Robert N Barker

University of Aberdeen, Aberdeen, Scotland, United Kingdom

Are you Robert N Barker?

Claim your profile

Publications (74)341.46 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Macrophages are key cells in inflammation and repair, and their activity requires close regulation. The characterization of cues coordinating macrophage function has focused on biologic and soluble mediators, with little known about their responses to physical stimuli, such as the electrical fields that are generated naturally in injured tissue and which accelerate wound healing. To address this gap in understanding, we tested how properties of human monocyte-derived macrophages are regulated by applied electrical fields, similar in strengths to those established naturally. With the use of live-cell video microscopy, we show that macrophage migration is directed anodally by electrical fields as low as 5 mV/mm and is electrical field strength dependent, with effects peaking ∼300 mV/mm. Monocytes, as macrophage precursors, migrate in the opposite, cathodal direction. Strikingly, we show for the first time that electrical fields significantly enhance macrophage phagocytic uptake of a variety of targets, including carboxylate beads, apoptotic neutrophils, and the nominal opportunist pathogen Candida albicans, which engage different classes of surface receptors. These electrical field-induced functional changes are accompanied by clustering of phagocytic receptors, enhanced PI3K and ERK activation, mobilization of intracellular calcium, and actin polarization. Electrical fields also modulate cytokine production selectively and can augment some effects of conventional polarizing stimuli on cytokine secretion. Taken together, electrical signals have been identified as major contributors to the coordination and regulation of important human macrophage functions, including those essential for microbial clearance and healing. Our results open up a new area of research into effects of naturally occurring and clinically applied electrical fields in conditions where macrophage activity is critical.
    No preview · Article · Dec 2015 · Journal of leukocyte biology
  • Source
    Caitlin Hughes · Mark A Vickers · Robert N Barker · Lindsay S Hall

    Preview · Article · Apr 2015 · Journal of Inflammation
  • Source

    Preview · Article · Apr 2015 · Journal of Inflammation
  • [Show abstract] [Hide abstract]
    ABSTRACT: Bullous pemphigoid is a blistering skin disease characterized by autoantibodies against the NC16a domain of bullous pemphigoid 180. This study was performed to characterize and map the fine specifity of T cell responses to NC16a. PBMC from a total of 28 bullous pemphigoid patients and 14 matched controls were tested for proliferative and cytokine responses to recombinant NC16a and a complete panel of 21 overlapping peptides spanning this region of BP180. Proliferative responses to NC16A and the peptide panel in the patients with active disease were similar in frequency and magnitude to those in healthy donors, and included late responses typical of naive cells in about 60% of each group. IL-4 responses were slightly stronger for 6 peptides, and significantly stronger to Nc16a, in patients than controls. Factor analysis identified factors that separate responses to the peptide panel discretely into IL-4(Th2 pattern), IFNγ(Th1 pattern) and IL-10 or TGFβ(Treg pattern). Factors segregating IL-10 versus IFNγ were predicted by active blistering or remission, and TGFβ or IL-10 versus IFNγ by age. Finally, we confirmed a significant upregulation of IgE responses to BP180 in the patients with pemphigoid. This shows the complexity of T cell phenotype and fine autoreactive specificity in responses to NC16A, in patients and normal controls. Important disease associated factors determine the balance of cytokine responses. Of these, specific IL-4 and IgE responses show the strongest associations with pemphigoid, pointing to an important contribution by Th2 cytokines to pathogenesis. This article is protected by copyright. All rights reserved.
    No preview · Article · Dec 2014 · Clinical & Experimental Immunology
  • I. Kotb · B. Lewis · A. Ormerod · R. Barker

    No preview · Conference Paper · Dec 2014

  • No preview · Article · Dec 2014 · Immunology

  • No preview · Article · Dec 2014 · Immunology

  • No preview · Article · Dec 2014 · Immunology

  • No preview · Article · Dec 2014 · Immunology
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Macrophages are antigen presenting cells that can adopt different activation states as directed by microenvironmental stimuli. It is well-recognised how CD4+ T helper (Th) signals drive macrophage activation, but the ability of differentially activated human macrophages to stimulate the major types of CD4+ T helper (Th) response by presenting antigen have not been well defined. Previous studies have focussed on murine cells, undifferentiated human monocytes, or macrophage products, and have been limited to non-physiological mitogenic Th responses. The aim was therefore to compare the Th cell polarising abilities of different human macrophage subsets when presenting specific antigen. We demonstrate for the first time that the way macrophages are activated, while naturally presenting antigen, has profound effects on downstream adaptive immune responses. In autologous co-cultures, LPS-activation was the most potent stimulus for antigen-loaded macrophages to drive Th17 polarisation from both unfractionated CD4+ T-cells and the CD45RO+ memory population, while IFNγ/LPS activated macrophages preferentially induced a Th1 phenotype. By contrast, IL-4-activated macrophages were ineffective in inducing responses by either Th subset. Although antigen-loaded dendritic cells were superior to macrophages in driving Th1 responses, the Th17 polarising capacity of the two antigen-presenting cell types was equivalent, and was strongly dependent on IL-1β secretion. Taken together, these results clearly demonstrate for the first time how differentially activated human macrophages present antigen to bias specific, rather than mitogen-driven, Th responses and lead us to propose that they impact adaptive immunity in vivo, particularly in determining Th17 polarisation within inflamed tissues.
    Full-text · Article · Oct 2014 · Immunobiology
  • [Show abstract] [Hide abstract]
    ABSTRACT: CTLA-4 is an inhibitory protein that contributes to immune homeostasis and tolerance, a role that has led to its exploitation as a therapeutic in several clinical settings including cancer and autoimmune disease. Development of CTLA-4 therapies focused largely on the full-length receptor isoform but other CTLA-4 isoforms are also expressed, including a secretable form of CTLA-4 (soluble CTLA-4 [sCTLA-4]). The contribution of sCTLA-4 to immune regulation has been less well studied, primarily because it was identified some years after the original description of CTLA-4. Here, we examine how sCTLA-4 might contribute to immune regulation and ask whether it might be a biomarker to inform current CTLA-4 therapies or represent a novel CTLA-4 target for future therapeutics.
    No preview · Article · Oct 2014 · Immunotherapy
  • [Show abstract] [Hide abstract]
    ABSTRACT: Although it is widely believed that IL-27 is anti-inflammatory, its role in controlling human immune responses is not fully established. In particular, its interactions with Th17 cytokines are unclear. Our aims were to establish the relationships between IL-27 and pro-inflammatory cytokines, including IL-17A, in human sera and cultures of peripheral blood mononuclear cells. Plasma IL-27 levels in 879 healthy humans from 163 families varied widely, but with relatively low heritability (19%). Despite IL-27 including a subunit encoded by Epstein-Barr virus-induced gene 3 (EBI3), there was no correlation of levels with serological evidence of infection with the virus. Although IL-27 has been reported to inhibit IL-17A production, we demonstrated a strong positive correlation in sera, but lower correlations of IL-27 with other pro-inflammatory cytokines. We verified that IL-27 inhibited IL-17A production by human peripheral blood T cells in vitro, but not that it stimulated IL-10 secretion. Importantly, addition of IL-17A decreased IL-27 production by stimulated T cells but had the opposite effect on resting T cells. Together, these data suggest a model whereby IL-27 and IL-17A exerts complex reciprocal effects to boost inflammatory responses, but restrain resting cells to prevent inappropriate activation.
    No preview · Article · Jun 2014 · Clinical & Experimental Immunology
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Candida albicans remains the fungus most frequently associated with nosocomial bloodstream infection. In disseminated candidiasis, the role of Foxp3(+) regulatory T (Treg) cells remain largely unexplored. Our aims were to characterize Foxp3(+) Treg-cell activation in a murine intravenous challenge model of disseminated C. albicans infection, and determine the contribution to disease. Flow cytometric analyses demonstrated that C. albicans infection drove in vivo expansion of a splenic CD4(+) Foxp3(+) population that correlated positively with fungal burden. Depletion from Foxp3(hCD2) reporter mice in vivo confirmed that Foxp3(+) cells exacerbated fungal burden and inflammatory renal disease. The CD4(+) Foxp3(+) population expanded further after in vitro stimulation with C. albicans antigens, and included at least three cell types. These arose from proliferation of the natural Treg-cell subset, together with conversion of Foxp3(-) cells to the induced Treg-cell form, and to a cell type sharing effector Th17-cell characteristics, expressing ROR-γt and secreting IL-17A. The expanded Foxp3(+) T cells inhibited Th1 and Th2 responses, but enhanced Th17-cell responses to C. albicans antigens in vitro, and in vivo depletion confirmed their ability to enhance the Th17-cell response. These data lead to a model for disseminated candidiasis whereby expansion of Foxp3(+) T cells promotes Th17-cell responses that drive pathology. This article is protected by copyright. All rights reserved.
    Full-text · Article · Apr 2014 · European Journal of Immunology
  • Lekh N Dahal · Robert N Barker · Frank J Ward
    [Show abstract] [Hide abstract]
    ABSTRACT: Autoreactive CD4(+) helper T cells specific for a range of nucleoprotein-derived autoantigens are an important feature of systemic lupus erythematosus, driving B cell differentiation and autoantibody production and contributing to the inflammatory lesions caused by immune complex deposition. Several peptide epitopes from nucleoprotein antigens have been identified and offer a means selectively to manipulate T cell responses by skewing toward a profile of cytokines that is less pro-inflammatory.Antigen-specific T cell lines and clones can be useful in the study of helper T cell subsets because their life span is prolonged and many individual cells can be generated, allowing particular phenotypes to be studied in detail. Magnetic beads offer a robust and convenient method for the isolation, polarization, and expansion of T cells, which can be adapted for a broad range of applications.
    No preview · Article · Feb 2014 · Methods in molecular biology (Clifton, N.J.)
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The offspring from pregnancies of women who have developed anti-D blood group antibodies are at risk of hemolytic disease of the newborn. We have previously mapped four peptides containing immunodominant T-helper cell epitopes from the RhD protein and the purpose of the work was to develop these into a product for suppression of established anti-D responses. A panel of each of the four immunodominant RhD peptides was synthesized with modifications to improve manufacturability and solubility, and screened for retention of recognition by human T-helper cells. A selected version of each sequence was combined in a mixture (RhDPmix), which was tested for suppressive ability in a humanized murine model of established immune responses to RhD protein. After HLA-DR15 transgenic mice had been immunized with RhD protein, a single dose of RhDPmix, given either intranasally (p=0.008, Mann-Whitney rank sum test) or subcutaneously (p=0.043), rapidly and significantly suppressed the ongoing antibody response. This was accompanied by reduced T-helper cell responsiveness, although this change was less marked for subcutaneous RhDPmix delivery, and by the recruitment of cells with a regulatory T-cell phenotype. The results support human trials of RhDPmix peptide immunotherapy in women with established antibody responses to the RhD blood group.
    Full-text · Article · Jan 2014 · Haematologica
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Macrophages respond to their microenvironment and develop polarized functions critical for orchestrating appropriate inflammatory responses. Classical (M1) activation eliminates pathogens while alternative (M2) activation promotes regulation and repair. M1 macrophage activation is strongly associated with suppressor of cytokine signalling 3 (SOCS3) expression in vitro, but the functional consequences of this are unclear and the role of SOCS3 in M1-macrophage polarization in vivo remains controversial. To address these questions, we defined the characteristics and function of SOCS3-expressing macrophages in vivo and identified potential mechanisms of SOCS3 action. Macrophages infiltrating inflamed glomeruli in a model of acute nephritis show significant up-regulation of SOCS3 that co-localizes with the M1-activation marker, inducible nitric oxide synthase. Numbers of SOCS3hi-expressing, but not SOCS1hi-expressing, macrophages correlate strongly with the severity of renal injury, supporting their inflammatory role in vivo. Adoptive transfer of SOCS3-short interfering RNA-silenced macrophages into a peritonitis model demonstrated the importance of SOCS3 in driving production of pro-inflammatory IL-6 and nitric oxide, while curtailing expression of anti-inflammatory IL-10 and SOCS1. SOCS3-induced pro-inflammatory effects were due, at least in part, to its role in controlling activation and nuclear accumulation of nuclear factor-κB and activity of phosphatidylinositol 3-kinase. We show for the first time that SOCS3 also directs the functions of human monocyte-derived macrophages, including efficient M1-induced cytokine production (IL-1β, IL-6, IL-23, IL-12), attenuated signal transducer and activator of transcription 3 activity and ability of antigen-loaded macrophages to drive T-cell responses. Hence, M1-associated SOCS3 was a positive regulator of pro-inflammatory responses in our rodent models and up-regulated SOCS3 is essential for effective M1-macrophage activation and function in human macrophages.
    Full-text · Article · Jan 2014 · Immunology
  • Mark A. Vickers · Robert N. Barker
    [Show abstract] [Hide abstract]
    ABSTRACT: Autoimmune hemolytic anemia (AIHA) is a classic example of type II hypersensitivity, caused by autoantibodies that bind red blood cells (RBC). The autoantibodies can be divided into cold or warm types, depending on the optimum temperature at which they bind, and AIHA can also be classified as either primary or secondary if there is an underlying disease such as neoplasia or infection. Hemolysis results from complement fixation leading to membrane attack complex formation, or phagocytosis mediated by receptors for IgGFc and C3. The pathogenicity of cold autoantibodies depends on their thermal amplitude, and on titer, isotype, and phagocyte activation state for warm antibodies. Multiple genetic and environmental factors contribute to susceptibility. Cold autoantibodies most commonly recognize the I blood group, and warm autoantibodies the Rh complex. Studies of responses to RBC autoantigens indicate that warm antibody production is helper dependent and associated with failure of peripheral self-tolerance mechanisms.
    No preview · Article · Dec 2013
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Low maternal dietary vitamin E (but not vitamin C) intake during pregnancy has been associated with increased in vitro cord blood mononuclear cell (CBMC) proliferative responses, childhood wheezing and asthma. We investigated whether these associations reflect direct effects of vitamin E by investigating the effects of supplementing CBMC cultures with physiological concentrations of vitamin E. CBMC from seventy neonates were cultured supplemented with either nothing, α-tocopherol or ascorbic acid. Proliferative, IFN-γ, IL-4, IL-10 and TGF-β responses were measured. In general, vitamin E supplementation was associated with a trend for reduced proliferative responses after stimulation with antigens and house dust mite, and with increased proliferation after stimulation with timothy grass allergen. There was a trend for CBMC cultures to exhibit decreased secretion of IFN-γ, IL-10 and IL-4. Supplementation with vitamin C had no effect on CBMC proliferation, but increased IFN-γ and IL-4 production, and decreased IL-10 production. In conclusion, in vitro vitamin E and C supplementation of CBMC modifies neonatal immune function, but not in a manner predicted by observational epidemiological studies. The observed associations between vitamin E and childhood respiratory disease are complex, and the nature and form of nutritional intervention need to be carefully considered before inclusion in trials.
    Full-text · Article · Sep 2013 · Nutrients
  • [Show abstract] [Hide abstract]
    ABSTRACT: CTLA-4 is a crucial immune regulator that mediates both negative costimulation signals to T cells, and regulatory T (Treg)-cell extrinsic control of effector responses. Here we present evidence supporting a novel mechanism for this extrinsic suppression, executed by the alternatively spliced soluble CTLA-4 isoform (sCTLA-4). Analyses of human T cells in vitro show that sCTLA-4 secretion can be increased during responses, and has potent inhibitory properties, since isoform-specific blockade of its activity significantly increased Ag-driven proliferation and cytokine (IFN-γ, IL-17) secretion. Treg cells were demonstrated to be a prominent source of sCTLA-4, which contributed to suppression in vitro when their numbers were limiting. The soluble isoform was also produced by, and inhibited, murine T cells responding to Ag in vitro, and blockade of its activity in vivo protected against metastatic spread of melanoma in mice. We conclude that sCTLA-4 is an important immune regulator, responsible for at least some of the inhibitory effects previously ascribed to the membrane-bound isoform. These results suggest that the immune system exploits the different CTLA-4 isoforms for either intrinsic or extrinsic regulation of T-cell activity.
    No preview · Article · May 2013 · European Journal of Immunology
  • Source
    Lekh N Dahal · Lindsay S Hall · Robert N Barker · Frank J Ward
    [Show abstract] [Hide abstract]
    ABSTRACT: Autoimmune haemolytic anaemia (AIHA) is caused by autoantibodies against RBC surface antigens that render RBC susceptible to Fc-mediated phagocytosis and complement-mediated lysis. Experimental AIHA can be induced by injection of rat RBC to naïve mice but a lymphocyte-mediated regulatory mechanism eventually suppresses the production of autoantibodies specific for mouse RBC. Critically, this tolerogenic response can be transferred to naïve mice by splenocytes from the rat RBC immunized mouse. Here we investigate whether Indoleamine 2,3 dioxygenase (IDO) or the initiators of IDO cascade, including the CTLA-4 receptor and its soluble isoform contribute to this tolerogenic mechanism. Splenocytes from experimental AIHA mice were adoptively transferred to naive mice under the cover of anti-CTLA-4, anti-soluble CTLA-4 antibodies or IDO inhibitor 1-Methyl Tryptophan (1-MT). Recipient mice were immunized with rat RBC and levels of antibody against self-RBC and rat-RBC were monitored. Our results indicate that transfer of tolerance to naïve recipients is dependent on IDO mediated immunosuppression as mice receiving previously tolerized splenocytes under the cover of 1-MT were refractory to tolerance and developed haemolytic disease upon further challenge with rat RBC. Initiators of IDO activity, CTLA-4 or soluble CTLA-4 did not mediate this tolerogenic process but on their blockade, boosted antigen-specific effector immune responses.
    Full-text · Article · Feb 2013 · Clinical & Experimental Immunology

Publication Stats

2k Citations
341.46 Total Impact Points

Institutions

  • 1997-2015
    • University of Aberdeen
      • • Institute of Medical Sciences
      • • Division of Applied Medicine
      Aberdeen, Scotland, United Kingdom
  • 1993-1996
    • University of Bristol
      • • School of Veterinary Sciences
      • • Medical School
      Bristol, England, United Kingdom