• Longitudinal proliferation mapping in vivo reveals NADPH oxidase-mediated dampening of Staphylococcus aureus growth rates within neutrophils.

      Seiß, Elena A; Krone, Anna; Formaglio, Pauline; Goldmann, Oliver; Engelmann, Susanne; Schraven, Burkhart; Medina, Eva; Müller, Andreas J; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Nature publishing group, 2019-04-05)
      Upon the onset of inflammatory responses, bacterial pathogens are confronted with altered tissue microenvironments which can critically impact on their metabolic activity and growth. Changes in these parameters have however remained difficult to analyze over time, which would be critical to dissect the interplay between the host immune response and pathogen physiology. Here, we established an in vivo biosensor for measuring the growth rates of Staphylococcus aureus (S. aureus) on a single cell-level over days in an ongoing cutaneous infection. Using intravital 2-photon imaging and quantitative fluorescence microscopy, we show that upon neutrophil recruitment to the infection site and bacterial uptake, non-lethal dampening of S. aureus proliferation occurred. This inhibition was supported by NADPH oxidase activity. Therefore, reactive oxygen production contributes to pathogen containment within neutrophils not only by killing S. aureus, but also by restricting the growth rate of the bacterium.
    • Filamin A Phosphorylation at Serine 2152 by the Serine/Threonine Kinase Ndr2 Controls TCR-Induced LFA-1 Activation in T Cells.

      Waldt, Natalie; Seifert, Anke; Demiray, Yunus Emre; Devroe, Eric; Turk, Benjamin E; Reichardt, Peter; Mix, Charlie; Reinhold, Annegret; Freund, Christian; Müller, Andreas J; et al. (Frontiers, 2018-01-01)
      The integrin LFA-1 (CD11a/CD18) plays a critical role in the interaction of T cells with antigen presenting cells (APCs) to promote lymphocyte differentiation and proliferation. This integrin can be present either in a closed or in an open active conformation and its activation upon T-cell receptor (TCR) stimulation is a critical step to allow interaction with APCs. In this study we demonstrate that the serine/threonine kinase Ndr2 is critically involved in the initiation of TCR-mediated LFA-1 activation (open conformation) in T cells. Ndr2 itself becomes activated upon TCR stimulation and phosphorylates the intracellular integrin binding partner Filamin A (FLNa) at serine 2152. This phosphorylation promotes the dissociation of FLNa from LFA-1, allowing for a subsequent association of Talin and Kindlin-3 which both stabilize the open conformation of LFA-1. Our data suggest that Ndr2 activation is a crucial step to initiate TCR-mediated LFA-1 activation in T cells.
    • Characterization of mice with a platelet-specific deletion of the adapter molecule ADAP.

      Rudolph, Jochen Michael; Guttek, Karina; Weitz, Gabriele; Meinke, Clara Antonia; Kliche, Stefanie; Reinhold, Dirk; Schraven, Burkhart; Reinhold, Annegret; HZI, Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig Germany. (ASM, 2019-03-04)
      The adhesion and degranulation-promoting adapter protein (ADAP) is expressed in T cells, NK cells, myeloid cells, and platelets. The involvement of ADAP in the regulation of receptor-mediated inside-out signaling leading to integrin activation is well characterized, especially in T cells and in platelets. Due to the fact that animal studies using conventional knock-out mice are limited by the overlapping effects of the different ADAP-expressing cells, we generated conditional ADAP knock-out mice (ADAPfl/fl PF4-Cretg). We observed that loss of ADAP restricted to the megakaryocytic lineage has no impact on other hematopoietic cells even after stimulation conditions. ADAPfl/fl PF4-Cretg mice showed thrombocytopenia in combination with reduced plasma levels of PF4 and TGF-β1. In vitro, platelets from these mice revealed reduced P-selectin expression, lower TGF-β1 release, diminished integrin αIIbβ3 activation and decreased fibrinogen binding after stimulation with podoplanin, the ligand of the C-type lectin-like receptor-2 (CLEC-2). Furthermore, loss of ADAP was associated with impaired CLEC-2-mediated activation of PLCγ2 and Erk1/2. Induction of experimental autoimmune encephalomyelitis (EAE) in mice lacking ADAP expression in platelets caused a more severe disease. In vivo administration of TGF-β1 early after T cell transfer improved EAE severity in mice with loss of ADAP restricted to platelets. Our results reveal a regulatory function of ADAP in platelets in vitro and during autoimmune disease EAE in vivo.
    • Memantine potentiates cytarabine-induced cell death of acute leukemia correlating with inhibition of K1.3 potassium channels, AKT and ERK1/2 signaling.

      Lowinus, Theresa; Heidel, Florian H; Bose, Tanima; Nimmagadda, Subbaiah Chary; Schnöder, Tina; Cammann, Clemens; Schmitz, Ingo; Seifert, Ulrike; Fischer, Thomas; Schraven, Burkhart; et al. (BMC, 2019-01-16)
      Treatment of acute leukemia is challenging and long-lasting remissions are difficult to induce. Innovative therapy approaches aim to complement standard chemotherapy to improve drug efficacy and decrease toxicity. Promising new therapeutic targets in cancer therapy include voltage-gated K We analyzed acute lymphoid (Jurkat, CEM) and myeloid (HL-60, Molm-13, OCI-AML-3) leukemia cell lines and patients' acute leukemic blasts after treatment with either drug alone or the combination of cytarabine and memantine. Patch-clamp analysis was performed to evaluate inhibition of K Our study demonstrates that memantine inhibits K Our study underlines inhibition of K
    • CD11c-expressing Ly6C+CCR2+ monocytes constitute a reservoir for efficient Leishmania proliferation and cell-to-cell transmission.

      Heyde, Sandrina; Philipsen, Lars; Formaglio, Pauline; Fu, Yan; Baars, Iris; Höbbel, Guido; Kleinholz, Corinna L; Seiß, Elena A; Stettin, Juliane; Gintschel, Patricia; et al. (2018-10-01)
      The virulence of intracellular pathogens such as Leishmania major (L. major) relies largely on their ability to undergo cycles of replication within phagocytes, release, and uptake into new host cells. While all these steps are critical for successful establishment of infection, neither the cellular niche of efficient proliferation, nor the spread to new host cells have been characterized in vivo. Here, using a biosensor for measuring pathogen proliferation in the living tissue, we found that monocyte-derived Ly6C+CCR2+ phagocytes expressing CD11c constituted the main cell type harboring rapidly proliferating L. major in the ongoing infection. Synchronization of host cell recruitment and intravital 2-photon imaging showed that these high proliferating parasites preferentially underwent cell-to-cell spread. However, newly recruited host cells were infected irrespectively of their cell type or maturation state. We propose that among these cells, CD11c-expressing monocytes are most permissive for pathogen proliferation, and thus mainly fuel the cycle of intracellular proliferation and cell-to-cell transfer during the acute infection. Thus, besides the well-described function for priming and activating T cell effector functions against L. major, CD11c-expressing monocyte-derived cells provide a reservoir for rapidly proliferating parasites that disseminate at the site of infection.
    • Activated protein C protects from GvHD via PAR2/PAR3 signalling in regulatory T-cells.

      Ranjan, Satish; Goihl, Alexander; Kohli, Shrey; Gadi, Ihsan; Pierau, Mandy; Shahzad, Khurrum; Gupta, Dheerendra; Bock, Fabian; Wang, Hongjie; Shaikh, Haroon; et al. (2017-08-21)
      Graft-vs.-host disease (GvHD) is a major complication of allogenic hematopoietic stem-cell(HSC) transplantation. GvHD is associated with loss of endothelial thrombomodulin, but the relevance of this for the adaptive immune response to transplanted HSCs remains unknown. Here we show that the protease-activated protein C (aPC), which is generated by thrombomodulin, ameliorates GvHD aPC restricts allogenic T-cell activation via the protease activated receptor (PAR)2/PAR3 heterodimer on regulatory T-cells (Tregs, CD4(+)FOXP3(+)). Preincubation of pan T-cells with aPC prior to transplantation increases the frequency of Tregs and protects from GvHD. Preincubation of human T-cells (HLA-DR4(-)CD4(+)) with aPC prior to transplantation into humanized (NSG-AB°DR4) mice ameliorates graft-vs.-host disease. The protective effect of aPC on GvHD does not compromise the graft vs. leukaemia effect in two independent tumor cell models. Ex vivo preincubation of T-cells with aPC, aPC-based therapies, or targeting PAR2/PAR3 on T-cells may provide a safe and effective approach to mitigate GvHD.Graft-vs.-host disease is a complication of allogenic hematopoietic stem cell transplantation, and is associated with endothelial dysfunction. Here the authors show that activated protein C signals via PAR2/PAR3 to expand Treg cells, mitigating the disease in mice.
    • A highly conserved redox-active Mx(2)CWx(6)R motif regulates Zap70 stability and activity.

      Thurm, Christoph; Poltorak, Mateusz P; Reimer, Elisa; Brinkmann, Melanie M; Leichert, Lars; Schraven, Burkhart; Simeoni, Luca; Helmholtz Centre of infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-05-09)
      ζ-associated protein of 70 kDa (Zap70) is crucial for T-cell receptor (TCR) signaling. Loss of Zap70 in both humans and mice results in severe immunodeficiency. On the other hand, the expression of Zap70 in B-cell malignancies correlates with the severity of the disease. Because of its role in immune-related disorders, Zap70 has become a therapeutic target for the treatment of human diseases. It is well-established that the activity/expression of Zap70 is regulated by post-translational modifications of crucial amino acids including the phosphorylation of tyrosines and the ubiquitination of lysines. Here, we have investigated whether also oxidation of cysteine residues regulates Zap70 functions. We have identified C575 as a major sulfenylation site of Zap70. A C575A substitution results in protein instability, reduced activity, and increased dependency on the Hsp90/Cdc37 chaperone system. Indeed, Cdc37 overexpression reconstituted partially the expression but fully the function of Zap70C575A. C575 lies within a Mx(2)CWx(6)R motif which is highly conserved among almost all human tyrosine kinases. Mutation of any of the conserved amino acids, but not of a non-conserved residue preceding the cysteine, also results in Zap70 instability. Collectively, we have identified a new redox-active motif which is crucial for the regulation of Zap70 stability/activity. We believe that this motif has the potential to become a novel target for the development of therapeutic tools to modulate the expression/activity of kinases.
    • HIPP neurons in the dentate gyrus mediate the cholinergic modulation of background context memory salience.

      Raza, Syed Ahsan; Albrecht, Anne; Çalışkan, Gürsel; Müller, Bettina; Demiray, Yunus Emre; Ludewig, Susann; Meis, Susanne; Faber, Nicolai; Hartig, Roland; Schraven, Burkhart; et al. (2017-08-04)
      Cholinergic neuromodulation in the hippocampus controls the salience of background context memory acquired in the presence of elemental stimuli predicting an aversive reinforcement. With pharmacogenetic inhibition we here demonstrate that hilar perforant path-associated (HIPP) cells of the dentate gyrus mediate the devaluation of background context memory during Pavlovian fear conditioning. The salience adjustment is sensitive to reduction of hilar neuropeptide Y (NPY) expression via dominant negative CREB expression in HIPP cells and to acute blockage of NPY-Y1 receptors in the dentate gyrus during conditioning. We show that NPY transmission and HIPP cell activity contribute to inhibitory effects of acetylcholine in the dentate gyrus and that M1 muscarinic receptors mediate the cholinergic activation of HIPP cells as well as their control of background context salience. Our data provide evidence for a peptidergic local circuit in the dentate gyrus that mediates the cholinergic encoding of background context salience during fear memory acquisition.Intra-hippocampal circuits are essential for associating a background context with behaviorally salient stimuli and involve cholinergic modulation at SST(+) interneurons. Here the authors show that the salience of the background context memory is modulated through muscarinic activation of NPY(+) hilar perforant path associated interneurons and NPY signaling in the dentate gyrus.
    • Immunomodulation by memantine in therapy of Alzheimer's disease is mediated through inhibition of Kv1.3 channels and T cell responsiveness.

      Lowinus, Theresa; Bose, Tanima; Busse, Stefan; Busse, Mandy; Reinhold, Dirk; Schraven, Burkhart; Bommhardt, Ursula H H; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016-07-22)
      Memantine is approved for the treatment of advanced Alzheimer´s disease (AD) and reduces glutamate-mediated neuronal excitotoxicity by antagonism of N-methyl-D-aspartate receptors. In the pathophysiology of AD immune responses deviate and infectious side effects are observed during memantine therapy. However, the particular effects of memantine on human T lymphocytes are unresolved. Here, we provide evidence that memantine blocks Kv1.3 potassium channels, inhibits CD3-antibody- and alloantigen-induced proliferation and suppresses chemokine-induced migration of peripheral blood T cells of healthy donors. Concurrent with the in vitro data, CD4+ T cells from AD patients receiving therapeutic doses of memantine show a transient decline of Kv1.3 channel activity and a long-lasting reduced proliferative response to alloantigens in mixed lymphocyte reactions. Furthermore, memantine treatment provokes a profound depletion of peripheral blood memory CD45RO+ CD4+ T cells. Thus, standard doses of memantine profoundly reduce T cell responses in treated patients through blockade of Kv1.3 channels. This may normalize deviant immunopathology in AD and contribute to the beneficial effects of memantine, but may also account for the enhanced infection rate.
    • Early changes in the metabolic profile of activated CD8(+) T cells.

      Cammann, Clemens; Rath, Alexander; Reichl, Udo; Lingel, Holger; Brunner-Weinzierl, Monika; Simeoni, Luca; Schraven, Burkhart; Lindquist, Jonathan A; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016)
      Antigenic stimulation of the T cell receptor (TCR) initiates a change from a resting state into an activated one, which ultimately results in proliferation and the acquisition of effector functions. To accomplish this task, T cells require dramatic changes in metabolism. Therefore, we investigated changes of metabolic intermediates indicating for crucial metabolic pathways reflecting the status of T cells. Moreover we analyzed possible regulatory molecules required for the initiation of the metabolic changes.
    • NMDA-receptor antagonists block B-cell function but foster IL-10 production in BCR/CD40-activated B cells.

      Simma, Narasimhulu; Bose, Tanima; Kahlfuss, Sascha; Mankiewicz, Judith; Lowinus, Theresa; Lühder, Fred; Schüler, Thomas; Schraven, Burkhart; Heine, Martin; Bommhardt, Ursula; et al. (2014)
      B cells are important effectors and regulators of adaptive and innate immune responses, inflammation and autoimmunity, for instance in anti-NMDA-receptor (NMDAR) encephalitis. Thus, pharmacological modulation of B-cell function could be an effective regimen in therapeutic strategies. Since the non-competitive NMDAR antagonist memantine is clinically applied to treat advanced Alzheimer`s disease and ketamine is supposed to improve the course of resistant depression, it is important to know how these drugs affect B-cell function.
    • Negative interactions and feedback regulations are required for transient cellular response.

      Mobashir, Mohammad; Madhusudhan, Thati; Isermann, Berend; Beyer, Tilo; Schraven, Burkhart; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2014)
      Signal transduction is a process required to conduct information from a receptor to the nucleus. This process is vital for the control of cellular function and fate. The dynamics of signaling activation and inhibition determine processes such as apoptosis, proliferation, and differentiation. Thus, it is important to understand the factors modulating transient and sustained response. To address this question, by applying mathematical approach we have studied the factors which can alter the activation nature of downstream signaling molecules. The factors which we have investigated are loops (feed forward and feedback loops), cross-talk of signal transduction pathways, and the change in the concentration of the signaling molecules. Based on our results we conclude that among these factors feedback loop and the cross-talks which directly inhibit the target protein dominantly controls the transient cellular response.
    • miR-20a inhibits TCR-mediated signaling and cytokine production in human naïve CD4+ T cells.

      Reddycherla, Amarendra V; Meinert, Ines; Reinhold, Annegret; Reinhold, Dirk; Schraven, Burkhart; Simeoni, Luca; Helmholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany. (2015)
      Upon TCR stimulation by peptide-MHC complexes, CD4+ T cells undergo activation and proliferation. This process will ultimately culminate in T-cell differentiation and the acquisition of effector functions. The production of specific cytokines by differentiated CD4+ T cells is crucial for the generation of the appropriate immune response. Altered CD4+ T-cell activation and cytokine production result in chronic inflammatory conditions and autoimmune disorders. miRNAs have been shown to be important regulators of T-cell biology. In this study, we have focused our investigation on miR-20a, a member of the miR-17-92 cluster, whose expression is decreased in patients suffering from multiple sclerosis. We have found that miR-20a is rapidly induced upon TCR-triggering in primary human naïve CD4+ T cells and that its transcription is regulated in a Erk-, NF-κB-, and Ca++-dependent manner. We have further shown that overexpression of miR-20a inhibits TCR-mediated signaling but not the proliferation of primary human naïve CD4+ T cells. However, miR-20a overexpression strongly suppresses IL-10 secretion and moderately decreases IL-2, IL-6 and IL8 production, which are crucial regulators of inflammatory responses. Our study suggests that miR-20a is a new player in the regulation of TCR signaling strength and cytokine production.
    • TCR-triggered extracellular superoxide production is not required for T-cell activation.

      Belikov, Aleksey V; Schraven, Burkhart; Simeoni, Luca (2014)
      In the last decade, reactive oxygen species (ROS) production has been shown to occur upon T-cell receptor (TCR) stimulation and to affect TCR-mediated signalling. However, the exact reactive species that are produced, how ROS are generated and their requirement for T-cell activation, proliferation or cytokine production remain unclear, especially in the case of primary human T cells. Moreover, several groups have questioned that ROS are produced upon TCR stimulation.
    • Clinically relevant doses of FLT3-kinase inhibitors quizartinib and midostaurin do not impair T-cell reactivity and function.

      Wolleschak, Denise; Mack, Thomas S; Perner, Florian; Frey, Stephanie; Schnöder, Tina M; Wagner, Marie-Christine; Höding, Christine; Pils, Marina C; Parkner, Andreas; Kliche, Stefanie; et al. (2014-06)
    • PAG/Cbp suppression reveals a contribution of CTLA-4 to setting the activation threshold in T cells.

      Smida, Michal; Cammann, Clemens; Gurbiel, Slavyana; Kerstin, Nadja; Lingel, Holger; Lindquist, Sabine; Simeoni, Luca; Brunner-Weinzierl, Monika C; Suchanek, Miloslav; Schraven, Burkhart; et al. (2013)
      PAG/Cbp represents a ubiquitous mechanism for regulating Src family kinases by recruiting Csk to the plasma membrane, thereby controlling cellular activation. Since Src kinases are known oncogenes, we used RNA interference in primary human T cells to test whether the loss of PAG resulted in lymphocyte transformation.
    • Simulated evolution of signal transduction networks.

      Mobashir, Mohammad; Schraven, Burkhart; Beyer, Tilo; Institute of Molecular and Clinical Immunology, Otto-von-Guericke University, Magdeburg, Germany. (2012)
      Signal transduction is the process of routing information inside cells when receiving stimuli from their environment that modulate the behavior and function. In such biological processes, the receptors, after receiving the corresponding signals, activate a number of biomolecules which eventually transduce the signal to the nucleus. The main objective of our work is to develop a theoretical approach which will help to better understand the behavior of signal transduction networks due to changes in kinetic parameters and network topology. By using an evolutionary algorithm, we designed a mathematical model which performs basic signaling tasks similar to the signaling process of living cells. We use a simple dynamical model of signaling networks of interacting proteins and their complexes. We study the evolution of signaling networks described by mass-action kinetics. The fitness of the networks is determined by the number of signals detected out of a series of signals with varying strength. The mutations include changes in the reaction rate and network topology. We found that stronger interactions and addition of new nodes lead to improved evolved responses. The strength of the signal does not play any role in determining the response type. This model will help to understand the dynamic behavior of the proteins involved in signaling pathways. It will also help to understand the robustness of the kinetics of the output response upon changes in the rate of reactions and the topology of the network.
    • The adapter protein ADAP is required for selected dendritic cell functions.

      Togni, Mauro; Engelmann, Swen; Reinhold, Dirk; Schraven, Burkhart; Reinhold, Annegret; Institute for Molecular and Clinical Immunology, Otto von Guericke University Magdeburg, Leipziger Strasse 44, 39120 Magdeburg, Germany. annegret.reinhold@med.ovgu.de. (2012)
      ABSTRACT:
    • TCR-mediated Erk activation does not depend on Sos and Grb2 in peripheral human T cells.

      Warnecke, Nicole; Poltorak, Mateusz; Kowtharapu, Bhavani S; Arndt, Boerge; Stone, James C; Schraven, Burkhart; Simeoni, Luca; Institute of Molecular and Clinical Immunology, Otto-von-Guericke University, Leipziger Strasse 44, Magdeburg 39120, Germany. (2012-04)
      Sos proteins are ubiquitously expressed activators of Ras. Lymphoid cells also express RasGRP1, another Ras activator. Sos and RasGRP1 are thought to cooperatively control full Ras activation upon T-cell receptor triggering. Using RNA interference, we evaluated whether this mechanism operates in primary human T cells. We found that T-cell antigen receptor (TCR)-mediated Erk activation requires RasGRP1, but not Grb2/Sos. Conversely, Grb2/Sos—but not RasGRP1—are required for IL2-mediated Erk activation. Thus, RasGRP1 and Grb2/Sos are insulators of signals that lead to Ras activation induced by different stimuli, rather than cooperating downstream of the TCR.
    • Integrating signals from the T-cell receptor and the interleukin-2 receptor.

      Beyer, Tilo; Busse, Mandy; Hristov, Kroum; Gurbiel, Slavyana; Smida, Michal; Haus, Utz-Uwe; Ballerstein, Kathrin; Pfeuffer, Frank; Weismantel, Robert; Schraven, Burkhart; et al. (2011-08)
      T cells orchestrate the adaptive immune response, making them targets for immunotherapy. Although immunosuppressive therapies prevent disease progression, they also leave patients susceptible to opportunistic infections. To identify novel drug targets, we established a logical model describing T-cell receptor (TCR) signaling. However, to have a model that is able to predict new therapeutic approaches, the current drug targets must be included. Therefore, as a next step we generated the interleukin-2 receptor (IL-2R) signaling network and developed a tool to merge logical models. For IL-2R signaling, we show that STAT activation is independent of both Src- and PI3-kinases, while ERK activation depends upon both kinases and additionally requires novel PKCs. In addition, our merged model correctly predicted TCR-induced STAT activation. The combined network also allows information transfer from one receptor to add detail to another, thereby predicting that LAT mediates JNK activation in IL-2R signaling. In summary, the merged model not only enables us to unravel potential cross-talk, but it also suggests new experimental designs and provides a critical step towards designing strategies to reprogram T cells.