthanasia. A number of observations suggested that this was not simply the result of dysregulation of immunity in the colon as pDC-depleted mice infected intravenously with C. rodentium also exhibited exacerbated weight loss, arguing that pDC influence systemic immune responses. Overall, these data indicate that pDC contribute at multiple levels to immunity to C. rodentium including control of bacterial numbers in the colon, maintenance of colon barrier function and regulation of immune responses to disseminated bacteria.
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    The Salmonella Effector SseK3 Targets Small Rab GTPases
    Gan, J ; Scott, NE ; Newson, JPM ; Wibawa, RR ; Wong Fok Lung, T ; Pollock, GL ; Ng, GZ ; van Driel, I ; Pearson, JS ; Hartland, EL ; Giogha, C (FRONTIERS MEDIA SA, 2020-08-19)
    During infection, Salmonella species inject multiple type III secretion system (T3SS) effector proteins into host cells that mediate invasion and subsequent intracellular replication. At early stages of infection, Salmonella exploits key regulators of host intracellular vesicle transport, including the small GTPases Rab5 and Rab7, to subvert host endocytic vesicle trafficking and establish the Salmonella-containing vacuole (SCV). At later stages of intracellular replication, interactions of the SCV with Rab GTPases are less well defined. Here we report that Rab1, Rab5, and Rab11 are modified at later stages of Salmonella infection by SseK3, an arginine N-acetylglucosamine (GlcNAc) transferase effector translocated via the Salmonella pathogenicity island 2 (SPI-2) type III secretion system. SseK3 modified arginines at positions 74, 82, and 111 within Rab1 and this modification occurred independently of Rab1 nucleotide binding. SseK3 exhibited Golgi localization that was independent of its glycosyltransferase activity but Arg-GlcNAc transferase activity was required for inhibition of alkaline phosphatase secretion in transfected cells. While SseK3 had a modest effect on SEAP secretion during infection of HeLa229 cells, inhibition of IL-1 and GM-CSF cytokine secretion was only observed upon over-expression of SseK3 during infection of RAW264.7 cells. Our results suggest that, in addition to targeting death receptor signaling, SseK3 may contribute to Salmonella infection by interfering with the activity of key Rab GTPases.
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    Application of a chemical probe to detect neutrophil elastase activation during inflammatory bowel disease
    Anderson, BM ; Poole, DP ; Aurelio, L ; Ng, GZ ; Fleischmann, M ; Kasperkiewicz, P ; Morissette, C ; Drag, M ; van Driel, IR ; Schmidt, BL ; Vanner, SJ ; Bunnett, NW ; Edgington-Mitchell, LE (NATURE PUBLISHING GROUP, 2019-09-16)
    Neutrophil elastase is a serine protease that has been implicated in the pathogenesis of inflammatory bowel disease. Due to post-translational control of its activation and high expression of its inhibitors in the gut, measurements of total expression poorly reflect the pool of active, functional neutrophil elastase. Fluorogenic substrate probes have been used to measure neutrophil elastase activity, though these tools lack specificity and traceability. PK105 is a recently described fluorescent activity-based probe, which binds to neutrophil elastase in an activity-dependent manner. The irreversible nature of this probe allows for accurate identification of its targets in complex protein mixtures. We describe the reactivity profile of PK105b, a new analogue of PK105, against recombinant serine proteases and in tissue extracts from healthy mice and from models of inflammation induced by oral cancer and Legionella pneumophila infection. We apply PK105b to measure neutrophil elastase activation in an acute model of experimental colitis. Neutrophil elastase activity is detected in inflamed, but not healthy, colons. We corroborate this finding in mucosal biopsies from patients with ulcerative colitis. Thus, PK105b facilitates detection of neutrophil elastase activity in tissue lysates, and we have applied it to demonstrate that this protease is unequivocally activated during colitis.