Biochemistry and Pharmacology - Research Publications
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ItemIncreased endogenous antigen presentation in the periphery enhances susceptibility to inflammation-induced gastric autoimmunity in mice(WILEY-BLACKWELL, 2017-01)How the immune system maintains peripheral tolerance under inflammatory conditions is poorly understood. Here we assessed the fate of gastritogenic T cells following inflammatory activation in vivo. Self-reactive T cells (A23 T cells) specific for the gastric H+ /K+ ATPase α subunit (HKα) were transferred into immunosufficient recipient mice and immunised at a site distant to the stomach with adjuvant containing the cognate HKα peptide antigen. Activation of A23 T cells by immunisation did not impact on either immune tolerance or protection from gastric autoimmunity in wild-type BALB/c mice. However, increased presentation of endogenously derived HKα epitopes by dendritic cells (DCs) in the gastric lymph node of IE-H+ /K+ β transgenic mice (IEβ) reduces A23 T-cell tolerance to gastric antigens after inflammatory activation, with subsequent development of gastritis. While HKα-specific A23 T cells from immunised wild-type mice were poorly responsive to in vitro antigen specific activation, A23 T cells from immunised IEβ transgenic mice were readily re-activated, indicating loss of T-cell anergy. These findings show that DCs of gastric lymph nodes can maintain tolerance of pathogenic T cells following inflammatory stimulation and that the density of endogenous antigen presented to self-reactive T cells is critical in the balance between tolerance and autoimmunity.
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ItemIFNγ receptor down-regulation facilitates Legionella survival in alveolar macrophages(OXFORD UNIV PRESS, 2020-02)Legionella pneumophila is an opportunistic human pathogen and causative agent of the acute pneumonia known as Legionnaire's disease. Upon inhalation, the bacteria replicate in alveolar macrophages (AM), within an intracellular vacuole termed the Legionella-containing vacuole. We recently found that, in vivo, IFNγ was required for optimal clearance of intracellular L. pneumophila by monocyte-derived cells (MC), but the cytokine did not appear to influence clearance by AM. Here, we report that during L. pneumophila lung infection, expression of the IFNγ receptor subunit 1 (IFNGR1) is down-regulated in AM and neutrophils, but not MC, offering a possible explanation for why AM are unable to effectively restrict L. pneumophila replication in vivo. To test this, we used mice that constitutively express IFNGR1 in AM and found that prevention of IFNGR1 down-regulation enhanced the ability of AM to restrict L. pneumophila intracellular replication. IFNGR1 down-regulation was independent of the type IV Dot/Icm secretion system of L. pneumophila indicating that bacterial effector proteins were not involved. In contrast to previous work, we found that signaling via type I IFN receptors was not required for IFNGR1 down-regulation in macrophages but rather that MyD88- or Trif- mediated NF-κB activation was required. This work has uncovered an alternative signaling pathway responsible for IFNGR1 down-regulation in macrophages during bacterial infection.
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ItemInterferon-induced GTPases orchestrate host cell-autonomous defence against bacterial pathogens(PORTLAND PRESS LTD, 2021-06)Interferon (IFN)-induced guanosine triphosphate hydrolysing enzymes (GTPases) have been identified as cornerstones of IFN-mediated cell-autonomous defence. Upon IFN stimulation, these GTPases are highly expressed in various host cells, where they orchestrate anti-microbial activities against a diverse range of pathogens such as bacteria, protozoan and viruses. IFN-induced GTPases have been shown to interact with various host pathways and proteins mediating pathogen control via inflammasome activation, destabilising pathogen compartments and membranes, orchestrating destruction via autophagy and the production of reactive oxygen species as well as inhibiting pathogen mobility. In this mini-review, we provide an update on how the IFN-induced GTPases target pathogens and mediate host defence, emphasising findings on protection against bacterial pathogens.
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ItemNo Preview AvailableSculpting the immune response to infection.(Springer Science and Business Media LLC, 2011-06-20)This report describes advances in the understanding of how microbes elicit and evade immune responses and the sensing of pathogens by host cells that leads to the activation and production of intra- and extracellular signaling molecules.
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ItemNo Preview AvailableImmune control of Legionella infection: an in vivo perspective(FRONTIERS RESEARCH FOUNDATION, 2011)Legionella pneumophila is an intracellular pathogen that replicates within alveolar macrophages. Through its ability to activate multiple host innate immune components, L. pneumophila has emerged as a useful tool to dissect inflammatory signaling pathways in macrophages. However the resolution of L. pneumophila infection in the lung requires multiple cell types and abundant cross talk between immune cells. Few studies have examined the coordination of events that lead to effective immune control of the pathogen. Here we discuss L. pneumophila interactions with macrophages and dendritic cell subsets and highlight the paucity of knowledge around how these interactions recruit and activate other immune effector cells in the lung.
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ItemA method for quantifying pulmonary Legionella pneumophila infection in mouse lungs by flow cytometry.(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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ItemA Convenient Model of Severe, High Incidence Autoimmune Gastritis Caused by Polyclonal Effector T Cells and without Perturbation of Regulatory T Cells(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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ItemThe Golgi apparatus in the endomembrane-rich gastric parietal cells exist as functional stable mini-stacks dispersed throughout the cytoplasm(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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ItemAN AUTOIMMUNE-DISEASE WITH MULTIPLE MOLECULAR TARGETS ABROGATED BY THE TRANSGENIC EXPRESSION OF A SINGLE AUTOANTIGEN IN THE THYMUS(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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ItemCooperation between Monocyte-Derived Cells and Lymphoid Cells in the Acute Response to a Bacterial Lung Pathogen(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.