science day 2013 - immunosensation · prof. gunther hartmann institute of clinical chemistry &...

Science Day2013

ImmunoSensation Cluster

Science Day 2013 October 22, 2013

Meeting venue: caesar – center of advanced european studies and research Ludwig-Erhard-Allee 2 53175 Bonn

Welcome to Cluster Science Day 2013 A warm welcome to all of you attending the first Cluster Science Day of the ImmunoSensation Cluster of Excellence! This day is completely dedicated to the scientific contributions of students and young postdocs in the cluster. A total of 71 abstracts were submitted which altogether represent a broad spectrum of outstanding science in immunology and its intersections with neurology, genetics, metabolism, biophysics and mathematics. Individually, all 71 abstracts represent valuable contributions to the field of immunology. Collectively, they also form an important resource on the scope of the cluster in the year 2013. The cluster steering committee has selected 22 abstracts for short oral presentations. At the end of this day, a total of 5000 € in prize money will be awarded to presenters. As part of our program, Astrid Draffehn and Eva Bartok of the Cluster Coordination Office will provide information about the activities for young investigator support in the cluster. A survey has been conducted among students, and their answers will form an important basis for the structure of the upcoming International Immunology Training Program Bonn (IITB), which will be launched shortly. As part of our exchange with Edmond and Lily Safra Center of Brain Sciences at the Hebrew University in Jerusalem, we are also very happy to host five Israeli students and their group leader, Hermona Soreq. Now let us have a day full of fascinating science! We are looking forward to your active participation in the discussion of all of the projects in this booklet, whether orally presented or not. You will see that there is much more excellent science than could possibly be presented in one day. Please use the collection of abstracts in this booklet to learn about the scientific techniques and topics of other groups. We hope that the Cluster Science Day will also serve as a platform for fruitful interactions and new collaborations. To encourage further discussion and exchange among young researchers in the Cluster, we would like to invite everyone to stay at the CAESAR after the talks for beer and an informal dinner. With best wishes, Gunther Hartmann Waldemar Kolanus Speaker Vice-Speaker

The Cluster of Excellence „ImmunoSensation: the immune

sensory system“ was founded in November 2012 as a central

hub for integrated, multidisciplinary research in the field

innate immunity. It is as a joint collaborative project of the

Medical Faculty and the Faculty of Mathematics and Life

Sciences (Life & Medical Sciences and Institute of Applied

Mathematics), the Max-Planck-associated Institute caesar

(Center of Advanced European Studies) and the DZNE

(German Center for Neurodegenerative Diseases) of the

Helmholtz-Society.

ImmunoSensation members include not only renowned

immunologists but also experts in sensory systems research,

neurobiology and mathematics. The German Research

Foundation (Deutsche Forschungsgemeinschaft, DFG) has

granted the ImmunoSensation Cluster over 28 Mio € in

funding for an initial period of 5 years, which will be devoted to

innovative research in immunology and its connections to

metabolism, the nervous system and beyond.

Contact

Speaker Prof. Gunther HartmannInstitute of Clinical Chemistry & Clinical PharmacologyUniversity Hospital BonnSigmund-Freud-Straße 25D-53127 Bonn

Vice-SpeakerProf. Waldemar KolanusLIMES Life & Medical Sciences InstituteMolecular Immune & Cell Biology UnitUniversity of BonnCarl-Troll-Straße 31D-53115 Bonn

Cluster Coordination Office (CCO)Central Coordination and Gender Support: Nicole DahmsFinancial Administration: Ida BartzSpeaker Office: Verena JohannYoung Investigator Support: Astrid Draffehn, Eva BartokPublic Relations: Diana SiglIT: Andriy Kubarenko

[email protected]

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Program Science Day 2013

Program Science Day 2013

9:00 - 9:15 Opening remarks and welcome Prof. Gunther Hartmann

9:15 - 9:45 Presentation of the IITB program Eva Bartok and Astrid Draffehn

9:45 - 10:00 Albayram A., Zimmer A. …………………….Page 12 Role of Cannabinoid 1 Receptors on Hippocampal GABAergic Neurons in Brain Aging

10:00 - 10:15 Bald T., Tüting T. …………………………….Page 14 Ultraviolet radiation-induced neutrophilic inflammation promotes angiotropism and metastasis in melanoma

10:15 - 10:30 Bodea L.G., Neumann H. …………………..Page 17 Trem2/Dap12 pathway involvement in neurodegeneration

10:30 - 11:00 Coffee break

11:00 - 11:15 Brosseron F., Heneka M.T. ………………...Page 20 Correlation of cerebrospinal fluid cytokines with clinical markers of dementia in mild cognitive impairment and Alzheimer´s disease

11:15 – 11:30 Brückner M., Halle A. ……………………......Page 21 Microglial dysfunction in Alzheimer’s disease

11:30 – 11:45 Daßler J., Coch C. ……………………...........Page 24

RIG-I induced Melanoma-derived Exosomes carry BAG6 and 3pRNA thereby triggering an innate immune response in vitro and a potent anti-tumor response in vivo

11:45 - 12:00 Eppler F., Kolanus W. ……………………….Page 28 Role of Dynamin 2 in Rap1-dependent lymphocyte adhesion


12:30 - 12:45 Goldeck M., Hornung V. …………………….Page 31

cGAS produces a 2’-5’-linked cyclic dinucleotide second messenger that activates STING

12:45 - 13:00 Hanin G., Soreq H. …………………………..Page 35

Competing targets of microRNA-608 modulate risks of hypertension and anxiety

13:00 - 13:15 Hückesfeld S., Pankratz M. …………………Page 38 Central Neurons modulate Feeding Behavior in Drosophila: Potential Role in Pathogen Avoidance

13:15 - 13:30 Kim S., Schumacher J. ……………………...Page 42

Unraveling the genetic basis of innate immune response in TLR4-activated human monocytes

13:30 – 14:30 Lunch

14:30 – 14:45 Krebs W., Schultze J.L. ……………………..Page 46 Characterization of epigenetic marks establishes specific patterns during macrophage activation

14:45 - 15:00 Kreutzberg T., Garbi N. ……………………...Page 47 Sensing of self by T cells promotes awareness towards cognate antigen and T helper cell differentiation

15:00 - 15:15 Labzin L., Latz E. ……………………………..Page 49

HDL inhibits TLR induced pro-inflammatory gene expression via the transcriptional repressor ATF3

15:15 - 15:30 Mass E., Hoch M. …………………….............0 Creld1 regulates NFATc1 localization and activation through interaction with calcineurin B

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15:30 - 15:45 Mertens C., Barchet W. ……………………...Page 53 Oxidative damage of DNA confers resistance to TREX1 degradation and potentiates STING dependent immune sensing

15:45 – 16:00 Piotrowitz K., Thiele C. ……………………...Page 59 How inflammation affects liver lipid metabolism


16:30 - 16:45 Schmid-Burgk J., Hornung V. ……………...8 High throughput functional genomics using designer nucleases assembled by ligation-independent cloning

16:45 - 17:00 Stutz A., Latz E. ……………………………….Page 73 The inflammasome component NLRP3 is regulated by phosphorylation

17:00- 17:15 Vorac J., Förster I. ……………………...........Page 75 The role of the Aryl hydrocarbon Receptor Repressor (AhRR) in innate immunity and infection

17:15- 17:30 Waiskopf N., Soreq H. ……………………….Page 76 Semiconductor Nanoparticles for Biological Applications: The Promise and Challenges

17:30- 17:45 Zehner M., Burgdorf S. ………………………Page 81 Mannose receptor – a receptor for cross-presentation surrounded by ER components

17:45 - 18:15 Meeting of the Science Day committee, refreshment break for the audience

18:15 - 18:45 Closing remarks Prof. Waldemar Kolanus, announcement of the prize winners

18:45 - 21:00 Get together for PhDs and young postdocs with dinner and beer

Session chairs

Regina Betz (Institute of Human Genetics)

Irmgard Förster (LIMES)

Natalio Garbi (IMMEI)

Annett Halle (caesar)

Joachim L. Schultze (LIMES)

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Table of Content Contents

Program Science Day 2013 .............................................................................. 4

Albayram O., Zimmer A. ..................................................................................12

Role of Cannabinoid 1 Receptors on Hippocampal GABAergic Neurons in Brain Aging ....................................................................................................................... 12

Asdonk T., Zimmer S. ......................................................................................13

Impact of the innate immune system on function and mobilization of endothelial progenitor cells (EPC) .............................................................................................. 13

Bald T., Tüting T. ..............................................................................................14

Ultraviolet radiation-induced neutrophilic inflammation promotes angiotropism and metastasis in melanoma .......................................................................................... 14

Becker J., Schumacher J. ...............................................................................15

An eight amino acid insertion in the cytoplasmic tail and two additional amino acid substitutions in the extracellular domain of HLA-DQ confer risk for idiopathic achalasia ................................................................................................................. 15

Bertheloot D., Latz E. .......................................................................................16

RAGE is a cell surface nucleic acid sensor .............................................................. 16

Bodea L.G., Neumann H. .................................................................................17

Trem2/Dap12 pathway involvement in neurodegeneration ...................................... 17

Böhnert V., Barchet W. ....................................................................................18

Developing a whole cell vaccine in a syngeneic mouse model of ovarian cancer .... 18

Brandstätter O., Förster I. ...............................................................................19

Expression of the arylhydrocarbon receptor repressor (AhRR) in the immune system of the gut ................................................................................................................. 19

Brosseron F., Heneka M.T. ..............................................................................20

Correlation of cerebrospinal fluid cytokines with clinical markers of dementia in mild cognitive impairment and Alzheimer´s disease ........................................................ 20

Brückner M., Halle A. .......................................................................................21

Microglial dysfunction in Alzheimer’s disease .......................................................... 21

Cavlar T., Hornung V. ......................................................................................22

STING-dependent type I IFN induction by the small-molecule compound CMA ....... 22

Claude J., Neumann H. ....................................................................................23

Neuroprotective properties of the microglial receptor Siglec-E ................................. 23

Daßler J., Coch C. ............................................................................................24

RIG-I induced Melanoma-derived Exosomes carry BAG6 and 3pRNA thereby triggering an innate immune response in vitro and a potent anti-tumor response in vivo .......................................................................................................................... 24

Dixit A., Engel D. ..............................................................................................25

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Role of Gr1+ inflammatory macrophages in pyelonephritis-induced fibrosis ............ 25

Eckerle I, Drosten C. ........................................................................................26

Epithelial cell lines from bats and rodents as a novel tool for the study of zoonotic viruses ..................................................................................................................... 26

Engel C., van den Boorn J.G...........................................................................27

Retinoic acid-inducible gene-I (RIG-I)-mediated immune activation is attenuated by hypoxia in murine melanoma cells ........................................................................... 27

Eppler F., Kolanus W. ......................................................................................28

Role of Dynamin 2 in Rap1-dependent lymphocyte adhesion .................................. 28

Gioran A., Bano D. ...........................................................................................29

Neuronal branching associated with mitochondrial deficiency in C. elegans ............ 29

Glodde N., Hölzel M. ........................................................................................30

A “modular” murine melanoma model to study determinants of multimodal immunotherapeutic regimens ................................................................................... 30

Goldeck M., Hornung V. ..................................................................................31

cGAS produces a 2’-5’-linked cyclic dinucleotide second messenger that activates STING ..................................................................................................................... 31

Gondorf F., Hübner M.P. .................................................................................32

Chronic exposure to filaria and its endosymbiotic Wolbachia bacteria improves E. coli induced sepsis in a TLR2 dependent manner .......................................................... 32

Görgen S., Latz E. ............................................................................................33

Identification of novel TAM receptor interactors via label transfer: Tagging cross-linker to bait proteins via Sortase A .......................................................................... 33

Grebe A., Latz E. ..............................................................................................34

Cyclodextrin promotes atherosclerosis regression by dissolution of cholesterol crystals and LXR activation ...................................................................................... 34

Hanin G., Soreq H. ...........................................................................................35

Competing targets of microRNA-608 modulate risks of hypertension and anxiety ... 35

Hemmerling I., Hornung V. ..............................................................................36

Establishment of a STING reporter cell line for studying the molecular mechanism of intracellular DNA sensing......................................................................................... 36

Herzner A.M., Schlee M. ..................................................................................37

Post-transcriptional modulation of the type I interferon response by minimal-motif oligodeoxynucleotides ............................................................................................. 37

Hückesfeld S., Pankratz M. .............................................................................38

Central Neurons modulate Feeding Behavior in Drosophila: Potential Role in Pathogen Avoidance ................................................................................................ 38

Jakobs C., Hornung V. ....................................................................................39

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Impact of AIM2-Inflammasome activation on adaptive immune responses after cutaneous DNA-Vaccination .................................................................................... 39

Janas M., Wohlleber D. ....................................................................................40

Mitochondria play a central role in a novel innate immune sensing mechanism of viral infection ................................................................................................................... 40

Kaczmarek J., Diehl L. .....................................................................................41

Regulation of CD8 T cell function by the small GTPase Arl4d .................................. 41

Kim S., Schumacher J. ....................................................................................42

Unraveling the genetic basis of innate immune response in TLR4-activated human monocytes ............................................................................................................... 42

Kokordelis P., Nattermann J. ..........................................................................43

An effective IFN-γ mediated inhibition of HCV replication by NK cells is associated with spontaneous clearance of acute hepatitis C in HIV(+) patients ......................... 43

Kopatz J., Neumann H. ....................................................................................44

Siglec-h on M1-polarized microglia antagonizes glioma cell growth ......................... 44

Krämer B., Nattermann J. ................................................................................45

NKp46High expression defines a natural killer cell subset that is potentially involved in control of hepatitis C virus replication and modulation of liver fibrosis .................. 45

Krebs W., Schultze J.L. ...................................................................................46

Characterization of epigenetic marks establishes specific patterns during macrophage activation ............................................................................................. 46

Kreutzberg T., Garbi N. ....................................................................................47

Sensing of self by T cells promotes awareness towards cognate antigen and T helper cell differentiation ..................................................................................................... 47

Kuepper J.M., Schumak B. ..............................................................................48

Inflammatory monocytes play a decisive role in the immunopathology of experimental cerebral malaria ....................................................................................................... 48

Labzin L., Latz E. ..............................................................................................49

HDL inhibits TLR induced pro-inflammatory gene expression via the transcriptional repressor ATF3 ........................................................................................................ 49

Mass E., Hoch M. .............................................................................................50

Creld1 regulates NFATc1 localization and activation through interaction with calcineurin B ............................................................................................................ 50

Mathews M., Neumann H. ................................................................................51

Expression and function of the late-onset Alzheimer’s disease associated CD33 in human microglia ...................................................................................................... 51

Mayer H., Bovier A. ..........................................................................................52

Stochastic modeling of T-Cell activation .................................................................. 52

Mertens C., Barchet W. ....................................................................................53

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Oxidative damage of DNA confers resistance to TREX1 degradation and potentiates STING dependent immune sensing ......................................................................... 53

Muth D., Drosten C. .........................................................................................54

European SARS-related bat coronaviruses carry interferon antagonists active in human cells ............................................................................................................. 54

Nadorp B., Soreq H. .........................................................................................55

From mice to men: MicroRNA regulators of acetylcholine packaging and degradation ................................................................................................................................ 55

Niemeyer D., Müller M.A. .................................................................................56

Middle East respiratory syndrome coronavirus accessory protein 4a is a type I interferon antagonist ................................................................................................ 56

Niepmann S., Hornung V. ................................................................................57

TNFα regulates NLRP3 activity and contributes to age related inflammasome activation and metabolic disease ............................................................................. 57

Pelka K., Latz E. ...............................................................................................58

The activity of human endosomal TLRs is differentially regulated by the tyrosine-based motif of UNC93B1 ......................................................................................... 58

Piotrowitz K., Thiele C. ....................................................................................59

How inflammation affects liver lipid metabolism ....................................................... 59

Raju D., Wachten D. .........................................................................................60

Glucosylceramide controls sperm-head formation ................................................... 60

Redler S., Betz R.C. .........................................................................................61

High-density genotyping in alopecia areata identifiesTNSF4 and HLA-C as two new susceptibility loci with genome-wide significance ..................................................... 61

Renn M., Tüting T. ............................................................................................62

Hgf-Cdk4R24C mouse melanomas resist adoptive cell transfer therapy with CTL targeting the melanocytic antigen gp100 through inflammation-induced reversible dedifferentiation ....................................................................................................... 62

Rieck M., Kolanus W. .......................................................................................63

Arl4d complexes with cytohesin-3 and controls T effector function by limiting IL-2 expression ............................................................................................................... 63

Riesenberg S., Hölzel M. .................................................................................64

Determinants of TNF responsiveness in melanoma ................................................. 64

Rogava M., Tüting T. ........................................................................................65

Sensing of UVB-induced DNA damage in the skin by the innate immune system involves the TLR4- and MyD88-dependent signaling pathway ................................. 65

Ruschel J., Bradke F. ......................................................................................66

Systemic Administration of Epothilone B Promotes Axon ........................................ 66

Sander J., Schultze J.L. ...................................................................................67

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Signal integration of macrophage activation is substantially conserved on transcriptional level between human and mice ......................................................... 67

Schmid-Burgk J., Schmidt T., Hornung V. .....................................................68

High throughput functional genomics using designer nucleases assembled by ligation-independent cloning .................................................................................... 68

Schmidt T., Hornung V. ...................................................................................69

High throughput generation of knockout cell lines with designer nucleases using deep sequencing .............................................................................................................. 69

Schonauer S., Wachten D. ..............................................................................70

Investigating the cross-talk between GBA1 and GBA2 in Gaucher disease ............. 70

Schuette V., Burgdorf S. .................................................................................71

The Mannose receptor induces T cell tolerance via inhibition of CD45 and up-regulation of CTLA-4 ................................................................................................ 71

Shahraz A., Neumann H. .................................................................................72

Function of the human-specific sialic acid binding receptor Siglec-11 in amyloid-β mediated neurotoxicity ............................................................................................. 72

Stutz A., Latz E. ................................................................................................73

The inflammasome component NLRP3 is regulated by phosphorylation .................. 73

Ulas T., Pallasch C.P., Hallek M., Schultze J.L. .............................................74

Comparative transcriptomics reveals gain of proliferative capacity, loss of phagocytic capacity and a distinct phenotype of tumor-associated macrophages in murine chronic lymphatic leukemia ...................................................................................... 74

Vorac J., Förster I. ...........................................................................................75

The role of the Aryl hydrocarbon Receptor Repressor (AhRR) in innate immunity and infection ................................................................................................................... 75

Waiskopf N., Banin U., Soreq H. .....................................................................76

Semiconductor Nanoparticles for Biological Applications: The Promise and Challenges .............................................................................................................. 76

Weisheit C., Engel D.R. ...................................................................................77

Crosstalk between sentinel and helper macrophages permits neutrophil migration into infected epithelium ................................................................................................... 77

Wittlich M., Wohleber D. ..................................................................................78

T helper 1 cells induce liver damage after adenoviral infection of hepatocytes ........ 78

Xue J., Schultze J.L. ........................................................................................79

Network engineering on transcriptional level reveals a multi-strata model of human macrophage activation ............................................................................................. 79

Yayon N., Soreq H. ...........................................................................................80

Intra-, Extra- and Intercellular miRNA-mediated Regulation of Cholinergic Synaptic Plasticity .................................................................................................................. 80

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Zehner M., Burgdorf S. ....................................................................................81

Mannose receptor – a receptor for cross-presentation surrounded by ER components ................................................................................................................................ 81

Zillinger T., Barchet W. ....................................................................................82

Structure-Function Analysis of STING Activation by c[G(2',5')pA(3',5')p] and Targeting by Antiviral DMXAA ................................................................................................. 82

ImmunoSensation Cluster Members ..............................................................83

Cluster members (in alphabetical order) .................................................................. 83

Cluster-associated scientists (in alphabetical order) ................................................ 85

Cluster-associated students (in alphabetical order) ................................................. 88

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Albayram O., Zimmer A.

Role of Cannabinoid 1 Receptors on Hippocampal GABAergic Neurons in Brain Aging Albayram O., Bilkei-Gorzo A., Zimmer A. Institute of Molecular Psychiatry, University Hospital, University of Bonn, Germany [email protected] BACKGROUND: Aging of the brain is associated with a state of elevated glial reactivity, which may contribute to cognitive dysfunctions and progression of neurodegenerative disorders. The endocannabinoid system (ECS) is involved in the regulation of glial activity; therefore we asked whether the early cognitive decline in animals lacking cannabinoid 1 (CB1) receptors is associated with enhanced neuroinflammation. METHODS: We examined the age-related neuroinflammation in CB1 receptor constitutive knockout (Cnr1−/−) mice and mice with a conditional deletion of CB1 receptor in GABA or glutamate-specific neurons. The cognition of mice was assessed in the Morris water maze. The optical fractionator method was used to quantify the total number of Iba1- and NeuN-positive cells. Total RNA was extracted from the hippocampi and mRNA expression was examined. RESULTS: The age-dependent increase in microglial cell numbers, and the levels of proinflammatory cytokines were elevated in the Cnr1−/− mice. This enhanced glial activity was accompanied by neuronal loss in the hippocampus and deficits in cognition. Deletion of CB1 receptors from the GABAergic, but not from the glutamatergic neurons, led to similar age-related changes as observed in the Cnr1−/− animals. CONCLUSIONS: Our results suggest that CB1 receptor activity on hippocampal GABAergic neurons is necessary for glial activity regulation, for protection against neuroinflammation and thus against age-dependent cognitive deficits.

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Asdonk T., Zimmer S.

Impact of the innate immune system on function and mobilization of endothelial progenitor cells (EPC) Asdonk T.1, Barchet W.2, Hartmann G.2, Nickenig G.1, Zimmer S.1

1Department of Medicine/Cardiology, University Hospital, University of Bonn, Germany 2Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital, University of Bonn, Germany [email protected] BACKGROUND: Endothelial dysfunction is a crucial part of the chronic Inflammatory Atherosclerotic process and mediated by innate immune mechanisms. Pattern recognition receptors (PRR) specialized in immunorecognition of nucleic acids may play an important role in endothelial biology in a proathersclerotic manner. Damaged endothelium is not only restored by adjacent mature endothelial cells but also by circulating endothelial progenitor cells (EPC) which are of prognostic value in atherosclerotic patients. Here, we studied the impact of TLR-3 activation on function and mobilization of EPC as well as endothelial biology. METHODS and RESULTS: In vitro experiments show that EPC express TLR-3 and receptor activation results in enhanced oxidative stress, apoptosis and impaired migration and proliferation. FACS analysis show that in wild type mice TLR-3 stimulation leads to enhanced EPC numbers in the peripheral blood and spleen, associated with endothelial dysfunction and advanced atherosclerotic plaque burden in vivo. However, regeneration capacity of mobilized EPC is significantly impaired upon TLR-3 activation resulting in reduced endothelial regeneration. CONCLUSIONS: We could demonstrate that TLR-3 activation by viral or self RNA fragments significantly harms endothelial biology in a proatheroclerotic manner. Inhibiting PRR dependant innate immune pathways could therefore provide a novel therapeutic target to relieve inflammation and atherosclerosis.

mailto:[email protected]

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Bald T., Tüting T.

Ultraviolet radiation-induced neutrophilic inflammation promotes angiotropism and metastasis in melanoma Bald T., Rogava M., Landsberg J., Glodde N., Lopez-Ramos D., Gaffal E., Tüting T. Laboratory of Experimental Dermatology, Department of Dermatology and Allergy, University Hospital, University of Bonn, Germany [email protected] BACKGROUND: The ability of UV radiation to cause tumour-initiating DNA mutations in melanocytes is firmly established. Additionally it has been hypothesised that the effect of UV radiation on keratinocytes and immune cells can stimulate the survival, proliferation and migration of melanocytes. How these microenvironmental effects of UV-irradiation influence melanoma pathogenesis is incompletely understood. METHODS: We experimentally investigated the impact of UV-induced inflammatory responses on the progression of primary melanomas that were initiated by one epicutaneous application of DMBA in HGF-CDK4(R24C) mice as well as on serial HGF-CDK4(R24C) melanoma skin transplants. These model systems allowed us to study the tumour-promoting effects of UV-irradiation independent of its tumour-initiating effects. RESULTS: Repetitive UV-irradiation induces a neutrophil-rich skin inflammation that did not alter the incidence, multiplicity and growth kinetics of DMBA-induced HGF-CDK4(R24C) melanomas. However, detailed analysis revealed that UV-irradiation enhanced the expansion of tumour cells along endothelial cell surfaces in a pericyte-like location. This phenomenon was originally described as angiotropic growth by histopathologists in human melanomas. Consistent with clinical data, we found that enhanced angiotropism was associated with significantly increased numbers of lung metastases. UV-irradiation of mice bearing serial HGF-CDK4(R24C) melanoma skin transplants also showed enhanced angiotropism and increased number of lung metastases after UV-irradiation. Importantly, we found that antibody-mediated depletion of Ly6G+ neutrophils abrogated the metastasis-promoting effects of UV-irradiation. CONCLUSIONS: Our work provides evidence that repetitive UV-irradiation induces a neutrophilic inflammatory response which catalyses reciprocal melanoma-endothelial cell interactions, drives angiotropic growth and promotes metastatic spread.


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Becker J., Schumacher J.

An eight amino acid insertion in the cytoplasmic tail and two additional amino acid substitutions in the extracellular domain of HLA-DQ confer risk for idiopathic achalasia Becker J., Gockel I., Wouters M.M., Niebisch S., Gockel H.R, Ramonet D., Zimmermann J., González Vigo A., Trynka G., Ruiz de Leόn A., Pérez de la Serna J., Urcelay E., Kumar V., Franke L., Westra H-J., Drescher D., Kneist W., Marquardt J.U., Galle P.R., Mattheisen M., Annese V., Latiano A., Fumagalli U., Laghi L., Cuomo R., Sarnelli G., Müller M., Eckardt A.J., Tack J., Hoffmann P., Herms S., Kiesslich R., von Rahden B.H.A., Allescher H-D., Schulz H.G., Wijmenga C., Heneka M.T., Lang H., Hopfner K-P., de Bakker P.I.W., Boeckxstaens G.E., Nöthen M.M., Knapp M., Schumacher J.

Institute of Human Genetics, University Hospital, University of Bonn, Germany & Department of Genomics, Life and Brain Center, University Hospital, University of Bonn, Germany [email protected] BACKGROUND: Idiopathic achalasia is a rare esophageal motility disorder. It is characterized by a failure of the lower esophageal sphincter to relax due to a loss of neurons in the myenteric plexus. Although the cause of the degeneration is unknown, autoimmune processes seem to be involved in individuals with a genetic susceptibility. METHODS: To identify genetic factors involved in achalasia, we performed the first large-scale association study for achalasia (1,068 cases, 4,242 controls) using Illumina´s Immunochip (116,672 markers). Next, we imputed classical HLA-haplotypes and corresponding amino acids using a large reference panel followed by association and conditional analysis. RESULTS: Association analysis of the Immunochip markers yielded 33 markers - all located in the MHC region - reaching genome-wide significance (P<5x10-08). We followed up this signal by testing the imputed classical HLA-haplotypes and their amino acid changes for association. An 8-residue insertion in the cytoplasmic tail of HLA-DQβ1 (P=2.17x10-18) and two amino acid substitutions in the extracellular domain of HLA-DQα1 and HLA-DQβ1 (P<1.20x10-09) were independently associated with achalasia risk. CONCLUSIONS: Our data emphasise the hypothesis that autoimmune-relevant processes are playing an important role within the disease process of achalasia. However, the cellular function of the risk variants remains to be elucidated.

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Bertheloot D., Latz E.

RAGE is a cell surface nucleic acid sensor Bertheloot D.1, Sirois C.M.2,3, Miller A.L.4, Horvath G.1, Garbi N.5, Latz E.1,2 1Institute of Innate Immunity, University Hospital, University of Bonn, Germany 2Department of Medicine, Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, MA 01605, USA 3 Centers for Therapeutic Innovation, Pfizer Inc., Boston MA 02115, USA 4Division of Respiratory, Inflammation & Autoimmunity, MedImmune LLC, Gaithersburg, MD 20878, USA 5 Institutes of Molecular Medicine and Experimental Immunology (IMMEI), University Hospital, University of Bonn, Germany [email protected] BACKGROUND: RAGE is a multi-ligand receptor localized at the cell surface. RAGE ligands are structurally heterogeneous but can all bind to RAGE through electrostatic interaction with the positively charged extracellular domain of RAGE. METHODS: In this study, we used a broad panel of techniques including basic bio-chemistry, culture of stable and primary cell lines, confocal microscopy and in-vivo experiments on mice (nucleic acid inhalation). RESULTS: We show that RAGE ectodomain binds in a sequence unspecific manner to nucleic acids (NA) using shift assay and AlphaScreen methods. In co-culture, only RAGE expressing HEK293 cells bound fluorescently labeled RNA and DNA molecules while the mother cells shown no binding. To study the effect of RAGE expression on NA sensing by the endosomal TLRs, we used HEK293 cells stably expressing TLR9, TLR7 or TLR8 that we transfected with RAGE or a control plasmid together with a 5kB-gLuc reporter. We found that RAGE amplified the activation of NFkB after cell stimulation. Finally, while in WT mice, DNA inhalation induced a strong lung inflammation, RAGE-/- mice shown no sign of inflammation. CONCLUSIONS: We show that RAGE binds NAs at the cell surface, increases their uptake and thereby amplifies the endosomal TLRs activation. RAGE being highly expressed in the lung and in activated immune cells, our findings have a strong implication for the understanding of lung inflammation and its treatment.


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Bodea L.G., Neumann H.

Trem2/Dap12 pathway involvement in neurodegeneration Bodea L.G.1, Zhang B.2, Gaiteri C.2, Zhu J.2, Emilsson V.3, Neumann H.1

1Institute of Reconstructive Neurobiology, University Hospital, University of Bonn, Germany 2Ichan Institute of Genomics and Multiscale Biology, Ichan School of Medicine at Mount Sinai, USA 3Icelandic Heart Association and University of Iceland, Iceland [email protected] BACKGROUND: Loss of function of the Triggering receptor expressed on myeloid cells 2 (Trem2) or its adaptor molecule Dap12 are known to cause the human Nasu-Hakola disease, which is characterized by neuroinflammation and bone cysts. Recently, Trem2 was causally-linked to late-onset Alzheimer’s disease (LOAD) and frototemporal dementia. Mechanisms of Trem2/Dap12 mediated neurodegeneration are still unknown. METHODS: Human LOAD samples, together with in vitro and in vivo models were investigated. Microglial phenotype and cytokine profile in brains of Trem2 knock-out (KO) animals were evaluated. Genome-wide associated studies (GWAS) of LOAD and control brain samples were carried-out and microglia in vitro models were generated based on the GWAS out-come. Microglia overexpressing Dap12 variants (full length or without signaling motifs) were characterized in regard of signal transduction after amyloid-β (Aβ) application, Aβ phagocytosis capacity, neurotoxicity and superoxide production in response to Aβ. RESULTS: Trem2 KO animals presented activated-like microglial phenotype compared with appropriate wild type (WT) controls. However, only the old Trem2 KO mice showed altered cytokine profile when compared with WT controls. GWAS studies of LOAD and control brain samples revealed a specific enrichment of the immune-related module in which Dap12 was found to act as casual regulator. Next, modified microglia cell lines overexpressing full length or truncated non-functional Dap12 were generated and were able to recapitulate LOAD brains expression pattern. Full length Dap12 was specifically phagocyting Aβ, activating its signaling cascade and releasing neurotoxic reactive oxygen species. The non-functional Dap12 was unable to internalize Aβ, thus stopping the subsequent neurotoxic effects. CONCLUSIONS: Data are showing Trem2-related modulation of microglial activity, its absence leading to a proinflammatory milieu in aged animals. Based on GWAS study of LOAD brain samples, Trem2 adaptor molecule Dap12 emerged as key regulator of the disease. Functional analysis proved Dap12 involvement in Aβ-related neurotoxicity via superoxide production.

18

Böhnert V., Barchet W.

Developing a whole cell vaccine in a syngeneic mouse model of ovarian cancer Böhnert V., Stasch M., tho Pesch C., Schmalenströr M., Wigger K., Zillinger T., Hartmann G., Kübler K., Barchet W. Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital, University of Bonn, Germany [email protected] BACKGROUND: Epithelial ovarian cancer (EOC) is the deadliest gynecological cancer in developed countries, due to tumor relapse after initial surgery and chemotherapy. We have developed an EOC mouse model that allows removal of the primary tumor, and to evaluate vaccine success in tumor experienced mice. METHODS: We generated a C57Bl/6 syngeneic murine EOC cell line, expressing the Diphtheria toxin receptor and luciferase (ID8-DTRluc). By IVIS imaging, we monitor growth and recession of the tumor in vivo. We have established a whole cell vaccination protocol based on activation of the innate immune receptor RIG-I. We currently use depleting antibodies to determine the immune cell responsible for tumor rejection. RESULTS: Following three-week vaccination, we achieve long-term tumor free survival of mice. EOC cell rechallenge of vaccinated mice leads to rapid tumor rejection within one week. Effector T helper cells appear to play a crucial role for vaccination success. CONCLUSIONS: Our approach poses the opportunity to utilize patient-specific cancer antigens for vaccination. By using innate immune stimuli we overcome immune suppressive mechanisms, and direct the immune system against residual tumor cells, leading ultimately to relapse free survival.

Science Day 2013

19

Brandstätter O., Förster I.

Expression of the arylhydrocarbon receptor repressor (AhRR) in the immune system of the gut Brandstätter O., Weighardt H., Förster I.

Immunology and Environment, LIMES-Institute, University of Bonn, Germany

[email protected]

BACKGROUND: The aryl hydrocarbon receptor (AhR) represents a sensor of environmental chemicals and is expressed in many cell types, including macrophages, dendritic cells (DC), lymphocytes and innate lymphoid cells (ILC), which secrete IL-22 and control infection with intestinal pathogens. The activity of the AhR is regulated by the AhR-Repressor (AhRR) in a negative feedback loop. METHODS: Using AhRR-reporter mice, we analyzed the expression of the AhRR in the gut by flow cytometry. Furthermore, we investigated whether AhRR is functionally associated with intestinal immunity by performing dextran sodium sulfate (DSS)-evoked colitis in WT and AhRR-deficient mice. RESULTS: The AhRR is express in more than 75% of CD11c+ DC in the lamina propria (LP) of the small intestine, predominantly in the CD11c+CD103+ migratory DC subset. Moreover, the AhRR could also be detected in Foxp3+ regulatory T cells (Treg) and RORγt (retinoid-related orphan receptor γ) positive ILC in the LP. In Peyer’s patches and mesenteric lymph nodes approximately 20-30% of DC express the AhRR, whereas no AhRR+ Treg could be identified. In the colitis model, the AhRR-deficient mice were much more susceptible to DSS compared to WT littermates. CONCLUSIONS: The strong expression of the AhRR in DC, Treg and ILC of the LP and the high susceptibility of AhRR-deficient mice to DSS-induced colitis underline the importance of a feedback control of AhR activity by the AhRR for maintenance of gut homeostasis.


20

Brosseron F., Heneka M.T.

Correlation of cerebrospinal fluid cytokines with clinical markers of dementia in mild cognitive impairment and Alzheimer´s disease Brosseron F.1, Kummer M.2, Krauthausen M.2, Heneka M.T.1,2

1German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany 2Clinic and Polyclinic for Neurology, Clinical Neuroscience Unit, University Hospital, University of Bonn, Germany [email protected] BACKGROUND: Neuroinflammation is involved in many neurodegenerative diseases and suspected to play important roles at early stages of Alzheimer´s disease (AD). This study analyzed cytokine levels in cerebrospinal fluid (CSF) of patients with AD and mild cognitive impairment (MCI) and investigated their relation to diagnostic dementia markers. METHODS: CSF samples were obtained from the Clinical Neuroscience Unit. Commercial enzyme-linked-immunosorbent assays (ELISA) were used to quantify amyloid and tau markers, the cytokines IL-6, IL-18, TNF-α, MCP-1, MIF, MIG and IP-10 as well as Lipocalin-2 (NGAL). Data was analyzed using ANOVA and linear correlation analysis adjusted for multiple testing. RESULTS: Mild cognitive impairment (MCI) and AD patients were clearly distinguished from other neurological patients using the standard AD markers. Among the investigated cytokines, only TNF-α was significantly elevated in the group of MCI patients. Other cytokines did not differ between patient groups, but in part correlated to age, amyloid, tau or neuropsychological markers. CONCLUSIONS: Elevation of TNF-α in MCI indicates involvement of this cytokine in early stages of neurodegenerative diseases like AD. Further, several cytokines appear to correlate to disease progression. Our data indicate a need for in-depth longitudinal analysis of cytokines in CSF of individuals at risk of dementia.

Science Day 2013

21

Brückner M., Halle A.

Microglial dysfunction in Alzheimer’s disease Brückner M., Halle A. Max-Planck research group „Neuroimmunology“, center of advanced european studies and research (caesar), Bonn, Germany [email protected] BACKGROUND: In Alzheimer’s disease (AD) microglia, the resident immune cells of the CNS, get recruited to amyloid-β plaques and it has been shown that microglia become functionally impaired in Alzheimer’s disease mouse models. Purinergic receptors have been described as mediators of microglial chemotaxis and phagocytosis. Their role in AD remains elusive. The aim of this study is to investigate the role of microglial purinergic receptors in AD, with focus on functional changes of microglia during AD. METHODS: Microglial morphology and phagocytic capacity were assessed in acute cerebral slices of transgenic Alzheimer’s disease mice with eGFP-expressing microglia using confocal and multiphoton microscopy. This experimental set-up allowed evaluation of microglia in spatial relation to amyloid-β plaques in conjunction with pharmacological manipulation of purinergic receptors. In addition, transcriptional changes in AD were investigated by qPCR using microglial cells harvested by laser microdissection, again discriminating between plaque - associated and - distant microglia. RESULTS: Microglia from the AD mouse model showed impaired phagocytic capacity compared to wildtype animals with a striking difference between microglial cells associated with plaques and plaque-distant microglia. Phagocytic deficits in microglia could be induced by a pharmacological P2 receptor antagonist, suggesting that P2 receptor activation promotes phagocytosis. In agreement, phagocytic impairment of plaque-associated microglia was rescued by ADP, a P2 purinergic receptor agonist. Furthermore, qPCR revealed differential expression levels of purinergic receptors in microglia in spatial relation to plaques. CONCLUSIONS: The difference between the phagocytic capacity of plaque - associated and - distant microglia suggests that the activation status of these immune cells is heterogeneous and remains dynamic even during a pathologic disorder like AD. The asymmetry between plaque-associated and distant microglia was also reflected on the transcriptional level. Our data suggest that P2 microglial purinergic receptors are involved in dysregulation of microglial phagocytosis in AD. Thus, P2 purinergic receptors, may serve as a target for pharmacological treatment given that reduced phagocytic capacity of microglia in AD could promote formation of amyloid-β plaques.

22

Cavlar T., Hornung V.

STING-dependent type I IFN induction by the small-molecule compound CMA Cavlar T.1, Deimling T.2, Ablasser A.1, Hopfner K-P.2, Hornung V.1 1Institute for Clinical Chemistry and Pharmacology, Unit for Clinical Biochemistry, University Hospital, University of Bonn, Germany 2Gene Center and Department of Biochemistry, Ludwig-Maximilians-University Munich, Germany [email protected] BACKGROUND: The detection of conserved microbial structures by pattern recognition receptors of the innate immune system initiates antimicrobial defense mechanisms that are geared to initiate immediate effector functions and to induce adaptive immune responses. In this respect, type I interferons (IFNs) play an important role. Therefore, a well-defined type I IFN inducer is desirable, as this would allow targeted clinical applications. METHODS: We performed in vitro studies in primary cells to delineate the mode of action of the type I IFN inducer CMA. Results were validated in reconstitution assays and virus infection experiments. Differential scanning fluorimetry and crystallographic studies were conducted to characterize the receptor-ligand interaction. RESULTS: CMA strongly triggers type I IFN through robust activation of the STING/TBK1/IRF-3 route. Despite its extraordinary antiviral activity in the murine system, CMA fails to trigger immune responses in human cells. Binding studies indicate that this species-specific phenomenon originates from its inability to bind to the C-terminal domain of human STING. The co-crystal structure shows that two CMA molecules are located in the central ligand-binding pocket of the STING dimer. CONCLUSIONS: Altogether, these results reveal a novel ligand-sensing mode of STING, opening new avenues for therapeutic applications and, furthermore, they unravel unexpected species-specific differences of this innate sensor.

Science Day 2013

23

Claude J., Neumann H.

Neuroprotective properties of the microglial receptor Siglec-E Claude J., Linnartz-Gerlach B., Kudin A., Kunz W., Neumann H.

Institute of Reconstructive Neurobiology, University Hospital, University of Bonn, Germany [email protected] BACKGROUND: Sialic acid-binding immunoglobulin like lectins (Siglecs) are microglial surface recognition receptors to sense intact or lesioned cells. Siglec-E, a member of this family, has an immunoreceptor tyrosine based inhibitory motif to suppress activatory signals. METHODS: Ex vivo, primary and stem cell-derived microglia were analysed with qRT-PCR and flow cytometry for Siglec-E transcription and expression. For functional analysis, lentiviral knock-down and overexpression were performed. In a co-culture with neurons the implication of Siglec-E in neuroinflammation was studied. RESULTS: RT-PCR and flow cytometry showed Siglec-E expression on microglia and up-regulation following IFN-γ treatment. Overexpression of Siglec-E decreased whereas knock-down increased neural debris phagocytosis and superoxide production. A Siglec-E:Fc fusion protein bound to sialic acid residues of the neuronal glycocalyx. Therefore, primary neurons were co-cultured with transduced microglia. Overexpression and knock-down of Siglec-E led to an increase and decrease in relative neurite length, respectively. The neuroprotective effect of Siglec-E was abrogated after removal of the sialic acid residues on the neuronal glycocalyx. Treatment with the anti-oxidant Trolox abolished the neurotoxic effect of the Siglec-E knock-down. CONCLUSION: Data suggest an immunomodulatory function of Siglec-E on microglia leading to a neuroprotective phenotype by decreasing the production of reactive oxygen species and a reduced phagocytosis rate of neural debris.


24

Daßler J., Coch C.

RIG-I induced Melanoma-derived Exosomes carry BAG6 and 3pRNA thereby triggering an innate immune response in vitro and a potent anti-tumor response in vivo Daßler J.1, Reiners K.S.2, van den Boorn J.G.1, Schlee M.1, Hartmann G.1, Pogge von Strandmann E.2, Coch C.1

1Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital, University of Bonn, Germany 2Department of Internal Medicine I, University Hospital, University of Cologne, Germany [email protected] BACKGROUND: Exosomes are vesicles 50-120nm in size, derived from the endosomal compartment and have been shown to act as signaling vehicles by transferring functional active molecules (like miRNA and mRNA) to distant cells. Exosomes released from tumor cells promote tumor growth by influencing tumor cells and cells of the tumormicroenvironment. RIG-I is an innate immune receptor responsible for the detection of ssRNA viruses. It recognizes viral 5`-triphosphorylated RNA (3pRNA) present in the cytosol of infected cells. RIG-I is widely expressed in all nucleated cells, including tumor cells. METHODS: In this study we investigate the impact of RIG-I on the release and function of melanoma derived exosomes. Therefore we transfected 3pRNA into melanoma cells and isolated tumorexosomes (3p-exo) from the supernatant. Afterwards we analyzed 3p-exo regarding their immune-modulatory potential in vitro and in vivo. RESULTS: 3p-exo induced antiviral cytokines and chemokines, including type I IFN, in immune and tumor cells in vitro by transfer of RIG-I activating 3p-moieties. Additionally, 3p-exo expressed BAG6 which activated NK cells to lyse melanoma cells via NKp30. Intratumoral injections of these RIG-I ligand/BAG6 carrying melanoma exosomes into a subcutaneous transplanted mouse melanoma model led to an NK cell dependent suppression of tumor-growth in vivo. CONCLUSIONS: We show that activation of RIG-I turns immunosuppressive tumor exosomes into potent anti-tumor effectors transferring a number of functional molecules including RIG-I ligand and BAG6 to innate effector and tumor cells. Our results identify a novel pathway how antiviral danger signals spread between cells and demonstrate how this can be used as therapeutic strategy in the treatment of cancer.

Science Day 2013

25

Dixit A., Engel D.

Role of Gr1+ inflammatory macrophages in pyelonephritis-induced fibrosis

Dixit A., Krause T., Kurts C., Engel D.

Institute of Experimental Immunology and Molecular Medicine, University Hospital, University of Bonn, Germany

[email protected]

BACKGROUND: Urinary tract infections (UTI) such as cystitis, urethritis and pyelonephritis are one of the most common bacterial infections worldwide. They are caused by Uropathogenic strain of E. Coli (UPEC). Chronic pyelonephritis results in inflammation, acute fever, nausea and diarrhea and in long-term and inevitably renal fibrosis and furthermore kidney failure. Macrophages have been known to play an important role in fibrosis of other organs but their role in pyelonephritis- induced fibrosis is still unclear.

METHODS: We have developed a mouse model for pyelonephritis-induced fibrosis. Renal leukocytes were analyzed using flow cytometry or histology.

RESULTS: Using this model, we could show that Gr1+ inflammatory macrophages accumulate 1-day post infection and decrease until day 6 in the infected kidneys. In contrast, Gr1- macrophages do not accumulate on day 1, but increase until day 6 post infection. CCR2 KO animals, which lack Gr1+ macrophages, showed a reduced accumulation of Gr1- macrophages within the kidney. Such reduction was not due to reduced proliferation indicating that differentiation of Gr1+ macrophages into Gr1- macrophages took place. Furthermore, CCR2 KO mice showed increased bacterial load, reduced fibrosis and improved kidney function.

CONCLUSIONS: These data indicate an important role of Gr1+ macrophages in the defense against UPECs and in the development of pyelonephritis induced renal fibrosis.

26

Eckerle I, Drosten C.

Epithelial cell lines from bats and rodents as a novel tool for the study of zoonotic viruses Eckerle I., Ehlen L., Müller M.A., Kallies R., Drosten C. Institute of Virology, University Hospital, University of Bonn, Germany [email protected] BACKGROUND: Virus research on emerging zoonotic diseases has focused on small mammals over the past decade, leading to an increasing number of newly discovered pathogens from a variety of wildlife species, mainly bats, rodents and insectivores. However until now, attempts to isolate these novel viruses in cell culture have been largely unsuccessful. Moreover, experimental studies on them have been limited by the difficulty of rearing host species and the lack of suitable in-vitro model systems. Especially epithelial cells are an important target within the host organism involved in virus entry, replication and shedding. Even though several cell lines from wildlife species have been established recently, there has been no focus to selectively culture epithelial cells from important reservoir species such as bats or rodents. METHODS and RESULTS: We developed methods to establish epithelial cell lines of the respiratory and renal tract from a variety of tropical and temperate bat and rodent species that are associated with zoonotic viruses (examples of successfully established bat and rodents cell lines are Rhinolophus spp., associated with SARS-Coronavirus; Pipistrellus spp., associated with the novel MERS-Coronavirus; Myodes glareolus, reservoir of the Old World hantavirus Puumala or Sigmodon hispidus, reservoir of the New World hantavirus Black Creek Canal virus). Cells were successfully cultured under standardized conditions from both fresh and frozen organ specimens and immortalized for the generation of permanent cell lines. Preliminary results show the ability of a subset of cells to polarize in filter inserts, determined by increase of trans-epithelial resistance and expression of tight junction proteins. Virus infection experiments revealed susceptibility to virus species from important zoonotic virus families such as Bunya-, Rhabdo- and Flaviviridae and from newly identified viruses that were refractory to conventional cell culture. CONCLUSIONS: So far, the exciting field of species-specific cell lines is still in its early days. Our results show that species-specific cell culture models are a valuable tool for the characterization of zoonotic viruses and provide a new method to study pathogen-host interaction of wildlife-derived viruses.

Science Day 2013

27

Engel C., van den Boorn J.G.

Retinoic acid-inducible gene-I (RIG-I)-mediated immune activation is attenuated by hypoxia in murine melanoma cells Engel C.1, Horváth D.1, Wenzel D.2, Hartmann G.1, van den Boorn J.G.1 1Institute for Clinical Chemistry and Clinical Pharmacology, University Hospital,

University of Bonn, Germany 2Institute of Physiology I, Life and Brain Center, University of Bonn, Germany [email protected] BACKGROUND: A common factor negatively influencing anti-tumor chemo- and irradiation therapy outcome is the hypoxia in many solid tumors. Yet, the influence of hypoxia on innate immune activation is largely unknown, in particular considering intratumoral triggering of cytosolic pattern-recognition receptors like RIG-I. We here investigated the direct influence of hypoxia on RIG-I function. METHODS: B16.F10 melanoma cells were incubated at normoxia (pO2=21%) and hypoxia (pO2=2%) and stimulated with the RIG-I ligand 5’-triphosphorylated RNA (3pRNA). RESULTS: Hypoxic cells upregulated RIG-I and CXCL10 protein significantly less upon 3pRNA stimulation while producing more ROS than normoxic cells. This is in contrast to previous findings that ascribe a RIG-I-like receptor enhancing function to ROS. Further type-I interferon protein secretion was enhanced under hypoxia, but lost its autocrine stimulatory potential due to a reduction of the IFNα receptor in the hypoxic B16 cells. In co-cultures of melanoma cells with Pmel-1 splenocytes under hypoxia CD8+ T cells showed significantly depressed activation in response to previously 3pRNA-triggered melanoma cells. CONCLUSIONS: Our results for the first time indicate the direct modulating effect of hypoxia on RIG-I function. These findings are of pivotal importance to the development of cancer immunotherapies that rely on RIG-I triggering within the tumor milieu.

28

Eppler F., Kolanus W.

Role of Dynamin 2 in Rap1-dependent lymphocyte adhesion Eppler F., Kolanus W. Molecular Immunology, LIMES Institute, University of Bonn, Germany [email protected] BACKGROUND: Immune responses strongly rely on an accurate regulation of leukocyte adhesion and migration. These complex processes are controlled by numerous proteins. One of those is the small GTPase Rap1, which is known to regulate integrin-activity and cell-polarization in leukocytes. In contrast, the large GTPase Dynamin 2, mainly studied for its role in endocytosis, has not yet been implicated in the biology of immune cell adhesion. METHODS: We performed different in vitro analyses of lymphocyte dynamics based on live cell imaging techniques, including adhesion under flow and static conditions, migration assays in different environments and, using confocal laser scanning microscopy, dynamics and distribution of fluorescently labeled proteins. Additionally applied methods include FACS, qRT-PCR, pull-down assays and western blotting. As a model system we mainly used primary human resting CD4+ T cells. The GTPase-activity of Dynamin 2 was blocked using the Dynamin-specific inhibitor Dynasore. RESULTS: Dynasore-mediated inhibition of Dynamin 2 led to a massive adhesion-deficiency in T cells and integrin-dependent migration was strongly reduced as well. Interestingly, integrin-macroclusters found on control cells were absent on cells incubated with the Dynamin-inhibitor. Furthermore, we found that the activation of Rap1 was drastically reduced in Dynasore-treated lymphocytes and that their adhesion-deficiency could be rescued by the overexpression of a constitutively active Rap1 mutant.

CONCLUSIONS: Here, we could identify Dynamin 2 as an entirely new modulator of Rap1-activity and thereby of integrins, which serve not only as mechanical anchors, but also as environment-sensing receptors in leukocytes.


Science Day 2013

29

Gioran A., Bano D.

Neuronal branching associated with mitochondrial deficiency in C. elegans Gioran A., Nicotera P., Bano D. German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany [email protected] BACKGROUND: Mitochondria are important organelles in all cell types and particularly in neuronal cells. Mitochondria provide most of the energy required for proper neuronal function, remain dynamic through fusion and fission cycles and cover big distances in those highly polarized cells. Any decline of their normal functional properties can lead to neuronal dysfunction. Indeed, events affecting any of the above aspects of mitochondrial function can lead to severe damage of neurons and their dendrites. Although studies have correlated mitochondrial dysfunction with neurodegeneration, little is known about the molecular mechanisms underlying this correlation. In this study we focus on the impact of mitochondrial deficiency on the dendrite morphology of the neurons in order to elucidate the underlying molecular mechanisms. METHODS: In this study, we use Caenorhabditis elegans as a model to study neuronal arborization in conditions of oxidative phosphorylation (OXPHOS) impairments. C. elegans is a transparent nematode with a well characterized nervous system, and the use of a genetic approach along with high resolution confocal microscopy allows the dissection of the molecular mechanisms underlying alterations of neuronal branching caused by OXPHOS defects. RESULTS: Our data show that animals with defective OXPHOS display abnormal and disorganized neuronal branching pattern. Furthermore, our data suggest that the remodeling of neuronal protrusions requires the activity of the nutrient sensor AMP-activated-protein kinase (AMPK). CONCLUSIONS: Our findings indicate a signaling cascade involved in the outgrowth and maintenance of dendritic arborizations in conditions of compromised OXPHOS.


30

Glodde N., Hölzel M.

A “modular” murine melanoma model to study determinants of multimodal immunotherapeutic regimens Glodde N.2, v.d. Boorn-Konijnenberg D.1, Bald T.2, Tüting T.2, Hölzel M.1 1Institute for Clinical Chemistry and Clinical Pharmacology, University Hospital, University of Bonn, Germany 2Laboratory for Experimental Dermatology, University Hospital, University of Bonn, Germany [email protected] Malignant melanoma is a highly aggressive skin cancer notoriously known for its chemoresistance and poor prognosis upon metastatic dissemination. Significant breakthroughs in the treatment of this devastating disease have been achieved in the recent years. Pharmacological inhibition of the BRAF kinase harboring activating mutations (BRAFV600E) in about the half of all melanoma cases results in profound clinical responses and prolonged overall survival rates. Furthermore, antibody-mediate blockade of negative immune checkpoint molecules like CTLA-4 and in particular the PD1/PD-L1 axis demonstrated high efficacy in clinical trials. As such, malignant melanoma is a paradigm disease to study the synergism of combined tumorbiological and tumorimmunological multimodal treatment approaches that raises the hope to provide a more durable barrier to therapy resistance. We exploited the oncogene addiction of our HgfxCdk4R24C murine melanoma model and CRIPSR/Cas9 genome editing to establish a rapid pipeline to probe clinically relevant determinants of responsiveness to multimodal immunotherapeutic regimens. Preliminary results will be presented.


Science Day 2013

31

Goldeck M., Hornung V.

cGAS produces a 2’-5’-linked cyclic dinucleotide second messenger that activates STING Goldeck M.1*, Ablasser A.1*, Cavlar T.1, Deimling T.2, Witte G.2, Röhl I.3, Hopfner K-P.2,4, Ludwig J.1, Hornung V.1

1Institute for Clinical Chemistry and Clinical Pharmacology, University Hospital, University of Bonn, Germany 2Department of Biochemistry and Gene Center, Ludwig-Maximilians-University, Munich, Germany 3Axolabs GmbH, Kulmbach, Germany 4Center for Integrated Protein Sciences, Munich, Germany [email protected] BACKGROUND: Detection of cytoplasmic DNA represents one of the most fundamental mechanisms of the innate immune system to sense the presence of microbial pathogens. The ER-resident protein STING is critically required for the initiation of type I interferon signalling upon detection of cytosolic DNA of both exogenous and endogenous origin. Next to its pivotal role in DNA sensing, STING also serves as a direct receptor for the detection of bacteria-derived cyclic dinucleotides. DNA recognition, however, is triggered in an indirect fashion that depends on a recently characterized cytoplasmic nucleotidyl transferase, termed cGAMP synthase (cGAS), which upon interaction with DNA synthesizes a dinucleotide molecule that in turn binds to and activates STING. METHODS: The dinucleotide molecule produced by cGAS in in vivo and in vitro reactions was analysed by a combinatorial approach based on mass spectrometry, enzymatic digestion, chemical synthesis and NMR. RESULTS: We show that the cGAS-catalysed reaction product is distinct from previously characterized cyclic dinucleotides: cGAS produces a cyclic GMP-AMP dinucleotide, which is comprised of a 2’-5’ and a 3’-5’ phosphodiester linkage >Gp(2’-5’)Ap(3’-5’)>. We found that the presence of this 2’-5’ linkage was required to exert potent activation of human STING. Moreover, we show that cGAS first catalyzes the synthesis of a linear 2’-5’ linked dinucleotide, which is then subject to cGAS-dependent cyclization in a second step through a 3’-5’ phosphodiester linkage. CONCLUSIONS: Altogether, the characterization of this 13-membered ring structure defines a novel class of second messenger molecules, extending the family of 2’-5’ linked antiviral biomolecules.


32

Gondorf F., Hübner M.P.

Chronic exposure to filaria and its endosymbiotic Wolbachia bacteria improves E. coli induced sepsis in a TLR2 dependent manner Gondorf F., Hoerauf A., Hübner M.P. Institute for Medical Microbiology, Immunology and Parasitology, University Hospital, University of Bonn, Germany [email protected] BACKGROUND: Helminths modulate the immune system of their hosts and induce a regulatory, anti-inflammatory milieu that enables long-term parasite survival within the host, but also benefits the host. Thus, helminth infections have been shown to improve autoimmune diseases. METHODS: In the current study we investigated whether chronic infection with the filarial nematode Litomosoides sigmodontis (L.s.) improves the outcome of an acute systemic inflammation caused by i.p. E. coli injection in BALB/c mice. RESULTS: Chronic L.s. infection significantly improved E. coli-induced hypothermia, bacterial clearance and sepsis survival. The L.s.-mediated protection correlated with reduced concentrations of pro-inflammatory cytokines and chemokines in serum and peritoneum and increased numbers of alternative activated macrophage (AAM) after E. coli challenge. Improved bacterial clearance in L.s.-infected animals was dependent on macrophages but not AAM. Endosymbiotic Wolbachia bacteria, that are present in most human pathogenic filaria and L. sigmodontis, are sensed by TLR2 and may induce cross-tolerance to TLR4 stimuli. Accordingly, in vitro experiments revealed that pretreatment of macrophages with L.s. antigen reduced LPS-induced activation in a Wolbachia- and TLR2-dependent manner and L.s.-mediated protection against E. coli challenge was not given in TLR2-deficient mice. CONCLUSIONS: Chronic filarial infection reduces E. coli-induced severe systemic inflammation and improves sepsis survival in a TLR2-dependent manner. We propose that exposure to endosymbiotic Wolbachia bacteria prevents E. coli-induced excessive macrophage activation by a TLR cross-tolerance mechanism.


Science Day 2013

33

Görgen S., Latz E.

Identification of novel TAM receptor interactors via label transfer: Tagging cross-linker to bait proteins via Sortase A Görgen S., Latz E. Institute of Innate Immunity, University Hospital, University of Bonn, Germany [email protected] BACKGROUND: Label transfer is a powerful tool to identify weak or transient protein-protein interactions. Identifying novel ligands of the TAM receptor tyrosine kinases TYRO3, AXL, and MER is of immunologic interest as they regulate immune responses by inhibiting TLR signaling. METHODS: The cross-linker NVOC-Biotin-S-S-LPXTGG was tagged to TAM receptor ectodomain-Fc fusion proteins via Sortase A (Staphylococcus aureus). The NVOC tagged ectodomain fusion protein is then used for fishing in complex samples, where the biotin will be transferred to interacting molecules by photo-activation and reduction of the disulfide bond. RESULTS: Co-IP experiments using human plasma comparing untagged TAMecto-Fc proteins to Fc-only control have shown binding protein bands in silver stained SDS-PAGE gels. To identify transient interacting proteins and low abundance proteins too weak to be seen by Co-IP, we used Sortase A to tag the cross-linker NVOC to the TAMecto-Fc. The NVOC-TAMecto-Fc protein was then used for fishing in complex samples where the biotin is transferred via the cross-linker selectively to interacting molecules. The labeled proteins remain to be identified. CONCLUSIONS: To fully utilize the potential of the technique the labeled proteins will to be quantitatively analyzed and identified via mass spectrometry. In future this technique to identify interacting proteins will be applied to a broader range of immunologically interesting receptors.


34

Grebe A., Latz E.

Cyclodextrin promotes atherosclerosis regression by dissolution of cholesterol crystals and LXR activation

Grebe A.1*, Zimmer S,2*, Bode N.2*, De Nardo D.1, Labzin L.I.1, Kerksiek A.3, Niyonzima N.4, Espevik T.4, Hempel C.5, Heneka H.T.6, Gustafsson J-A.7, Ulas T.8, Schultze J.L.8, Wright S.D.9, Nickenig G.1, Lütjohann D.3, Latz E.1, 4,10,11 1 Institute of Innate Immunity, University Hospital, University of Bonn, Germany 2 Medizinische Klinik und Poliklinik II, University Hospital, University of Bonn, Germany 3 Institute of Clinical Chemistry und Clinical Pharmacology, University Hospital, University of Bonn, Germany 4 Centre of Molecular Inflammation Research at the Norwegian University of Science and Technology, Trondheim, Norway 5 Addi and Cassi Fund, Reno, NV, USA 6 Clinic and Polyclinic for Neurology, University Hospital, University of Bonn, Germany 7 Center for Nuclear Receptors and Cell Signaling, University of Houston, Houston, TX, USA 8 Genomics and Immunoregulation, Life and Medical Sciences Institute (LIMES), University of Bonn, Germany 9 CSL Behring, King of Prussia, PA, USA

10 Department of Infectious Diseases and Immunology, UMass Medical School, Worcester, MA, USA 11 German Center of Neurodegenerative Diseases (DZNE), Bonn, Germany *equal contribution

[email protected] BACKGROUND: Inflammation is the driving force in the pathogenesis of atherosclerosis. Cholesterol crystals (CCs) are a hallmark of atherosclerotic lesions and represent a potent inflammatory stimulus. 2-Hydroxypropyl-β-cyclodextrin (CD), a clinically used oligosaccharide, enhances cholesterol solubility and mediates cholesterol depletion from cell membranes. In an atherosclerosis mouse model, CD treatment was found to mediate atherosclerosis regression. Thus, we investigated the mechanism of CD function in murine atherosclerosis.

METHODS: Isotope labeled CC and GC-MS-SIM analyses were used to assess crystal-derived cholesterol metabolism and localization. Reverse cholesterol transport from macrophages loaded with isotope-labeled CC was analyzed in C57BL/6 mice. CD-induced transcriptional changes in CC loaded macrophages were assessed by genome-wide mRNA profiling.

RESULTS: In vitro, CD solubilized CCs and promoted cholesterol ester and oxysterol production in macrophages resulting in liver X receptor (LXR)-mediated transcriptional reprogramming. CD increased cholesterol efflux from macrophages and enhanced reverse cholesterol transport in vivo. Furthermore, CD had anti-inflammatory effects on macrophages.

CONCLUSIONS: These data suggest that CD’s beneficial effects on murine atherosclerosis are mediated by its ability to dissolve CCs and induce the metabolism and efflux of crystal-derived cholesterol from macrophages in vitro and in vivo. Thus, CD may be used in clinical trials to pharmacologically reduce atherosclerosis burden.

Science Day 2013

35

Hanin G., Soreq H.

Competing targets of microRNA-608 modulate risks of hypertension and anxiety UHanin G.UP

1P, Shenhar-Tsarfaty S.P

1P, Yayon N. P

1P, Yin Hoe Y. P

2P, Bennett E.R. P

1P, Sklan

E.H.P

3P, Rao D. C. P

4P, Rankinen T.P

5P, Bouchard C. P

5P, Geifman-Shochat S.P

2P, Shifman

S.P

1P, Greenberg D.S. P

1P ,USoreq H. UP

1

P

1PThe Silberman Institute of Life Sciences, The Hebrew University of Jerusalem,

Givat Ram, Jerusalem 91904, Israel, and The Edmond and Lily Safra Center for Brain Science. P

2 PSchool of Biological Sciences, Nanyang Technological University, 60 Nanyang

Avenue, Singapore 637551 P

3 PDepartment of Clinical Microbiology and Immunology, Sackler School of

Medicine, Tel Aviv University, Tel Aviv 69978, Israel, P

4 PDivision of Biostatistics, School of Medicine, Washington University in St.

Louis, St. Louis, MO, USA. P

5 PHuman Genomics Laboratory, Pennington Biomedical Research Center, Baton

Rouge, LA, USA [email protected] BACKGROUND: MicroRNAs (miRNAs) can repress multiple targets, but how a single de-balanced interaction affects others remained unclear. METHODS and RESULTS: Here, we report that changing a single miRNA-target interaction controlling cholinergic signaling can simultaneously affect multiple other miRNA-target interactions, increasing both anxiety and hypertension. We show that the primate-specific miR-608 targets acetylcholinesterase (AChE). Surface Plasmon Resonance (SPR) tests demonstrated weakened miR-608 interaction with the rs17228616 AChE allele having a single nucleotide polymorphism (SNP) in the 3’-untranslated region (3’UTR). In cultured human cells, this weakened interaction potentiated miR-608-mediated suppression of other targets, including the RhoGTPase CDC42 and the cytokine interleukin-6 (IL6). Moreover, post-mortem human cortical tissues from volunteer homozygotes for the minor rs17228616 allele showed AChE elevation and CDC42/IL6 decreases compared to major allele homozygotes, and young U.S. healthy volunteers carrying the minor rs17228616 allele showed reduced serum cortisol and elevated systolic and diastolic blood pressure, predicting heightened risk of anxiety and hypertension-related diseases. Supporting this notion, parallel suppression of the evolutionarily conserved brain CDC42 activity by intracerebroventricular ML141 injection caused acute anxiety in mice.

CONCLUSIONS: Our findings demonstrate that single SNPs in primate-specific miRNA-binding regions could cause expanded downstream effects changing important biological pathways, jointly elevating hypertension and anxiety and introducing risk of major diseases.


36

Hemmerling I., Hornung V.

Establishment of a STING reporter cell line for studying the molecular mechanism of intracellular DNA sensing Hemmerling I.1, Schmid-Burgk J.1, Horvath G.2, Latz E.2, Ablasser A.1, Hornung V.1 1 Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital, University of Bonn, Germany 2 Institute of Innate Immunology, University Hospital, University of Bonn,

Germany

[email protected] BACKGROUND: The cytosolic recognition of nucleic acids is central for the initiation of an innate immune response against a broad range of microbes. The nucleotidyltransferase cGAS, its 2nd messenger molecule cyclic GAMP(2´-5´) and the host protein STING have been recently identified as central components of a signaling pathway that links the sensing of intracellular DNA with the induction of antiviral and pro-inflammatory cytokines. METHODS: Cell culture, In vitro stimulation assays, Immunoblot, Confocal Microscopy RESULTS: We have generated a STING reporter cell line, which stably expresses a mCherry-tagged STING fusion construct. Upon stimulation STING re-localizes to perinuclear organelles, an event that could be tracked via fluorescence imaging and which correlated with the activation of downstream signaling. Of note, using STING reporter cells we were able to identify a novel intercellular route of signaling transduction: upon recognition of intracellular DNA, cGAS-produced cGAMP is transported via gap junctions to bystander cells, where it activates STING and thereby initiates an antiviral state*. CONCLUSIONS: HEK STING cells are highly valuable tool for studying STING activation at a single cell level in living cells. For future studies, we aim to implement STING reporter cells for image-based high-throughput screens to identify novel components and endogenous regulators of the STING-based intracellular recognition pathway.

Science Day 2013

37

Herzner A.M., Schlee M.

Post-transcriptional modulation of the type I interferon response by minimal-motif oligodeoxynucleotides Herzner A.M., Keßels S., Hartmann G., Schlee M. Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital, University of Bonn, Germany [email protected] BACKGROUND: Immunostimulatory nucleic acids (ISN) activate innate immune receptors in the endolysosome and the cytosol leading to the release of type I IFNs (IFN-α/β), chemokines and pro-inflammatory cytokines. We made the unexpected observation that cytosolic A-type oligodeoxynucleotide (ODN) CpG2216 (TLR9-ligand) suppressed type-I-IFN-secretion but not the release of the chemokine IP10 in human monocytes. METHODS: Monocytes were stimulated with combinations of cytosolic ISN and CpG2216-like ODN and cytokine mRNA expression and secretion were analysed by RT-PCR and ELISA. Furthermore, protein translation was monitored by polysome profiling. Phosphorylation of proteins involved in DNA damage and translation regulation was detected by western blot. RESULTS: Immunomodulation by CpG2216-like ODN (iODN) was independent of TLR9-activating CpG-motifs but required dG-rich ends. Unexpectedly, the induction of IFN-α mRNA was unaltered, indicating post-transcriptional regulation. Polysome profiles revealed a global translation inhibition induced by cytosolic iODN. Quantitative analysis of transcript distribution within the fractions indicated differential translation of type I IFN and IP10 transcripts. This immunomodulation was not accompanied by changes of eIF2-α-, 4EBP- or checkpoint kinase phosphorylation. CONCLUSIONS: Our study demonstrates (I) a novel, post-transcriptional regulation of the type I IFN-response and (II) the induction of a global translation inhibition induced by small ODN comprising dG-rich ends.

38

Hückesfeld S., Pankratz M.

Central Neurons modulate Feeding Behavior in Drosophila: Potential Role in Pathogen Avoidance Hückesfeld S., Schlegel P., Pankratz M. Department of Brain Physiology and Behavior, LIMES Institute, University of Bonn, Germany [email protected] BACKGROUND: Defense against pathogens can be brought about by immune response as well as changes in behavior. Active avoidance of pathogenic substrate was shown to be an important way to avoid harmful infections. A comparable avoidance behavior was observed in Drosophila larvae when manipulating activity of a central cluster of Hugin producing neurons. It’s mammalian homologue NeuromedinU was previously been shown to be involved inflammatory processes. METHODS: Behavioral and electrophysiological experiments were conducted to study the effects of manipulation of neuronal activity of the Hugin cluster on larval behavior. In order to create a connectivity map of the inputs and outputs of the Hugin circuitry, serial EM-based reconstruction of the entire CNS was used. RESULTS: In behavioral experiments, activation of Hugin neurons led to severe decrease in food intake, enhancement of locomotor activity and subsequent escape from the food source. In-depth electrophysiological recordings revealed a decrease in motor activity of the pharyngeal pump and increased activity of abdominal muscles. Connectomic analysis identified potential gustatory input onto parts of the Hugin circuit as well as direct connections to premotor neurons of the pharyngeal pump. CONCLUSIONS: We describe a cluster of central neurons whose activation lead to abolishment of feeding and escape from food source. This shows similarities to pathogen avoidance behavior in C.elegans. Next, we want to test the hypothesis of a potential involvement of Hugin in pathogen avoidance response in Drosophila.


Science Day 2013

39

Jakobs C., Hornung V.

Impact of AIM2-Inflammasome activation on adaptive immune responses after cutaneous DNA-Vaccination Jakobs C.1, Tüting T.2, Hornung V.1

1Institute of Clinical Chemistry and Pharmacology, Unit for Clinical Biochemistry, University Hospital, University of Bonn, Germany 2Department of Dermatology and Allergology, University Hospital, University of Bonn, Germany [email protected] BACKGROUND: The skin provides our first line of defense against microbial pathogens. Beside its function as a physical barrier, the broad expression of pattern recognition receptors, highlights its pivotal role as an organ of the immune system. In this context we are interested in the role of inflammasome activation in response to DNA recognition within the skin. METHODS: We used ballistic DNA vaccination to investigate the immune responses in wildtype and inflammasome knockout-mice. The innate immune response was assessed by histology and cytokine expression. Moreover, the use of plasmid-DNA encoding for antigens, allowed us to investigate adaptive immune responses by measuring CTL frequency and killing activity. RESULTS: DNA vaccination led to the induction of pro-inflammatory cytokines and infiltration of immune cells and induction of adaptive immune responses. Surprisingly, those effects were strongly enhanced in AIM2-deficient mice in comparison to wild type mice. CONCLUSIONS: Taken together these data show that cutaneous DNA vaccination leads to DNA-dependent cytokine production and immune cell infiltration, as well as induction of cellular immunity to plasmid-encoded antigens. We hypothesize that the increased innate and adaptive immune response in the absence of AIM2 is caused by a negative regulatory effect of the AIM2-ASC-caspase-1 axis on the IFN-pathway in keratinocytes.

40

Janas M., Wohlleber D.

Mitochondria play a central role in a novel innate immune sensing mechanism of viral infection Janas M.1, Kashkar H.2, Heikenwälder M.3, Krönke M.2, Knolle P.A.1, Wohlleber D.1

1Institute of Molecular Medicine, University Hospital, University of Bonn, Germany 2Institute of Medical Microbiology, Immunology and Hygiene, University Hospital, University of Cologne, Germany 3Institute of Virology, Technische Universität München, Germany [email protected] BACKGROUND: In a new CTL effector mechanism against viral infections of the liver, secreted TNF induces selective clearance of infected hepatocytes, whereas uninfected cells are protected. Here, we investigate the molecular mechanism(s) underlying the sensitization of infected hepatocytes towards the apoptosis-inducing effect of TNF. METHODS: In our model of viral hepatitis mice infected with a hepatotropic adenovirus are challenged with recombinant TNF. By using biochemical and histochemical methods we analyze checkpoints in TNF apoptosis and survival signaling. RESULTS: We found that only in infected hepatocytes an activation of the initiator caspase 8 was triggered by TNF, which subsequently induced Bid cleavage and culminated in effector caspase 3 activation. Furthermore viral infection directly interfered with mitochondrial vulnerability. It caused an imbalance of pro- and antiapoptotic Bcl-2 proteins that regulate mitochondrial integrity. This came along with increased sensitivity of mitochondria towards apoptotic stimuli like activated caspase 8. CONCLUSIONS: Viral infection sensitizes hepatocytes towards TNF induced apoptosis by interfering with TNFR signaling at the level of mitochondrial fragility as well as initiator caspase activation. Hence, we identified a novel innate immune sensing mechanism that is independent of antigen presentation on target cells and classical innate immune recognition pathways and thereby may overcome viral immune escape in infected cells.

Science Day 2013

41

Kaczmarek J., Diehl L.

Regulation of CD8 T cell function by the small GTPase Arl4d Kaczmarek J.1, Tolksdorf F.2, Rieck M.2, Metzger C.1, Kolanus W.2, Diehl L.1

1Institute of Molecular Medicine and Experimental Immunology 2Molecular Immunology, LIMES Institute, University of Bonn, Germany [email protected] BACKGROUND: The liver is known to induce tolerance rather than immunity. Liver sinusoidal endothelial cells (LSEC) can induce a non-responsive state in naïve CD8 T cells after antigen-specific stimulation. Members of the ADP-ribosylation factor (ARF) family of guanine-nucleotide-binding (G) proteins, including the ARF-like (ARL) proteins, regulate membrane traffic and organelle structure by recruiting cargo-sorting coat proteins, modulating membrane lipid composition and interacting with regulators of other G proteins. Arl4D is localized to the plasma membrane and can recruit cytohesin-2/ARNO and modulate actin remodeling via regulating ARF6 activity. METHODS: We performed T cell stimulation assays in vitro and analyzed IL-2 production and expression of Arl4d in relation to interleukin-2 inductions. We further studied in vivo T cell activity in response to an adenoviral infection in the presence or absence of Arl4d. RESULTS: We found Arl4d mRNA to be highly induced during CD8 T cell stimulation by LSEC. Using CD8 T cells from Arl4d-/- mice we found the lack of Arl4d to result in the overproduction of IL-2. Moreover, arl4d was involved in regulating expansion of anti-viral CD8 T cells, as Arl4d-deficient CD8 T cells expanded extensively during adenoviral infection and increased amounts of IL-2-producing T cells could be found accumulated in the spleen, but more pronounced in the liver of infected mice. CONCLUSIONS: Arl4d represses IL-2 production and expansion of naïve CD8 T cells during viral infection in vivo. Thus, the action of Arl4d may be central to dampen IL-2 production and for subsequent abrogation of immediate effector function in naïve CD8 T cells by LSEC.


42

Kim S., Schumacher J.

Unraveling the genetic basis of innate immune response in TLR4-activated human monocytes Kim S.1,2, Bechheim M.2, Becker J.1, Pütz B.3, Hornung V.2, Schumacher J.1

1Institute of Human Genetics, University Hospital, University of Bonn, Germany 2Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital, University of Bonn, Germany 3Statistical Genetics, Max-Planck-Institute of Psychiatry, Munich, Germany [email protected] BACKGROUND: Toll-like receptors (TLRs) play a key role in innate immunity. Apart from their function in host defense, dysregulation in TLR-signaling can confer risk to autoimmune diseases, septic shock or cancer. Despite major advancements in our understanding of how the innate immune system recognizes pathogens, the genetic basis for differences in innate immune responses is only poorly defined. This study was aimed to characterize the genetic basis of variation in gene expression in TLR4-stimulated human monocytes. METHODS: For this purpose we isolated monocytes of 136 individuals and stimulated them with lipopolysaccharide (LPS) to activate Toll-like receptor 4 (TLR4). From these donors, we performed transcriptome profiling at three time points (0 min/90 min/6 h) and genome-wide SNP-genotyping. RESULTS: Using the differential expression upon LPS treatment revealed 222 genes that are regulated by expression quantitative trait loci (eQTLs) at 90 min. and 213 genes at 6 hours. Among these, we show that SNPs conferring risk to primary biliary cirrhosis (PBC), inflammatory bowel disease (IBD) and celiac disease are immune response eQTLs for novel candidate genes, bringing new insights into the pathophysiology of these disorders in the context of TLR4-activation. CONCLUSIONS: This study is the first to map eQTL under immune stimulation and significantly enhance our knowledge on the innate immune system and its genetic determinants.

Science Day 2013

43

Kokordelis P., Nattermann J.

An effective IFN-γ mediated inhibition of HCV replication by NK cells is associated with spontaneous clearance of acute hepatitis C in HIV(+) patients Kokordelis P.1,2, Krämer B.1,2, Körner C.1, Boesecke C.1,2, Voigt E.3, Ingiliz P.4, Glässner A.1,2, Eisenhardt M.1,2, Wolter F.1,2, Kaczmarek D.1,2

, Nischalke H-D.1,2, Rockstroh J.K.1,2, Spengler U.1,2, Nattermann J.1,2

1Department of Internal Medicine I, University Hospital, University of Bonn, Germany 2German Center for Infection Research (DZIF), Bonn, Germany 3Praxis am Ebertplatz, Cologne, Germany 4Medical Center for Infectious Diseases (MID), Berlin, Germany [email protected] BACKGROUND: HCV co-infection is an increasing health problem in HIV(+) individuals. However, a considerable proportion of HIV(+) patients manage to overcome acute hepatitis C spontaneously. In the present study, we analyzed the role of natural killer (NK) cells in modulating the course of acute hepatitis C in HIV(+) patients. METHODS: 27 HIV(+) patients with acute hepatitis C (self-limited course: n=10; chronic course: n=17) , 12 HIV(+) patients with chronic hepatitis C, 8 HIV mono-infected individuals, and 12 healthy controls were studied. NK cells were phenotypically analyzed by flow cytometry. IFN-γ secretion, degranulation (CD107a) and anti-HCV activity of NK subpopulations were analyzed using the HuH7A2HCVreplicon cells system. RESULTS: NK cell frequency did not differ significantly between HIV(+) patients with chronic and self-limited course of acute hepatitis C. However, we found NK cells from patients with self-limiting infection to be significantly more effective in inhibiting HCV replication in vitro than NK cells from patients developing chronic hepatitis C. Of note, anti-viral NK cell activity showed no significant correlation with NK cell degranulation but was positively correlated with IFN-γ secretion, and blocking experiments confirmed an important role for IFN-γ in NK cell-mediated inhibition of HCV replication. Accordingly, NK cells from patients that spontaneously cleared the virus displayed a stronger IFN-γ secretion than those developing chronic infection. Finally, we observed high expression of NKG2D and NKp46, respectively, to be associated with self-limiting course of acute hepatitis C. Accordingly, we found that blocking of these NK cell receptors significantly impaired anti-viral NK cell activity. CONCLUSIONS: Our data suggest a strong IFN-γ -mediated antiviral NK cell response to be associated with a self-limited course of acute hepatitis C in HIV(+) patients.

44

Kopatz J., Neumann H.

Siglec-h on M1-polarized microglia antagonizes glioma cell growth Kopatz J., Beutner C., Welle K., Bodea L.G., Reinhardt J., Claude J., Linnartz-Gerlach B., Neumann H. Institute of Reconstructive Neurobiology, University Hospital, University Bonn, Germany [email protected] BACKGROUND: Sialic-acid-binding immunoglobulin-like lectin-h (Siglec-h) is a recently identified mouse-specific CD33-related receptor that signals via the immunoreceptor tyrosine-based activation motif (ITAM) linked adaptor protein DAP12. So far the function of Siglec-h as well as its ligand are unknown. METHODS: Microglia/glioma co-culture, bead phagocytosis, sh-RNA interference mediated receptor knock down, proliferation and apoptosis assays. RESULTS: We demonstrate gene transcription and protein expression of Siglec-h by mouse microglia after interferon-γ (IFN-γ) treatment or polarization into a M1-subtype. Siglec-h on IFN-γ stimulated microglia was involved in bead phagocytosis by increasing the uptake. A Fc fusion protein generated by our laboratory selectively bound to different glioma cells, but not to control cells. M1-polarized microglia phagocytosed glioma cells without prior induction of apoptosis and also limited their proliferation in a co-culture system. Reduced level of microglial phagocytosis of glioma cells was found after shRNA interference mediated Siglec-H knock down in microglia. Furthermore, microglial clearance of glioma in vitro is dependent on the Siglec-h interaction with its adaptor protein DAP12. CONCLUSIONS: Our data show that M1-polarized microglial cells can engulf glioma cells via a DAP12-mediated Siglec-h dependent uptake.

Science Day 2013

45

Krämer B., Nattermann J.

NKp46High expression defines a natural killer cell subset that is potentially involved in control of hepatitis C virus replication and modulation of liver fibrosis Krämer B.1*, Körner K.1*, Kebschull M.2, Glässner A.1, Eisenhardt M.1, Nischalke H-D.1, Alexander M.3, Sauerbruch T.1, Spengler U.1, Nattermann J.1 1Department of Internal Medicine I, University Hospital, University of Bonn, Germany 2Department of Periodontology, University Hospital,University of Bonn, Germany 3Institute of Human Genetics, University Hospital, University of Bonn, Germany *contributed equally [email protected] BACKGROUND: Natural killer (NK) cells play a role in the early control and natural course of hepatitis C virus (HCV) infection. NK cell activation is regulated by a multitude of receptors, including NKp46 receptor. However, reports on NKp46 in hepatitis C are controversial. Therefore, we investigated the hepatic phenotype and function of NK cells, considering differential surface expression of NKp46 resulting in NKp46(High) and NKp46(Dim) subsets. METHODS: Intra- and extrahepatic NK-cell subsets from HCV-infected patients were characterized by flow cytometry. Cytotoxic activity and interferon-gamma (IFN-γ) secretion were studied using K-562, P815, and primary hepatic stellate cells as targets. Anti-HCV activity of NK-cell subsets was studied using the replicon system. RESULTS: Importantly, NKp46(High) NK cells showed a higher cytolytic activity and stronger IFN-γ secretion than NKp46(Dim) NK cells. Accordingly, NKp46(High) NK cells efficiently blocked HCV replication in vitro. Blocking experiments confirmed an important role for the NKp46 receptor. Furthermore, we found an intrahepatic accumulation of NKp46(High) NK cells. Of note, high cytolytic activity of NKp46(High) NK cells was also confirmed in the intrahepatic NK-cell population, and the frequency of intrahepatic NKp46(High) was inversely correlated with HCV-RNA levels and fibrosis stage. CONCLUSIONS: NKp46(High) expression defines a specific NK-cell subset that may be involved in both the suppression of HCV replication and HCV-associated liver damage underpinning the role of NK cells in the immunopathogenesis of HCV.

46

Krebs W., Schultze J.L.

Characterization of epigenetic marks establishes specific patterns during macrophage activation Krebs W., Beyer M., Ulas T., Schmidt S.V., Mallmann M., Schultze J.L. Genomics and Immunoregulation, LIMES Institute, University of Bonn, Germany [email protected] BACKGROUND: Activation of macrophages is guided by transcriptional programs inducing specific macrophage phenotypes. Histone modification (HM) highly correlates with gene expression and allows a prediction of transcriptional regulation in macrophage phenotypes, like classically (M1) and alternatively (M2) activated macrophages. METHODS: Applying multiplex next generation sequencing (ChIP-seq) for the macrophage lineage determining transcription factor (TF) PU.1 and HM, we assessed differential epigenetic regulation of M1 and M2 at a global transcriptional level. RESULTS: Genomic areas with increased permissive HM positively correlated with gene expression. Using k-means algorithm, genes of M1 and M2 macrophages were clustered into groups according their enrichment patterns of HM. Groups of genes with permissive HM in close proximity to the TSS were related to known biological functions typical for M1 and M2. Only 10% of differentially expressed genes between M1 and M2 showed significant HM changes suggesting other regulatory mechanisms participating in gene expression, like TFs. Comparing the global distribution of PU.1 binding between M1 and M2 revealed increased enrichment of PU1 binding in M2 close to the promoter TSS region. CONCLUSIONS: Integration of ChIP-seq and transcriptome data supports a model, where M2 macrophages are mainly influenced by differences at transcription factor level allowing for fast changes in gene expression, while transcriptional programs in M1 macrophages are induced and maintained through slower epigenetic changes.

Science Day 2013

47

Kreutzberg T., Garbi N.

Sensing of self by T cells promotes awareness towards cognate antigen and T helper cell differentiation Kreutzberg T., Garbi N. Department of Molecular Immunology, Institutes of Molecular Medicine and Experimental Immunology, University Hospital, University of Bonn, Germany [email protected] BACKGROUND: Naïve T cells entering secondary lymphoid organs encounter residential dendritic cells (DCs) and frequently scan the antigenic content of multiple DCs. Recognition of MHC/self-peptide complexes displayed on DCs induces basal T cell activation, referred to as tonic T cell receptor (TCR) signaling. Our aim was to characterize the consequences of sensing MHC/self-peptide on DCs by T cells regarding their sensitivity to subsequent encounter with foreign antigen and differentiation. METHODS: Administration of diphtheria toxin to CD11c.DOG mice permits the depletion of DCs and thereby deprives T cells of tonic TCR signaling. This allows us to examine the consequences of MHC/self-peptide recognition for T cell function. RESULTS: Steady state interactions between DCs and T cells are required to enhance T cell sensitivity towards cognate antigen. Furthermore, CD4 T cells derived from DC-depleted mice had impaired capacity to differentiate into T helper cell types when stimulated under polarizing conditions. CONCLUSIONS: These results demonstrate that sensing of DCs by T cells during the steady state lowers the threshold of TCR activation upon challenge with cognate antigen and promotes T cell differentiation.

48

Kuepper J.M., Schumak B.

Inflammatory monocytes play a decisive role in the immunopathology of experimental cerebral malaria Kuepper J.M.1, Klocke K.1, Borsche M.1, Dunay I.R.2, Hoerauf A.1, Schumak B.1

1Institute for Medical Microbiology, Immunology and Parasitology (IMMIP), University Hospital, University of Bonn Germany 2Insitute for Medical Microbiology, Otto-von-Guericke-Universität Magdeburg, Germany [email protected] BACKGROUND: Infections with Plasmodium berghei ANKA parasites lead to experimental cerebral malaria (ECM) in C56BL/6 mice; such immune-mediated damage is commonly associated with strong Th1 responses. In other parasitic diseases, inflammatory monocytes were shown to regulate Th1 reactions; in ECM their role remains poorly defined. METHODS: Due to the development of specific antibodies we depleted in vivo inflammatory monocytes (anti-Gr1 or anti CCR2) or neutrophils (anti-Ly6G) during infection of B6 mice using an OVA transgenic strain (PbTg). We monitored survival (analyzed by log-rank Kaplan-Meier test) and performed ex vivo analysis at day 6 such as flow cytometry, histology and PCR to determine cellular recruitment to the brain, T cell responses and IFN-g production (analyzed by one-way ANOVA and posthoc tests). RESULTS: The application of anti-Gr-1 or anti-CCR2 but not of anti-Ly6G to PbTg-infected mice hindered the recruitment of lymphocytes into the CNS and moreover prevented the development of ECM symptoms resulting in prolonged survival. Depletion of inflammatory monocytes but not neutrophils led to decreased IFN-g levels and IFN-g+-CD8+-T effector cells in the brains. Importantly, the generation of PbTg specific T cell responses was not influenced by any depletion approach. CONCLUSIONS: We provide initial evidence that inflammatory monocytes (Ly6Chigh) are substantially involved in detrimental immuno-pathology of ECM during Plasmodium infection.


Science Day 2013

49

Labzin L., Latz E.

HDL inhibits TLR induced pro-inflammatory gene expression via the transcriptional repressor ATF3 Labzin L.1, De Nardo D.1, Schultze J.L.2, Latz E.1

1Institute of Innate Immunity, University Hospital, University of Bonn, Germany 2Life and Medical Sciences Institute (LIMES), University of Bonn, Germany [email protected] BACKGROUND: High density lipoprotein (HDL) is protective against the inflammatory condition atherosclerosis. Considering the important role of macrophages and pattern recognition receptors, such as Toll Like Receptors (TLRs) in atherosclerosis, we investigated the effect of HDL on TLR-induced macrophage activation and inflammation. METHODS: A mouse model of TLR-induced liver inflammation was used to assess HDL's effect on TLR activation in vivo. Meanwhile, the effects of HDL on TLR signaling, gene expression and cytokine secretion were analyzed in vitro in primary mouse macrophages. A systems biology approach utilizing microarray and ChIP sequencing technology was used to identify and validate candidate transcription factors mediating HDL's effects. RESULTS: HDL displayed striking anti-inflammatory properties under normocholesterolemic conditions, protecting against TLR-driven liver damage in vivo and reducing macrophage cytokine production in vitro.. Mechanistically, HDL did not affect upstream TLR signaling, but instead inhibited pro-inflammatory cytokine expression at the transcriptional level. The transcriptional repressor ATF3 was identified as the key mediator of HDL’s effects in macrophages. Consistent with these findings, HDL's anti-inflammatory effects were fully dependent on ATF3 expression both in vitro and in vivo. CONCLUSIONS: HDL has strong anti-inflammatory effects on macrophages via the induction of ATF3. These findings further explain the protective actions of HDL in cardiovascular and other inflammatory diseases.

50

Mass E., Hoch M.

Creld1 regulates NFATc1 localization and activation through interaction with calcineurin B Mass E.1, Wachten D.2, Hoch M.1 1LIMES-Institute, Program Unit Development, Genetics & Molecular Physiology, Molecular Developmental Biology, University of Bonn, Germany 2Center of Advanced European Studies and Research (caesar), Department of Molecular Sensory Systems, Molecular Physiology Group, Bonn, Germany [email protected] BACKGROUND: The calcineurin/nuclear factor of activated T cells (NFAT) signaling pathway is critical for numerous biological processes in vertebrates including the regulation of inflammatory responses, angiogenesis, cancer metastasis and cardial development. Natural product inhibitors of the calcineurin phosphatase such as Cyclosporine A (CsA) are widely used as immunosuppressants in transplantation medicine. Unfortunately, the continuous use of CsA is accompanied by severe adverse effects and, therefore, there is a need to identify regulators of calcineurin activity and new targets to downregulate calcineurin-dependent signaling pathways. METHODS: We generated and analyzed Cysteine-rich with EGF-like domains 1 (Creld1) knockout mice (Creld1KO), performed cell culture studies in murine and human cells and analyzed the interaction of Creld1 with calcineurin in vitro using various biochemistry techniques. Methods that we used include immunohistochemistry in cells and tissues, qRT-PCR, co-immunoprecipitation assays, fluorescence protein protection assays, NFAT- phosphorylation, translocation and luciferase assays. RESULTS: Our study identifies the Creld1 gene as an essential regulator of calcineurin/NFAT signaling. Its overexpression is sufficient to promote calcineurin-dependent NFATc1 activation. In contrast, NFATc1 translocation and target gene expression fail in mCreld1 knockout mice, resulting in defective cardiac valve development and embryonic lethality. We further found that Creld1 interacts with the regulatory subunit of calcineurin, CnB, which controls the phosphatase activity of the catalytic calcineurin subunit, CnA. Localization of Creld1 to the ER is important to exert its action on calcineurin. Furthermore, we found impaired calcineurin/NFAT signaling as the likely cause for atrioventricular septal defects in patients with CRELD1 mutations. CONCLUSIONS: Our data indicate that an important calcineurin-containing microdomain is formed at the ER through Creld1 serving as an anchor for the recruitment of the CnB subunit. Ca2+-dependent proteins, including calcineurin, could thus be exposed to high concentrations of ER-derived Ca2+, which is required for their activation. Together, our work opens a new avenue for elucidating the architecture of a Creld1-containing signaling platform required for calcineurin/NFAT activation at the ER and for developing new Creld1-interacting drugs to modulate calcineurin/NFAT signaling. Next steps include the analysis of Creld1 function in immune cells using a conditional loss-of-function allele.

Science Day 2013

51

Mathews M., Neumann H.

Expression and function of the late-onset Alzheimer’s disease associated CD33 in human microglia Mathews M., Roy K., Grobe Einsler M., Neumann H. Institute of Reconstructive Neurobiology, University Hospital, University of Bonn, Germany [email protected] BACKGROUND: Genome wide association studies have associated cd33 polymorphisms with late-onset Alzheimer’s disease (LOAD). CD33 (Siglec3) is a rapidly-evolving, species specific sialic-acid binding Immunoglobulin-like lectin expressed on myeloid-lineage cells like microglia. CD33 is often masked by cis-interacting sialic acids (sias) which regulate the receptor. METHODS: Human microglial cell lines, generated from induced pluripotent stem cells,were validated via flow cytometry, proliferation and phagocytosis assays as a model to study CD33. RT-PCR, immunocytochemistry and flow cytometry showed CD33 transcription and expression on microglia. Effect of sias on CD33 expression and phagocytosis were measured by flow cytometry. Immunohistochemistry (IHC) was used to measure CD33 expression in LOAD brains. RESULTS: Pluripotent stem cells were successfully differentiated into microglial cell lines (iPSdM) and showed expression of microglial surface markers, proliferation capacity and phagocytosis. Transcription and protein expression of CD33 was observed on iPSdM. Expression was increased after removal of cis-interacting sias. Furthermore, addition of non-human sias lead to an increase in CD33 expression but loss of phagocytosis capability. IHC showed an increase of CD33 in LOAD brain slices corroborating their association. CONCLUSIONS: A successful model for human microglia was established and used to study CD33 which provides a strong base for functional analysis of CD33 with respect to LOAD.


52

Mayer H., Bovier A.

Stochastic modeling of T-Cell activation Mayer H., Bovier A. Institute for Applied Mathematics, University of Bonn; Germany [email protected] BACKGROUND: Mathematical, in particular stochastic, models are frequently used to investigate biological systems in order to figure out the key aspects and mechanisms of the system. METHODS: Stochastic tools, especially large deviation techniques, are used to analyse the key quantities characterizing the behaviour of the modelled system. RESULTS: We consider a model of the interaction of T-cells and Antigen presenting cells and the probability of T-cell activation, i.e. the frequency of activated T-cells, in three different scenarios. This model was originally proposed by van den Berg, Rand and Burroughs and further investigated by Zint, Baake and den Hollander. It can be shown that the probability of interest grows exponentially with the number of presented foreign peptides for a certain range of parameters. CONCLUSIONS: For this very simple model the key task of having a low risk for autoimmune responses and a significantly higher probability for the detection of pathogens is satisfied.

Science Day 2013

53

Mertens C., Barchet W.

Oxidative damage of DNA confers resistance to TREX1 degradation and potentiates STING dependent immune sensing Mertens C.1, Gehrke N.1, Zillinger T.1, Wenzel J.2, Bald T.2, Zahn S.2, Tüting T.2, Hartmann G.1, Barchet W.1 1Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital, University of Bonn, Germany 2Laboratory of Experimental Dermatology, Department of Dermatology and Allergology, University Hospital, University of Bonn, Germany [email protected] BACKGROUND: Viruses are detected by the innate immune system via their nucleic acid genomes. It is thought that the mere presence of DNA in the cytosol is sufficient to activate the receptor cGAMP synthase (cGAS), but reactivity based on location alone poses the risk of erroneous recognition of self DNA that can result in autoimmune disease such as lupus erythematosus (LE). METHODS: Myeloid cells were stimulated with DNA derived from increasingly UV-damaged cells, and cytokine production was analyzed by ELISA. Reactive oxygen species (ROS) levels were determined after UV-irradiation and correlated with 8-OHG, a marker of oxidatively damaged DNA. Unmodified and oxidized DNA was injected into earlobes of lupus prone mice, followed by ear swelling measurement and immunohistochemistry. RESULTS: Oxidative DNA modifications caused by ROS (produced by macrophages and neutrophils or during UV-light absorption) greatly enhanced the immunogenicity of DNA in the cytosol. Oxidative modifications conferred resistance to DNA degradation by the exonuclease TREX1, leading to accumulation of oxidized DNA that activated the cGAS/STING signaling pathway. LE prone mice formed skin lesions at the sites where oxidized DNA was injected, which suggests that enhanced immune stimulation by UV damaged DNA might cause UV phototoxicity observed in LE patients. CONCLUSIONS: We provide evidence for the involvement of a damage-associated DNA modification in the immune detection of DNA in the cytosol with important implications for infection, sterile inflammation and autoimmunity.

54

Muth D., Drosten C.

European SARS-related bat coronaviruses carry interferon antagonists active in human cells Muth D.1, Niemeyer D.1, Drexler J.F.1, Gloza-Rausch F.1, Binger T.1, Ritz D.1,Pfefferle S.1, Zimmermann K.2, Pfeifer A.2, Müller M.A.1, Drosten C.1

1Institute of Virology, University Hospital, University of Bonn, Germany 2Institute of Pharmacology and Toxicology, University Hospital, University of Bonn, Germany [email protected] BACKGROUND: Asian Rhinolophid bats were found to be the most likely natural host of SARS coronavirus (CoV). Sampling and analysis of bats in Europe revealed that SARS-related coronaviruses are also present in European Rhinolophidae. In order to determine the zoonotic potential of these viruses functional genetic markers need to be identified and assessed. METHODS: Full genome sequencing of newly found viruses revealed the presence of homologous interferon (IFN) antagonists. Two selected antagonists were cloned and used in overexpression analysis to determine their potential mode of action. Additionally, chimeric recombinant SARS-CoV carrying the selected bat CoV genes were generated. For cell culture experiments in a host context a Rhinolophus bat embryonic lung cell line susceptible to SARS-CoV was established. RESULTS: Both selected bat coronavirus derived antagonists showed similar features as compared to their SARS-CoV homologs in terms of subcellular localization and inhibitory effects on the cellular IFN response. Replication analysis of chimeric recombinant SARS-CoV compared to wild type recombinant SARS-CoV showed that all viruses grew similarly in primate and bat cells. CONCLUSIONS: These data suggest that both homologous proteins are able to antagonize IFN response in primate cells without prior adaptation and are fully functional in the context of a replicating genome of a closely related coronavirus.

Science Day 2013

55

Nadorp B., Soreq H.

From mice to men: MicroRNA regulators of acetylcholine packaging and degradation Nadorp B., Soreq H. The Edmond and Lily Safra Center for Brain Science, The Department of Biological Chemistry and The Center for Bioengineering, The Hebrew University of Jerusalem, Israel 91904 [email protected] BACKGROUND: MicroRNAs (miRNAs) may dim complete signaling pathways, but their control over neurotransmission processes is largely unknown. Here, we report that predicted miRNA suppressors of ACh packaging, much more than ACh synthesis, show massive overlap with those regulating ACh degradation. METHODS: Four different bioinformatics algorithms were used to identify miRNAs predictably binding to the 3’ un-translated regions (3’UTR) of the vesicular-ACh-transporter (VAChT), the choline-acetyl-transferase (ChAT), two different splicing variants of the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). RESULTS: We identified a total of 411 miRNAs, with 76 of those targeting more than one ACh metabolism-related transcript. Over half of the 67 VAChT-targeting miRNAs (55%) are predicted to target the ACh hydrolyzing genes AChE and/or BChE, half of those (49%) being primate-specific. Of the 76 dual targeting miRNAs 28 associate with inflammation, anxiety, brain damage, cardiac or neurodegenerative diseases. Altogether, 67% and 43% of these miRNAs were linked with inflammation and with more than one disease group, respectively. CONCLUSIONS: Overlapping miRNA regulation emerges from our findings as a recently evolved surveillance mechanism over cholinergic neurotransmission in health and disease. The large number of identified, primate-specific miRNAs demonstrates the need of new mouse models with knocked-in primate-specific miRNAs, such as miR-608.

56

Niemeyer D., Müller M.A.

Middle East respiratory syndrome coronavirus accessory protein 4a is a type I interferon antagonist Niemeyer D.1, Zillinger T.2, Muth D.1, Zielecki F.3, Horvath G4, Suliman T.1, Barchet W.2, Weber F3, Drosten C.1, Müller M.A.1 1Institute of Virology, University Hospital, University of Bonn, Germany 2Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital, University of Bonn, Germany 3Institute for Virology, Philipps-University Marburg, Germany 4Institute of Innate Immunity, University Hospital, University of Bonn, Germany [email protected] BACKGROUND: The emerging Middle East respiratory syndrome coronavirus (MERS-CoV) causes acute respiratory tract infections similar to severe acute respiratory syndrome (SARS). MERS-CoV counteracts parts of the innate immune response in human bronchiolar cells but mechanistic details are unclear. Whereas several SARS-CoV accessory proteins were shown to inhibit the type I interferon (IFN) response, functions of MERS-CoV accessory proteins are unknown. Here we analyzed accessory proteins (p)3, 4a, 4b and 5 for their abilities to inhibit the IFN response. METHODS and RESULTS: In an IFN-beta promoter reporter assay only p4a inhibited a general activation of the IFN response. Targeted IFN stimulation at the level of RIG-I helicases revealed that p4a inhibited MDA5- but not RIG-I-driven IFN activation in a dose-dependent manner. In addition, p4a blocked the nuclear translocation of a co-expressed GFP-IRF-3 and reduced the secretion of bioactive IFN confirming its intervention at the IFN induction level. An in-silico analysis predicted a double-stranded (ds)RNA-binding motif for p4a. In pull-down experiments p4a bound specifically to poly(I:C) beads. Finally, it could be shown that p4a co-localized with viral dsRNA molecules in MERS-CoV infected cells. CONCLUSIONS: Accessory p4a was found to block interferon induction at the level of MDA5 activation presumably by direct interaction with dsRNA.


Science Day 2013

57

Niepmann S., Hornung V.

TNFα regulates NLRP3 activity and contributes to age related inflammasome activation and metabolic disease Niepmann S.1, Bauernfeind F.1,2, Hornung V.1

1Institute of Clinical Chemistry and Pharmacology, Unit for Clinical Biochemistry, University Hospital, University of Bonn, Germany 2Department of Internal Medicine III, University Hospital, University of Bonn, Germany [email protected] BACKGROUND: De novo protein synthesis of NLRP3 induced by TLR signaling is a prerequisite for NLRP3 mediated caspase-1 activation. However, also the pro-inflammatory cytokine TNFα is sufficient to induce NLRP3 expression in vitro. In aged mice, the serum levels of TNFα were found to be elevated, possibly arising from macrophages located in the peripheral adipose tissue. We thus speculated, whether TNFα could also contribute to caspase-1 activation in vivo and thereby influence inflammasome mediated disturbances in glucose tolerance. METHODS: We analyzed cytokine levels, metabolic parameters and the expression of NLRP3 and caspase-1 activity in several tissues of young and aged mice. Additionally, wild type mice receiving a TNFα antagonizing therapy with Infliximab were investigated. RESULTS: As expected, TNFα and IL-18 levels were elevated in old mice. Increased NLRP3 activation in the tissues analyzed correlated with the presence of cleaved caspase-1. In contrast, TNF antagonizing treatment with infliximab in aged mice reduced NLRP3 expression, inflammasome activation, the levels of inflammasome processed cytokines and improved the glucose tolerance. CONCLUSIONS: Our results suggest that TNFα indeed controls NLRP3 expression and inflammasome activation in vivo and significantly contributes to inflammasome mediated impaired glucose tolerance in old age.

58

Pelka K., Latz E.

The activity of human endosomal TLRs is differentially regulated by the tyrosine-based motif of UNC93B1 Pelka K.1, Labzin L.1, Phulphagar K.1, Stahl R.1, Zimmermann J.2, Höning S2, Latz E.1,3,4

1Institute of Innate Immunity, University Hospital, University of Bonn, Germany 2Biochemistry-Center, University Hospital, University of Cologne, Germany 3UMass Medical School, Department of Infectious Diseases and Immunology, Worcester, MA, USA 4German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany [email protected] BACKGROUND: UNC93B1 regulates the trafficking of nucleic acid (NA)-sensing TLRs from the ER to endolysosomes. Recently, a tyrosine-based motif (Yxx[L/I]) of UNC93B1 was shown to bind to the adapter protein complex AP2. The motif was essential for murine TLR9 activity, but dispensable for other murine endosomal TLRs. Endosomal TLRs greatly differ between mouse and human. We therefore investigated the role of the Yxx[L/I] motif in human endosomal TLR trafficking. METHODS: HEK cells stably expressing human TLRs were reconstituted with wild type or Yxx[L/I] mutant UNC93B1. The localization of UNC93B1 was analyzed by confocal microscopy and TLR responses were analyzed by ELISA. The role of AP2 was investigated using RNA interference. RESULTS: Destruction of the Yxx[L/I] motif of human UNC93B1 caused the mislocalization of UNC93B1 to the cell surface and the depletion of UNC93B1 from AP2-dependent, but not other endosomes. Human TLR9 activity was abolished while TLR8 responses were increased. This differential regulation occured independent of the AP2-dependent pathway as knockdown of AP2 increased both TLR8 and TLR9 responses. CONCLUSIONS: Our study reveales that the Yxx[L/I] motif of UNC93B1 differentially regulates human TLRs, shows that both TLR8 and TLR9 signal from AP2-independent endolysosomes and provides new insights into the complex regulation of endosomal TLR trafficking.


Science Day 2013

59

Piotrowitz K., Thiele C.

How inflammation affects liver lipid metabolism Piotrowitz K., Vorac J., Unterweger I., Hettich H., Kuerschner L., Foerster I., Thiele C. Life & Medical Sciences (LIMES) Institute, University of Bonn, Germany [email protected] BACKGROUND: Non alcoholic fatty liver disease (NAFLD) is characterized by an accumulation of triglycerides within hepatocytes which ultimately develops into liver fibrosis. Increasing evidence suggests a link between NAFLD and risc factors of the metabolic syndrome such as obesity, type 2 diabetes and insulin resistance. Elucidating the underlying mechanisms that link lipid metabolism to inflammation will allow for insights into NAFLD pathogenesis. METHODS: Our group developed a novel click-chemistry based method which allows to trace lipid metabolism in freshly isolated hepatocytes and intact livers of mice without using radioactivity. With this method we compared liver lipid metabolism with or without LPS challenge in pulse chase experiments. RESULTS: In hepatocytes from untreated or LPS treated mice a detailed time resolved analysis of the essential initial phase of lipid metabolism including a lipid class resolution was performed. We observed a significant changed metabolism of triacylglycerol, phosphatidylcholine and phosphatidylethanolamin. Additionally, these cells took up reproducibly more fatty acids after LPS treatment. These studies were corroborated by fluorogenic microscopy experiments analyzing the lipid droplet number and average size, both of which increased upon LPS treatment. CONCLUSIONS: Our data demonstrates an influence of inflammation on lipid metabolism in the liver.


60

Raju D., Wachten D.

Glucosylceramide controls sperm-head formation Raju D., Wachten D. Center for Advanced European Studies and Research (caesar), Bonn, Germany [email protected] BACKGROUND: GBA2, a non-lysosomal β-glucosidase, degrades the glycosphingolipid glucosylceramide (GlcCer) to glucose and ceramide and is highly expressed in mouse testis. GBA2-deficient mice accumulate GlcCer in testis, which affects sperm-head formation and, thereby, male fertility. RESULTS: We could show that in testis, GBA2 is expressed in Sertoli cells. Sertoli cells contain the ectoplasmic spezialisation (ES), which is composed of F-actin and embraces the developing sperm head. We demonstrate that the F-actin (ES) around sperm heads is misaligned in GBA2-deficient mice. Interestingly, cytoskeletal dynamics in GBA2-deficient dermal fibroblasts are also altered: GBA2-deficient cells display higher turnover rate of G-actin to F-actin (45%) and, therefore, higher numbers of different actin structures. Preliminary results suggest that the accumulation of GlcCer affects the function of focal adhesion complexes in the plasmamembrane, which feeds-back on the cytoskeleton. CONCLUSIONS: Lack of GBA2 and the resulting accumulation of GlcCer alter cytoskeletal dynamics. Our results indicate that a change in the membrane composition of Sertoli cells due to accumulation of GlcCer might affect actin structures at the ES, resulting in a defect of sperm-head formation. Elucidating the hitherto unknown mechanism behind disruption of spermatogenesis due to lack of a GBA2/accumulation of GlcCer would be ground-breaking.


Science Day 2013

61

Redler S., Betz R.C.

High-density genotyping in alopecia areata identifiesTNSF4 and HLA-C as two new susceptibility loci with genome-wide significance Redler S.1, Angisch M.2, Wolf S.1, Heilmann S.1,3, Giehl K.A.4, Hanneken S.5, Eigelshoven S.5, Kruse R.6, Blaumeiser B.7, Böhm M.8,

Garcia Bartels N.9, Lutz G.10

, Wolff H.4, Blume-Peytavi U.9, Nöthen M.M.1,3

, Becker T.2,11, Betz R.C.1

1 Institute of Human Genetics, University Hospital, University of Bonn, Germany 2 Institute for Medical Biometry, Informatics and Epidemiology, University of Bonn, Germany

3 Department of Genomics, Life & Brain Center, University of Bonn, Germany 4 Department of Dermatology, University of Munich, Germany

5 Department of Dermatology, University of Düsseldorf, Germany 6 Dermatological Practice, Paderborn, Germany 7 Department of Medical Genetics, University of Antwerp, Belgium 8 Department of Dermatology, University of Münster, Germany

9 Clinical Research Center for Hair and Skin Science, Department for Dermatology and Allergy, Charité-Universitätsmedizin Berlin, Germany 10 Hair & Nail, Dermatological Practice, Wesseling, Germany 11 German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany [email protected] BACKGROUND: Alopecia areata (AA) is a common hair loss disorder with a complex mode of inheritance. The etiopathogenesis of AA is not entirely understood. However, immunological and association studies generate accumulating support for the hypothesis that innate and acquired immunity are implicated. Previous studies reported an association between AA and specific HLA alleles, as well as genetic variants of other genes implicated in autoimmunity. METHODS: To provide further insight into the genetic basis of AA, we analyzed a sample of 774 AA patients and 1,488 controls on a custom Illumina BeadChip array (Immunochip), followed by replication in an independent sample of 1,016 cases and 1,060 controls of Central European origin. Furthermore, a stepwise logistic regression analysis of the HLA region was undertaken. RESULTS: We identified HLA-C and TNFSF4 as two new susceptibility loci with genome-wide significance. Furthermore, we could confirm the well established HLA alleles HLA-DQA1 and HLA-DQA. Nominal significant association was observed for an additional three interesting loci for follow-up studies – FASLG, THADA and C11orf30. CONCLUSIONS: Our results point to the involvement of HLA-C and TNFSF4 in the aetiology of AA. The more detailed enlightenment of the pathways specific to AA is a further step towards a deeper insight into the understanding of this immune-mediated disease.


62

Renn M., Tüting T.

Hgf-Cdk4R24C mouse melanomas resist adoptive cell transfer therapy with CTL targeting the melanocytic antigen gp100 through inflammation-induced reversible dedifferentiation Renn M., Landsberg J., Kohlmeyer J., Bald T., Rogava M., Cron M., Hölzel M., Tüting T. Laboratory of Experimental Dermatology, Department of Dermatology and Allergy, University Hospital, University of Bonn, Germany [email protected] BACKGROUND: Immunotherapy with adoptively transferred T-cells targeting melanocytic antigens can induce remissions in patients with metastatic melanoma, but tumors frequently relapse. METHODS: The genetically engineered HgfxCdk4R24C mouse melanoma model and an effective adoptive T-cell immunotherapy protocol targeting the melanocytic differentiation antigen gp100 were used to gain further insights in the mechanisms underlying tumor relapse. RESULTS: Adoptive T-cell therapy led to dedifferentiation in primary HgfxCdk4R24C melanoma. Loss of the target antigen gp100 was associated with increased immune cell infiltration. Using the novel HCmel3 cell line derived from our model in serial transplantation experiments, we could show, that tumor cells could switch between a differentiated and dedifferentiated phenotype in response to T-cell induced inflammation. In vitro, we identified the proinflammatory cytokine TNF-α as a major factor responsible for the loss of recognition by gp100 specific T-cells. TNF-α led to a reversible downregulation of differentiation markers like gp100 and the upregulation of neural crest progenitor markers like Ngfr, impairing T-cell recognition. CONCLUSIONS: We found that tumor relapse after initially successful T-cell immunotherapy involves reversible dedifferentiation driven by proinflammatory mediators such as Tnf-α, rather than the immunoselection of tumor cell variants with persistent genetic loss of the antigen. In our model, melanoma cells exist in a dynamic, interconvertible equilibrium between differentiated and dedifferentiated subpopulations that rapidly adapt to inflammatory signals in the environment.

Science Day 2013

63

Rieck M., Kolanus W.

Arl4d complexes with cytohesin-3 and controls T effector function by limiting IL-2 expression Rieck M.1, Tolksdorf F.1, Diehl L:2, Kaczmarek J.2, Grell J.1, Paul B.1, Knolle P.2, Kolanus W.1 1Molecular Immunology, LIMES Institute, University of Bonn, Germany 2Institutes of Molecular Medicine and Experimental Immunology, University Hospital, University of Bonn, Germany [email protected] BACKGROUND: Interleukin-2 is a key regulator of adaptive immune responses, but its regulation is incompletely understood. The cytohesin proteins are GEFs(guanine nucleotide exchange factors) for ARF GTPases, involved in the regulation of various signaling cascades, e.g. the insulin-, EGF- or integrin-pathway. Arl4d belongs to the family of Arf-like proteins and was revealed as a specific interactor of the cytohesins. Here we investigated the role of both proteins in the regulation of IL-2 production as hallmark of T cell activation. METHODS: Analysis of knockout mice, in vitro stimulation of isolated T cells, biochemical protein interaction studies, functional analysis of target protein overexpression in cell lines. RESULTS: We show that the expression of Arl4d and cytohesin-3 in T cells is dependent on PDL-1 signaling. Consequently, both proteins become differentially down-regulated upon T cell activation and up-regulated during T cell attenuation, e.g. by priming with LSECs (liver sinusoidal endothelial cells). Deficiency of either Arl4d or cytohesin-3 in stimulated primary T cells results in a robust IL-2 hyper production. Mechanistically, Arl4d and cytohesin-3 selectively suppress AKT and MAP kinase signaling, by forming a complex that competes with the counteracting cytohesin-1 for the binding to the CNK scaffolding platform. CONCLUSIONS: Our data reveal a novel type of PDL1-dependent regulatory circuitry that links Cytohesin-3 and its interaction partner Arl4d to specific signaling events important for limiting IL-2 production in both human and murine T cells. The Cytohesin-3/Arl4d complex plays a fundamental role in setting the sensitivity of signaling following TCR ligation and thus determines the activation threshold for T cells.


64

Riesenberg S., Hölzel M.

Determinants of TNF responsiveness in melanoma Riesenberg S.1, Kohlmeyer J.2, Reinhardt J.1, Schadendorf D.3, Tüting T.2, Hölzel M.1 1Institute for Clinical Chemistry and Clinical Pharmacology, University Hospital, University of Bonn, Germany 2Laboratory for Experimental Dermatology, University Hospital, University of Bonn, Germany 3Dermatology, University Hospital Essen, Germany [email protected] The proinflammatory cytokine TNF-alpha causes dedifferentiation of melanoma cells and links inflammation in the microenvironment to tumor cell plasticity. Recently we could show that such a phenotype switch drives resistance to a T-cell therapy targeting melanocytic antigens in a murine melanoma model through reversible loss of the melanocytic differentiation program. The response to inflammation (TNF-alpha) is conserved in human melanoma cells, but is variable due to genetic and epigenetic heterogeneity. It is conceivable that determinants of TNF-alpha responsiveness could be important for stratification of immunotherapies. Therefore we aimed to scrutinize TNF-alpha responsiveness in a panel of 17 human melanoma cells by multidimensional profiling. All cell lines have been characterized by whole-exome sequencing and represent a balanced selection of key oncogenic driver mutations found in human melanoma. Gene expression profiling identified subgroups of TNF-responsiveness linked to different phenotypic states. Preliminary results will be presented.


Science Day 2013

65

Rogava M., Tüting T.

Sensing of UVB-induced DNA damage in the skin by the innate immune system involves the TLR4- and MyD88-dependent signaling pathway Rogava M., Bald T., Glodde N., Gaffal E., Tüting T. Laboratory of Experimental Dermatology, Department of Dermatology and Allergy, University Hospital, University of Bonn, Germany [email protected] BACKGROUND: Excessive exposure of the skin to UVB radiation causes DNA damage in epidermal keratinocytes and initiates a reactive neutrophil-rich inflammatory response. Little is known how the innate immune system senses UVB-induced DNA damage in the skin. We hypothesized that TLR-dependent signalling pathways participate in this process. METHODS: We investigated how genetic or pharmacologic blockade of TLR signalling impacts upon skin inflammatory responses induced by two consecutive sunburning doses of 4.5 kJ/m2 UVB on the back skin. RESULTS: Histopathological analyses revealed reduced numbers of infiltrating immune cells and diminished reactive hyperproliferation of epidermal keratinocytes in UV-irradiated skin of Tlr4-/- and MyD88-/- mice, but not of Tlr3-/- and Trif-/- mice, when compared to wild-type controls. Flow cytometric and gene expression analyses confirmed that the systemic activation and local recruitment of neutrophils was largely absent in Tlr4-/- and MyD88-/- mice but not in Tlr3-/- and Trif-/- mice. The reactive skin inflammatory response following UV irradiation was also largely abrogated by the TLR4 inhibitor CLI-095 or by antibody-mediated depletion of neutrophils. CONCLUSIONS: Our results demonstrate that the TLR4- and MyD88-driven innate immune signaling pathway critically contributes to the reactive neutrophil-rich inflammatory response following UVB-induced DNA damage in the skin.

66

Ruschel J., Bradke F.

Systemic Administration of Epothilone B Promotes Axon Ruschel J.1, Hellal F.1†, Flynn K.C.1‡, Dupraz S.1, Blesch A.2, Weidner N.2, Bradke F.1 1Axonal Growth and Regeneration, German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany 2Spinal Cord Injury Center, Heidelberg University Center, Heidelberg, Germany † Current address: Institute for Stroke and Vascular Dementia Research, University of Munich Medical Center, Munich, Germany ‡ Current address: Department of Molecular Medicine, Max Planck Institute of Biochemistry, Martinsried, Germany [email protected] BACKGROUND: Spinal cord injury causes irreversible loss of motor function and sensation as a result of axon damage. Microtubules drive key processes during axon regeneration, including preserving the axon integrity, driving axon growth and restraining fibrotic scar formation after spinal cord injury. However, targeting microtubules after spinal cord injury is limited because many microtubule stabilizing drugs do not cross the blood brain barrier. METHODS and RESULTS: Here, we report that systemic and post-injury administration of the microtubule stabilizing and blood brain barrier permeable drug Epothilone B has beneficial effects after spinal cord injury. We found in spinal cord injured rodents and in videomicroscopic cell analysis that Epothilone B decreases scarring by interfering with meningeal fibroblast migration, through abrogation of microtubule dynamics and cell polarity. Furthermore, time lapse microscopy and in vivo live imaging revealed that Epothilone B causes the microtubular network to protrude into the distal tip of the axon to propel axon growth in an inhibitory environment. Finally, we identified by selective ablation serotonergic axons as a key mediator of the Epothilone B induced gait improvement. CONCLUSIONS: These data provide a promising translational perspective of Epothilones in enabling functional axon regeneration after central nervous system injury.

Science Day 2013

67

Sander J., Schultze J.L.

Signal integration of macrophage activation is substantially conserved on transcriptional level between human and mice Sander J., Schmidt S.V., Xue J., Mallmann M., Beyer M., Ulas T., Schultze J.L. Genomics and Immunoregulation, LIMES Institute, University of Bonn, Germany [email protected] BACKGROUND: In immunology the translation from mouse to human is a challenging process, not only due to the fundamental biological differences between the two species. However, the identification of genes showing highly conserved expression patterns across immune cell types facilitates to distinguish between clearly translatable gene sets and diverging ones. METHODS: Based on transcriptome data of four representative macrophage populations (unpolarized, stimulated with IFNγ, IL4 or TNFα plus PGE2 plus Pam3) generated from both human monocyte and murine bone-marrow derived macrophages we calculated conservation of expression (CoE) scores for annotated orthologous genes. The scores were combined with differential expression analysis to identify highly conserved common or macrophage subtype specific markers. RESULTS: We show that the global transcriptional profile across macrophage populations is substantially conserved between the two species. Specifically, most of the genes known or previously identified as important transcription factors and markers in macrophage biology showed highly conserved expression patterns. Additionally, we identified a yet unknown set of differentially expressed and subtype specific highly conserved genes. CONCLUSIONS: This study clarifies the overall conservation between murine and human macrophage populations integrating different stimulating signals and composes a fundament for translating findings in macrophages from one species to the other.

68

Schmid-Burgk J., Schmidt T., Hornung V.

High throughput functional genomics using designer nucleases assembled by ligation-independent cloning Schmid-Burgk J., Schmidt T., Hornung V. Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital, University of Bonn, Germany [email protected] BACKGROUND: Recent developments in the field of artificial nucleases open a novel path to study gene function in cultured cell lines. Innate immunity seems well-suited as a field of application, since the defense mechanisms studied often rely on non-redundant receptor-, signal transduction-, and effector-protein coding genes. METHODS: Ligation-independent cloning (LIC) allows to combine more than two fragments of DNA at high fidelity (1). We have developed LIC-based semi-automated high-throughput assembly platforms for TALE nuclease genes (2) as well as for CRISPR gRNA plasmids, aiming for the large-scale study of gene function. RESULTS: We could confirm high genome editing activity of large numbers of LIC-assembled TALE- and CRISPR nucleases in human cells. By using deep-sequencing, we could furthermore identify a bias towards microhomology-directed editing events in TALEN-targeted genomic loci, which could be pharmacologically controlled by inhibition of DNA-PK. We generated a clonal sequence-confirmed library of CRISPR plasmids covering 39% of the human genome utilizing microarray based synthesis of oligo pools, orthogonal mixing, and deep sequencing. CONCLUSIONS: The technologies for high-throughput synthesis and validation of nuclease libraries will pave the way to a novel class of forward genetic screens set to unravel genes involved in innate immunity. While paralleling the development of shRNA- and haploid screens, nuclease screens could provide the advantage of gaining more complete and more stable phenotypes to select for, while not being limited to specialized haploid cell lines.

Science Day 2013

69

Schmidt T., Hornung V.

High throughput generation of knockout cell lines with designer nucleases using deep sequencing Schmidt T.1*, Schmid-Burgk J.1*, Altmüller J.2, Becker K.2, Stemshorn K.2, Cathomen T.3, Nürnberg P.2, Hornung V.1 1Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital, University of Bonn, Germany 2Cologne Center for Genomics (CCG), University of Cologne, Germany 3Cell and Gene Therapy, Center for Chronic Immunodeficiency (CCI), University of Freiburg, Germany *Equally contributed to this work [email protected] BACKGROUND: Designer nucleases like TAL effector nucleases and the CRIPSR/Cas9 system have become widely used tools to induce double strand breaks and thus frame shifts at specific loci in eukaryotic genomes. Here we provide a robust screening workflow and analysis tool that allows the rapid generation of knockout cell lines. METHODS: Following transfection of designer nucleases, cell clones are obtained by limiting dilution. To enable a rapid screening for all-allelic frame shift mutations, a protocol consisting of a two step PCR and subsequent deep sequencing was developed. In addition we wrote an easy-to-use alignment and genotype calling software that allows a simple analysis of the deep sequencing data. Subsequently, the thus identified knockout clones can be recovered and analyzed for gene expression and evaluated in functional assays. RESULTS: By knocking out key players of the antiviral immunity (e.g. STAT1, RIG-I) we could show that this technique is highly applicable for many cell types and generates knockout cell lines within four weeks. CONCLUSIONS: We developed an easy and robust workflow and evaluation algorithm that allows rapid screening of genome-edited cell clones that can be easily employed by most laboratories. Knockout cell lines will provide new insights in cell biological and especially immune sensing processes.

70

Schonauer S., Wachten D.

Investigating the cross-talk between GBA1 and GBA2 in Gaucher disease Schonauer S., Koerschen H.G., Wachten D. Center of Advanced European Studies and Research, Department of Molecular Sensory Systems, Bonn, Germany [email protected] BACKGROUND: The beta-glucosidases GBA1 and GBA2 both degrade glucosylceramide to glucose and ceramide. However, they are localized in different cellular compartments and it is not known whether their localization points towards a different cellular function. Mutations in the GBA1-gene cause Gaucher disease, whereas loss of GBA2 is linked to male infertility in mice. Our results reveal a possible cross-talk between the two enzymes in patients suffering from Gaucher disease. METHODS: A cross-talk between GBA1 and GBA2 was analyzed in dermal fibroblasts from Gaucher disease and control patients on an expression and activity level. RESULTS: Quantitative real-time PCR experiments showed that both GBA1 and GBA2 mRNA-expression levels were similar between control patients and patients carrying a mutation in GBA1. Our fluorescence-based beta-glucosidase activity assay revealed that GBA1 activity was dramatically decreased in Gaucher disease patients with a mean activity of only 11%. Interestingly, GBA2 activity was also diminished with a mean remaining activity of only 40%. CONCLUSIONS: Our results reveal a cross-talk between GBA1 and GBA2 on the activity level. This finding adds a new aspect to the complex nature of Gaucher disease and presents a new potential target for its treatment.


Science Day 2013

71

Schuette V., Burgdorf S.

The Mannose receptor induces T cell tolerance via inhibition of CD45 and up-regulation of CTLA-4 Schuette V., Burgdorf S. Cellular Immunology, LIMES Institute, University of Bonn, Germany [email protected] BACKGROUND: To characterize the influence of the Mannose Receptor (MR) on T cell activation by mechanisms distinct from antigen internalization, we analyzed CD8+ DesTCR T cells, which recognize endogenous peptides on MHC I molecules and are thereby independent from MR-mediated antigen- uptake. METHODS: Utilization of in vitro and in vivo cytotoxicity assays, phosphatase activity assays, microarrays and FACS staining procedures enabled us to study the regulatory mechanism of the MR during T cell activation. RESULTS: We could demonstrate in vitro and in vivo that under steady state conditions, the presence of the MR on DCs impaired the cytotoxicity of activated CD8+ T cells. This regulatory effect was mediated by a direct interaction of the MR with CD45 on the T cells surface, abolishing its phosphatase activity and hence T cell signaling. Suppression of CD45 resulted in the up-regulation of the inhibitory molecule CTLA-4 on T cells in vitro and in vivo, which in turn was responsible for the reduced cytotoxic CD8+ T cell activity. Furthermore, we could demonstrate that all mentioned effects were circumvented under inflammatory conditions. CONCLUSIONS: Hitherto, the MR has mainly been associated with antigen uptake by DCs and resulting cross-presentation under inflammatory conditions. We describe a tolerogenic effect of the MR on T cells at steady state, which is overcome in the presence of inflammatory stimuli. This indicates that the MR contributes to the induction of both immunity and tolerance. Therefore, vaccination approaches which aim at immunity, depend on a strong immunogenic adjuvant during antigen-targeting towards the MR. Otherwise, tolerance is induced.

72

Shahraz A., Neumann H.

Function of the human-specific sialic acid binding receptor Siglec-11 in amyloid-β mediated neurotoxicity Shahraz A., Kopatz J., Neumann H. Institute of Reconstructive Neurobiology, University Hospital, University of Bonn, Germany [email protected] BACKGROUND: It has been shown that human microglia express inhibitory human lineage specific receptor sialic acid binding immunoglobulin-like lectin-11 (Siglec-11) and that murine microglia genetically modified to express the human lineage specific receptor Siglec-11 were less toxic to neurons. METHODS: Recently, we established a protocol for in vitro differentiation of induced pluripotent stem (iPS) cells into microglial precursors. In addition, we differentiate iPS cell derived neural stem cells into human neurons to obtain a human neuron-microglia co-culture system. RESULTS: The human microglial lines expressed Siglec-11, which is a potential receptor for polysialic acid (PSA). Microglial cells were responsive to amyloid-β by increasing reactive oxygen species (ROS) production. Data showed that treatment with PSA not only can prevent the amyloid-β-triggered production of ROS by the human microglial lines, but also can reduce amyloid-β phagocytosis by these cells. In the human microglia-neuron co-culture, amyloid-β treatment resulted in neurotoxicity with loss of neurites that was abrogated by treatment with PSA. CONCLUSIONS: The brain enriched carbohydrate PSA acted neuroprotective on Siglec-11 expressing human microglia and prevented the amyloid-β-mediated neurotoxicity in a human microglia-neuron co-culture model.


Science Day 2013

73

Stutz A., Latz E.

The inflammasome component NLRP3 is regulated by phosphorylation Stutz A.1, Horvath G.L.1, Stahl R.1, Meissner F.2,*, Latz E.1,3,4,* 1Institute of Innate Immunity, University Hospital, University of Bonn, Germany 2Dept. Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried/Munich, Germany 3Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, USA 4Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Bonn, Germany. * These authors contributed equally to this work. [email protected] BACKGROUND: NLRP3 is an intracellular pattern-recognition receptor that senses many different activators including bacterial toxins and endogenous danger signals. Upon activation, the NLRP3 inflammasome is formed, leading to the secretion of pro-inflammatory cytokines and cell death. NLRP3 activity is regulated on many levels, and possibly also on a post-translational level. METHODS: Post-translational modifications of NLRP3 were analyzed by mass spectrometry. Further investigation included mutating the modified residues in NLRP3 expression plasmids and reconstituting NLRP3-deficient cells. Analysis utilized biochemical readouts (ELISA, immunoprecipitation, immunoblots) and fluorescence microscopy. RESULTS: Three phosphorylated residues were identified. Mutation of the sites revealed that a phosphorylation-mimetic residue on one of these sites inhibited NLRP3 inflammasome activation. The residue is localized in the pyrin domain of NLRP3, which is responsible for protein-protein interaction. Further analysis found that self-interaction of the pyrin domain of NLRP3 as well as recruitment of its downstream adapter ASC were inhibited by the phosphorylation-mimetic residue. CONCLUSIONS: NLRP3 activity is likely regulated by phosphorylation of one residue in the pyrin domain. NLRP3 may need to be dephosphorylated before it can activate downstream signaling. Thus, this study adds a previously unknown layer of NLRP3 regulation, which may lead to novel strategies for treating diseases that involve NLRP3.

74

Ulas T., Pallasch C.P., Hallek M., Schultze J.L.

Comparative transcriptomics reveals gain of proliferative capacity, loss of phagocytic capacity and a distinct phenotype of tumor-associated macrophages in murine chronic lymphatic leukemia Ulas T.1;5, Pallasch C.P.2;5, Reinart N.2, Schwamb J.2, Ristovska V.2, Beyer M.1, Mallmann M.1, Heukamp L.3, Schmidt S.V.1 , Hallek M.2;4;6, Schultze J.L.1;6 1Genomics and Immunoregulation, LIMES Institute, University of Bonn, Germany 2Department I of Internal Medicine and Center of Integrated Oncology (CIO), University of Cologne, Germany 3Institute of Pathology, University of Cologne, Germany 4CECAD Cologne: Cellular Stress Responses in Aging-Associated Diseases 5Authors contributed equally 6sharing senior authorship [email protected] BACKGROUND: Tumor-associated macrophages (TAM) are a major component of the tumor microenvironment linked to reduced survival in most tumor entities. METHODS: Comparative transcriptomics in macrophages from spleen and bone marrow at pre-leukemic versus leukemic state was performed in a mouse model for chronic lymphocytic leukemia (CLL) and experimentally validated by assessing cell proliferation and phagocytosis. RESULTS: Comparative transcriptomics revealed profound re-programming of macrophages during leukemogenesis. Interestingly, gene set enrichment analysis (GSEA) showed little overlap with the prototype classical M1-like or alternative M2-like macrophage polarization patterns. Instead, major transcriptional changes indicated a gain of proliferative capacity and loss of phagocytic mechanisms as defined by gene ontology enrichment analysis (GOEA). These data clearly indicated that macrophages specifically integrate signals from the tumor microenvironment and the resulting transcriptional program is rather unique not following previous concepts of macrophage polarization. Indeed, increased proliferation of F4/80+ macrophages was demonstrated in leukemic animals at local tumor sites in vivo. Moreover, reduced phagocytic capacity of macrophages was validated in vivo in leukemic mice and in vitro using macrophages of CLL patients. CONCLUSIONS: Comparative transcriptomics of macrophages within the same tissue at pre-malignant versus malignant state allowed to identify important macrophage-mediated suppressive mechanisms in leukemia.

Science Day 2013

75

Vorac J., Förster I.

The role of the Aryl hydrocarbon Receptor Repressor (AhRR) in innate immunity and infection Vorac J., Weighardt H., Förster I. Immunology and Environment, LIMES Institute, University of Bonn, Germany [email protected] BACKGROUND: The Aryl hydrocarbon Receptor (AhR) is a ligand activated transcription factor, involved in the detoxification of xenobiotics and in innate immunity. The AhR is regulated by the AhRR through a negative feedback mechanism. METHODS: AhRR/EGFP-reporter (AhRRE/+) and knockout mice (AhRRE/E) were generated to determine expression and function of the AhRR. AhRR signaling was analysed after TLR stimulation in vitro. A LPS-induced septic shock model and Citrobacter rodentium infections were performed to specify the role of the AhRR in vivo. RESULTS: Analysis of naïve AhRRE/+ mice revealed constitutive AhRR/EGFP expression, mainly in immune cells of barrier organs. This AhRR/EGFP expression was enhanced by TLR stimulation in dendritic cells. Analysis of LPS-induced septic shock and Citrobacter rodentium infections revealed protection of AhRRE/E mice. Upon LPS challenge, AhRRE/E mice showed reduced levels of cytokines systemically and locally in the liver. Kupffer cells were strongly decreased in AhRRE/E mice, explaining the reduced cytokine production. Moreover, a decreased number of apoptotic cells and diminished levels of caspase-3 were detectable in the livers of AhRRE/E mice. CONCLUSIONS: AhRR deficiency decreases the immune response to LPS, suggesting a cross-talk between environmental sensing by the AhR/AhRR system and sensing of microbes by pattern recognition receptors.

76

Waiskopf N., Banin U., Soreq H.

Semiconductor Nanoparticles for Biological Applications: The Promise and Challenges Waiskopf N.1,2, Banin U.1, Soreq H.2 1The Institute of Chemistry and the Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat Ram, Jerusalem 2The Alexander Silberman Institute of Life Sciences and the Edmond and Lily Safra Center for Brain Sciences, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat Ram, Jerusalem [email protected] BACKGROUND: Synthesis and surface engineering of semiconductor nanocrystals (SC-NCs) with unique and controlled physical and chemical properties emerges as a powerful approach for biological and medical research. SC-NCs are characterized by high fluorescence quantum yield and large molar extinction coefficients, allowing fluorescence detection with high sensitivity. The absorbance spectrum of SC-NCs is broad and their photoluminescence spectrum is narrow, controlled and size-dependent, enabling performance of parallel multi-labeling experiments. Moreover, the high photochemical stability of SC-NCs make them most suitable for dynamic high-resolution imaging and direct follow-up in biological milieus. Nevertheless, constructing SC-NC-labeled macromolecules is challenging due to the need to ensure macromolecular stability, sustain the biological functions and avoiding cytotoxicity while achieving the desired application. Each of these demands requires specific design of the SC-NCs properties, their surface coating and the labeling process. METHODS and RESULTS: Here, I will present the construction of cholinesterase-bound SC-NCs as a specific example for demonstrating these promise and challenges of developing SC-NCs for biological applications. This will include SC-NCs synthesis, phase transfer, conjugation, purification and characterization of the conjugates. CONCLUSIONS: SC-NCs labeling of bio-molecules can open new venues to address key biological questions and improve understanding of the underlying mechanisms of diseases.

Science Day 2013

77

Weisheit C., Engel D.R.

Crosstalk between sentinel and helper macrophages permits neutrophil migration into infected epithelium Weisheit C.2, Schiwon M.1, Franken L.1, Gutweiler S.1, Dixit A.1, Thiebes S.1, Quast T.3, Fuhrmann M.4, Opdenakker G.5, Bernhagen J.6, Bucala R.7, Panzer U.8, Kolanus W3., Gröne H.J.9, Knolle P.A.1, Kurts C.1, Engel D.R.1

1Institutes for Molecular Medicine and Experimental Immunology, University Hospital, University of Bonn, Germany 2Clinic for Anesthesiology, University Hospital, University of Bonn, Germany 3Life and Medical Sciences Institute (LIMES), University of Bonn, Germany 4German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany 5Laboratory of Immunobiology, Rega Institute for Medical Research, University of Leuven, KU Leuven, Belgium 6Institute of Biochemistry and Molecular Cell Biology, RWTH Aachen University, Germany 7Yale University School of Medicine, New Haven, CT, USA 8Medizinische Klinik II, University Clinic Hamburg Eppendorf Germany 9Cellular and Molecular Pathology, German Cancer Research Center Heidelberg, Germany [email protected] BACKGROUND: The phagocytes of the innate immune system, macrophages and neutrophils, contribute to antibacterial defense, but their functional specialization and cooperation is unclear. METHODS: In a murine model of urinary tract infection we addressed the question how macrophages and neutrophils share the tasks during immune defense. RESULTS: Here we report that three distinct phagocyte subsets play highly coordinated roles in bacterial urinary tract infection. Ly6C- macrophages acted as tissue-resident sentinels that attracted circulating neutrophils and Ly6C+ macrophages. Such Ly6C+ macrophages played a previously undescribed helper role: once recruited to the site of infection, they produced the cytokine TNF, which caused Ly6C- macrophages to secrete CXCL2. This chemokine activated matrix metalloproteinase-9 in neutrophils, allowing their entry into the uroepithelium to combat the bacteria. CONCLUSIONS: In summary, the sentinel macrophages elicit the powerful antibacterial functions of neutrophils only after confirmation by the helper macrophages, reminiscent of the licensing role of helper T cells in antiviral adaptive immunity. These findings identify helper macrophages and TNF as critical regulators in innate immunity against bacterial infections.


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Wittlich M., Wohleber D.

T helper 1 cells induce liver damage after adenoviral infection of hepatocytes Wittlich M.1, Staudt V.2, Bopp T.2, Schmitt E.2, Knolle P.A.1, Wohleber D.1 1Institutes for Molecular Medicine and Experimental Immunology (IMMEI), University Hospital, University of Bonn, Germany 2Institute for Immunology, Johannes Gutenberg University Mainz, Germany [email protected] BACKGROUND: CD8+ cytotoxic T cells (CTL) are capable of releasing TNF and inducing apoptosis of infected hepatocytes after cross-presentation of viral antigen via liver sinusoidal endothelial cells. This novel non-canonical CTL effector function accounts for about 40% of the antiviral effector function. CD4+ T cells are necessary for an efficient licensing of dendritic cells and priming of CD8+ T cells. But since especially Th1 cells are a source of inflammatory cytokines like TNF, they could also be able to induce liver damage after viral infection. METHODS: Mice were infected with a recombinant hepatotropic adenovirus, which primarily infects hepatocytes and expresses ovalbumin, followed by adoptive transfer of in vitro differentiated CD4+ T cells. Protein fragments of ovalbumin can be presented to the OVA-specific H2-Ab1 (I-Ab)-restricted T helper cells. RESULTS: Preliminary findings so far support the ability of Th1 differentiated cells to induce liver damage, whereas Th2 differentiated cells, incapable of producing TNF, could not induce a comparable ALT elevation. This potential non-canonical effector function of Th1 cells is dependent on the amount of transferred cells. Future experiments will elucidate if other T helper cells with the potential to produce inflammatory cytokines, e.g. Th17 cells, are also capable of inducing liver injury.

Science Day 2013

79

Xue J., Schultze J.L.

Network engineering on transcriptional level reveals a multi-strata model of human macrophage activation Xue J.1, Schmidt S.V.1, Sander J.1, Draffehn A.1, Krebs W.1, Quester I.1, De Nardo D.2, Gohel T.D.1, Emde M.1, Ganesan H.1, Nino-Castro A.1, Mallmann M.1, Labzin L.2, Theis H.1, Kraut M.1, Beyer M.1, Latz E.2,3,4, Freeman T.C.5, Ulas T.1 , Schultze J.L.1,6 1Genomics and Immunoregulation, LIMES-Institute, University of Bonn,

Germany 2Institute of Innate Immunity, University Hospital, University of Bonn, Germany 3Division of Infectious Diseases and Immunology, UMass Medical School,

Worcester, MA 01605, USA 4German Center of Neurodegenerative Diseases (DZNE), Bonn, Germany 5The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Edinburgh, Midlothian EH25 9RG Scotland, UK 6Address correspondence and reprint requests to Prof. Dr. Joachim L. Schultze ([email protected]) [email protected] BACKGROUND: Macrophages respond to various environmental signals and their activation is associated with significant transcriptional reprogramming. While much progress has been made in understanding of macrophage activation and function, the transcriptional programs that regulate these processes remain poorly characterized. METHODS: We used monocyte-derived macrophages as a human model system and generated 384 transcriptome expression profiles including various activation signals from macrophages and other immune cell types (T-, B-, NK-cells, and DCs). Bioinformatics analyses such as co-regulation networks, Weighted Gene Correlation Network Analysis (WGCNA), reverse engineering of cellular networks, Gene Set Enrichment Analysis (GSEA) of alveolar macrophages were performed. RESULTS: We extend the current M1- and M2-polarization model into a multi-strata model of macrophage activation. Overlaying this model onto in vivo data leads to new models of human alveolar macrophage activation. Network engineering revealed a core of transcriptional regulators associated with macrophage activation complemented by sets of regulators leading to specific activation programs. Linking this information to the ImmGen data defined an activation-independent human and murine macrophage core signature. CONCLUSIONS: This large and unique transcriptome dataset of human macrophage activation is an important step forward to better understand how macrophages integrate and compute signals from their local microenvironment under inflammatory conditions.

80

Yayon N., Soreq H.

Intra-, Extra- and Intercellular miRNA-mediated Regulation of Cholinergic Synaptic Plasticity Yayon N., Soreq H. The Edmond and Lily Safra Center for Brain Science, The Department of Biological Chemistry and The Center for Bioengineering, The Hebrew University of Jerusalem, Israel 91904 [email protected] BACKGROUND: Synaptic plasticity contributes to learning, cognition and stress responses and involves modified clustering of ion channels or other key proteins at the synaptic cleft due to yet unclear delivery dynamics. Stimulus-driven changes in gene expression were conventionally considered too slow for controlling synaptic plasticity; however, small microRNAs (miRNA) may rapidly react to stimuli by binding to and blocking translation of existing synaptic protein-coding mRNAs. Correspondingly, rapid release of miRNA-mediated translational blockade over synaptic-delivered mRNAs was reported following electrical stimuli. Specifically, nicotinic induction of synaptic plasticity that may involve astrocyte-mediated signals depends on miRNA modulation. METHODS and RESULTS: MS2-mediated fluorescence signaling tools are constructed for live cell imaging with lentiviral and molecular beacon-derived real-time tracking of mRNAs and their regulating miRNAs. Human-originated cultured neurons subjected to modified external stimuli and cellular conditions will enable monitoring of synaptic changes in mRNA and miRNA transport and regulation. To address astrocyte contributions to cholinergic synaptic plasticity, we wish to combine these cells in co-cultures or in-vivo model systems. CONCLUSIONS: Development of real-time imaging systems could deepen our understanding of miRNA contributions to the dynamics and impact of synaptic plasticity and its implications to astrocyte-neuron communication in health and disease.

Science Day 2013

81

Zehner M., Burgdorf S.

Mannose receptor – a receptor for cross-presentation surrounded by ER components Zehner M., Burgdorf S. Cellular Immunology, Life and Medical Science Institute (LIMES), University of Bonn, Germany [email protected] BACKGROUND: If DCs internalize the model-antigen ovalbumin (OVA) by the mannose receptor (MR), such antigens are presented on MHC I molecules (cross-presentation) to antigen-specific cytotoxic T cells. MR-internalized antigens are targeted into a stable early-endosomal compartment, from which they need to be exported into the cytosol for proteasomal degradation. Thereby, the mechanisms for non-canonical protein transport across endosomal membranes and regulatory factors for this transport are poorly understood and in the center of my research.

METHODS: Beside the use of cloning and overexpression of different construct, western blotting and flow cytometry to analyze this mechanism of antigen export, we developed a novel method, which allowed a flow-cytometrical analysis of individual endosomes. RESULTS: We demonstrated that antigen translocation is controlled by the endocytic receptor and regulated by ubiquitination, which leads to the recruitment of p97, a member of the ER-associated degradation machinery that provided the driving force for antigen translocation. Furthermore, we identified TSG101 as a central regulator of MR ubiquitination and antigen translocation. In addition to this we were also able to identify sec61 as a promising candidate to build the pore that is needed for the export event itself. CONCLUSIONS: In this study, we were able to show that different factors of the ER are essential for cross-presentation and recruited to endosomal compartments. This brought us a closer insight into the critical events during cross-presentation and increased our understanding of this mechanism. In addition to this, we developed a method (endosomal flow cytometry), which will also be a helpful tool for future studies for different questions.


82

Zillinger T., Barchet W.

Structure-Function Analysis of STING Activation by c[G(2',5')pA(3',5')p] and Targeting by Antiviral DMXAA Zillinger T.1, Gao P.2, Hartmann G.1, Patel D.J.2, Barchet W.1

1 Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital, University of Bonn, Germany 2 Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, USA [email protected] BACKGROUND: Cytosolic Recognition of nucleic acids is a cornerstone of antimicrobial innate immunity. The cytosolic cGAMP synthase (cGAS) recognizes DNA and creates a unique second messenger, the cyclic dinucleotide (CDN) c[G(2’,5’)pA(3’,5’)p], which then activates the CDN-sensor STING, leading to type I Interferon production. The central role of STING in DNA sensing and the recent identification of mouse-specific STING-agonists such as DMXAA make it an attractive target for human drug-development. METHODS: Informed by X-ray cristallography data, point mutants of STING were designed. Primary human variants were amplified from cDNA of PBMCs. Structure-function relationships for STING mutants and variants were assessed by reconstitution of cGAS signaling, and by direct activation via CDNs or the small molecule drug DMXAA. RESULTS: All primary hSTING variants were 232R as opposed to the postulated consensus 232H. Human STING exhibited a preference for 2’5’ linked cGAMP and virtually no activity for c-di-GMP or the mouse-specific drug DMXAA. Point mutation of a single amino acid was sufficient to enable activation of human STING by DMXAA. CONCLUSIONS: The naturally predominant variants of hSTING exhibit higher versatility in CDN-isomer recognition compared to the 232H consensus, but are not activated by the small molecule DMXAA. DMXAA-responsive mutants S162A and S162G will guide the rational design of therapeutic activators of human STING.

Science Day 2013

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ImmunoSensation Cluster Members

ImmunoSensation Cluster members Cluster members (in alphabetical order) Name E-Mail Institute Ablasser, Andrea [email protected] Institute of Clinical Chemistry and

Clinical Pharmacology Al-Amoudi, Ashraf [email protected] German Center for Neurodegenerative

Diseases (DZNE) Bano, Daniele [email protected] German Center for Neurodegenerative

Diseases (DZNE) Barchet, Winfried [email protected]

bonn.de Institute of Clinical Chemistry and Clinical Pharmacology

Becker, Tim [email protected] German Center for Neurodegenerative Diseases (DZNE)

Betz, Regina [email protected] Institute of Human Genetics Bieber, Thomas [email protected]

bonn.de Clinic and Polyclinic for Dermatology and Allergy

Bovier, Anton [email protected] Institute for Applied Mathematics Bradke,Frank [email protected] German Center for Neurodegenerative

Diseases (DZNE) Brossart, Peter [email protected]

bonn.de Clinic and Polyclinic III

Buch, Susanne [email protected] Life and Medical Sciences (LIMES) Burgdorf, Sven [email protected] Life and Medical Sciences (LIMES) Dilloo, Dagmar [email protected]

bonn.de Department of Pediatric Hematology and Oncology

Drosten, Christian [email protected] Institute of Virology Fava, Eugenio [email protected] German Center for Neurodegenerative

Diseases (DZNE) Förster, Irmgard [email protected] Life and Medical Sciences (LIMES) Fröhlich, Holger [email protected] B-IT Bonn Garbi, Natalio [email protected] Institutes of Molecular Medicine and

Experimental Immunology (IMMEI) Gerzer, Rupert [email protected] Institute of Aerospace Medicine Gieselmann,Volkmar [email protected]

bonn.de Institute of Biochemistry and Molecular Biology

Halle, Annett [email protected] center of advanced european studies and research (caesar)

Hartmann, Gunther [email protected]

Institute of Clinical Chemistry and Clinical Pharmacology

Heneka, Michael [email protected]

Clinic and Polyclinic for Neurology

Hoch, Michael [email protected] Life and Medical Sciences (LIMES) Hölzel, Michael [email protected]

bonn.de Institute of Clinical Chemistry and Clinical Pharmacology

Hörauf, Achim [email protected]

Institute for Medical Microbiology, Immunology and Parasitology

Hornung, Veit [email protected] Institute of Clinical Chemistry and Clinical Pharmacology

Kalff, Jörg [email protected] Department of Surgery Kastenmüller, Wolfgang

[email protected] Institutes of Molecular Medicine and Experimental Immunology (IMMEI)

Kaupp, U. Benjamin [email protected] center of advanced european studies and research (caesar)

84

Knolle, Percy [email protected] Institutes of Molecular Medicine and Experimental Immunology (IMMEI)

Kolanus, Waldemar [email protected] Life and Medical Sciences (LIMES) Kurts, Christian [email protected] Institutes of Molecular Medicine and

Experimental Immunology (IMMEI) Lange, Christoph [email protected] Genomic Mathematics and

Bioinformatics Latz, Eicke [email protected] Institute of Innate Immunity Neumann, Harald [email protected] Institute of Reconstructive

Neurobiology Nickenig, Georg [email protected]

bonn.de Clinic and Polyclinic II

Nicotera, Pierluigi [email protected] German Center for Neurodegenerative Diseases (DZNE)

Nöthen, Markus [email protected] Institute of Human Genetics Novak, Natalja [email protected]

bonn.de Clinic and Polyclinic for Dermatology and Allergy

Oldenburg, Johannes

[email protected]

Institute of Transfusion Medicine and Immunohaematology

Pankratz, Michael [email protected] Life and Medical Sciences (LIMES) Sahl, Hans-Georg [email protected] Institute for Medical Microbiology,

Immunology and Parasitology Schultze, Joachim L. [email protected] Life and Medical Sciences (LIMES) Soreq, Hermona [email protected] Safra Center / Institute for Biological

Chemistry Spengler, Ulrich [email protected] Clinic and Polyclinic I Straßburg, Christian [email protected]

bonn.de Clinic and Polyclinic I

Thiele, Christian [email protected] Life and Medical Sciences (LIMES) Tüting, Thomas [email protected]

bonn.de Laboratory of Experimental Dermatology

Wachten, Dagmar [email protected] center of advanced european studies and research (caesar)

Weighardt, Heike [email protected] Life and Medical Sciences (LIMES) Zimmer,Andreas [email protected] Institute of Molecular Psychiatry

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Cluster-associated scientists (in alphabetical order) Name E-mail Institute Abdullah, Zeinab [email protected]

bonn.de Institutes of Molecular Medicine and Experimental Immunology (IMMEI)

Adjobimey, Tomabu [email protected] Institute for Medical Microbiology, Immunology and Parasitology

Albayram, Önder [email protected] Institute of Molecular Psychiatry Albert, Thilo [email protected] Institute of Transfusion Medicine and

Immunohaematology Alkhaldi, Weaam [email protected] German Center for

Neurodegenerative Diseases (DZNE) Asdonk, Tobias [email protected]

bonn.de Clinic and Polyclinic II

Bartok, Eva [email protected] Institute of Clinical Chemistry and Clinical Pharmacology

Bauernfeind, Franz [email protected] Institute of Clinical Chemistry and Clinical Pharmacology

Bekeredjian-Ding, Isabelle

[email protected]

Institute for Medical Microbiology, Immunology and Parasitology

Bennett, Estelle [email protected] Safra Center / Institute for Biological Chemistry

Beyer, Marc [email protected] Life and Medical Sciences (LIMES) Bilkei-Gorzo, Andras [email protected] Institute of Molecular Psychiatry Böttcher, Jan [email protected] Institutes of Molecular Medicine and

Experimental Immunology (IMMEI) Brenker, Christoph [email protected] German Center for

Neurodegenerative Diseases (DZNE) Casati , Anna [email protected] Department of Pediatric Hematology

and Oncology Coch, Christoph [email protected] Institute of Clinical Chemistry and

Clinical Pharmacology Craveiro, Rogerio [email protected]

bonn.de Department of Pediatric Hematology and Oncology

De Nardo, Christine [email protected] Institute of Innate Immunity De Nardo, Dominic [email protected] Institute of Innate Immunity Degen, Joachim [email protected] Life and Medical Sciences (LIMES) Diehl, Linda [email protected] Institutes of Molecular Medicine and

Experimental Immunology (IMMEI) Draffehn, Astrid [email protected] Life and Medical Sciences (LIMES) Driesen, Julia [email protected] Institute of Transfusion Medicine and

Immunohaematology Eckerle, Isabella [email protected] Institute of Virology Engel, Daniel [email protected] Institutes of Molecular Medicine and

Experimental Immunology (IMMEI) Engel, David [email protected] Life and Medical Sciences (LIMES) Faiz, Saqib [email protected] Clinic and Polyclinic for Neurology Franken, Lars [email protected] Institutes of Molecular Medicine and

Experimental Immunology (IMMEI) Franklin, Bernardo [email protected] Institute of Innate Immunity Fuss, Bernhard [email protected]> Life and Medical Sciences (LIMES) Gaffal, Evelyn [email protected] Laboratory of Experimental

Dermatology Gaikwad, Sadanand [email protected]

bonn.de Clinic and Polyclinic for Neurology

Ghanem, Alexander [email protected]

Clinic and Polyclinic II

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Göser, Felix [email protected] Clinic and Polyclinic I Gotot, Janine [email protected] Institutes of Molecular Medicine and

Experimental Immunology (IMMEI) Greenberg, David S. [email protected]

l Safra Center / Institute for Biological Chemistry

Heine, Annkristin [email protected]

Institutes of Molecular Medicine and Experimental Immunology (IMMEI)

Henrichfreise, Beate [email protected] Institute for Medical Microbiology, Immunology and Parasitology

Hochheiser, Katharina

[email protected]

Institutes of Molecular Medicine and Experimental Immunology (IMMEI)

Höchst, Bastian [email protected] Institutes of Molecular Medicine and Experimental Immunology (IMMEI)

Hoemig-Hoelzel, Cornelia

[email protected]


Horvath, Gabor [email protected] Institute of Innate Immunity Hübner, Marc [email protected] Institute for Medical Microbiology,

Immunology and Parasitology Junglen, Sandra [email protected] Institute of Virology Kaczmarek, Dominik [email protected]


Kastenmüller, Katrin [email protected] Institutes of Molecular Medicine and Experimental Immunology (IMMEI)

Kerstin , Nadja [email protected] Department of Pediatric Hematology and Oncology

Kim, Sarah [email protected] Institute of Human Genetics Kohlmeyer, Judith [email protected]


Kubarenko, Andriy [email protected]


Kübler, Kirsten [email protected]


Kummer, Markus [email protected]


Kümmerer, Beate [email protected] Institute of Virology Landsberg, Jenny [email protected]


Langhans, Bettina [email protected]

Clinic and Polyclinic I

Langhoff, Pia [email protected] Institute of Innate Immunity Layland, Laura laura.layland@microbiology-

bonn.de Institute for Medical Microbiology, Immunology and Parasitology

Linnartz-Gerlach, Bettina

[email protected] Institute of Reconstructive Neurobiology

Loch, Gerrit [email protected] Life and Medical Sciences (LIMES) Ludwig, Janos [email protected] Institute of Clinical Chemistry and

Clinical Pharmacology Ludwig-Portugall, Isis [email protected] Institutes of Molecular Medicine and

Experimental Immunology (IMMEI) Lutz, Philipp [email protected] Clinic and Polyclinic I Mankan, Arun [email protected] Institute of Clinical Chemistry and

Clinical Pharmacology Matzner, Uli [email protected] Institute of Biochemistry and

Molecular Biology Mirandola, Sandra [email protected] German Center for

Neurodegenerative Diseases (DZNE) Müller, Jens [email protected] Institute of Transfusion Medicine and

Immunohaematology

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Müller, Marcel [email protected] Institute of Virology Nischalke, Hans Dieter

[email protected]


Nolting, Jens [email protected] Institute of Clinical Chemistry and Clinical Pharmacology

Otte, David-Marian [email protected] Institute of Molecular Psychiatry Pradier, Bruno [email protected] Institute of Molecular Psychiatry Rácz, Ildiko [email protected] Institute of Molecular Psychiatry Renn, Marcel [email protected] Institute of Clinical Chemistry and

Clinical Pharmacology Reyes, Elisabet [email protected]

bonn.de Clinic and Polyclinic for Neurology

Schlee, Martin [email protected] Institute of Clinical Chemistry and Clinical Pharmacology

Schnaars, Mareike [email protected] center of advanced european studies and research (caesar)

Schröder, Matthias [email protected] Institutes of Molecular Medicine and Experimental Immunology (IMMEI)

Schuberth, Christine [email protected]


Schumacher, Johannes

[email protected] Institute of Human Genetics

Schumak, Beatrix [email protected] Institute for Medical Microbiology, Immunology and Parasitology

Shenhar-Tsarfaty, Shani

[email protected] Safra Center / Institute for Biological Chemistry

Specht, Sabine [email protected] Institute for Medical Microbiology, Immunology and Parasitology

Takahashi, Chihiro [email protected] Institute of Molecular Psychiatry Thelen, Melanie [email protected] Institute of Biochemistry and

Molecular Biology Van den Boorn, Jasper

[email protected] Institute of Clinical Chemistry and Clinical Pharmacology

Wehner, Sven [email protected] Department of Surgery Wohlleber, Dirk [email protected] Institutes of Molecular Medicine and

Experimental Immunology (IMMEI) Wojtalla, Alexandra [email protected] Institute of Molecular Psychiatry

88

Cluster-associated students (in alphabetical order) Name E-mail Institute Aschenbrenner, Anna [email protected] Life and Medical Sciences

(LIMES) Balbach, Melanie [email protected] center of advanced european

studies and research (caesar) Bald, Tobias [email protected] Laboratory of Experimental

Dermatology Barbash, Shahar [email protected] Safra Center / Institute for

Biological Chemistry Baumgart, Ann-Katrin [email protected] Institutes of Molecular Medicine

and Experimental Immunology (IMMEI)

Becker, Jessica [email protected] Institute of Human Genetics Beckert, Hannes [email protected] center of advanced european

studies and research (caesar) Beier, Esther [email protected] Institute of Clinical Chemistry and

Clinical Pharmacology Beinhauer, Anna [email protected] Life and Medical Sciences

(LIMES) Bertheloot, Damien [email protected] Institute of Innate Immunity Bodea, Liviu [email protected] Institute of Reconstructive

Neurobiology Böhnert, Volker [email protected]

m Institute of Clinical Chemistry and Clinical Pharmacology

Brandstätter, Olga [email protected] Life and Medical Sciences (LIMES)

Brosseron, Frederic [email protected]


Bruder, Ann Kristin [email protected] Institute of Clinical Chemistry and Clinical Pharmacology

Brueckner, Matthias [email protected] center of advanced european studies and research (caesar)

Buettner, Sven [email protected] German Center for Neurodegenerative Diseases (DZNE)

Cavlar, Taner [email protected] Institute of Clinical Chemistry and Clinical Pharmacology

Chauhan, Dhruv [email protected] Institute of Clinical Chemistry and Clinical Pharmacology

Cheng, Ru-Lin [email protected] Institutes of Molecular Medicine and Experimental Immunology (IMMEI)

Da Silva Santos , Telma Emanuela

[email protected] German Center for Neurodegenerative Diseases (DZNE)

Daßler, Juliane [email protected] Institute of Clinical Chemistry and Clinical Pharmacology

Dixit, Akanksha [email protected] Institutes of Molecular Medicine and Experimental Immunology (IMMEI)

Ebert, Thomas [email protected] Institute of Clinical Chemistry and Clinical Pharmacology

Echeverry Bautista, Fabio Andres

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center of advanced european studies and research (caesar)

Eisenhardt, Marianne [email protected]


Embgenbroich, Maria [email protected] Life and Medical Sciences (LIMES)

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Engel, Christina [email protected] Institute of Clinical Chemistry and Clinical Pharmacology

Eppler, Felix [email protected] Life and Medical Sciences (LIMES)

Fehlauer, Holger [email protected] center of advanced european studies and research (caesar)

Fornarelli, Alessandra [email protected] German Center for Neurodegenerative Diseases (DZNE)

Fülle, Lorenz [email protected] Life and Medical Sciences (LIMES)

Fußhöller, David M. [email protected] center of advanced european studies and research (caesar)

Gäbel, Yvonne [email protected] Institutes of Molecular Medicine and Experimental Immunology (IMMEI)

Gaidt, Moritz [email protected] Institute of Clinical Chemistry and Clinical Pharmacology

Gennequin, Benjamin [email protected]

Institute of Molecular Psychiatry

Gerbitzki, Nancy [email protected] Life and Medical Sciences (LIMES)

Ghule, Aishwarya [email protected] Institute of Molecular Psychiatry Gioran, Anna [email protected] German Center for

Neurodegenerative Diseases (DZNE)

Glässner, Andreas [email protected]


Glodde, Nicole [email protected] Laboratory of Experimental Dermatology

Göbel, Anna [email protected] Institutes of Molecular Medicine and Experimental Immunology (IMMEI)

Göhrs, Ramona [email protected] Institute of Molecular Psychiatry Goldeck, Marion [email protected] Institute of Clinical Chemistry and

Clinical Pharmacology Görgen, Simon [email protected] Institute of Innate Immunity Gosejacob, Dominic [email protected] Life and Medical Sciences

(LIMES) Gottschalk, Catherine catherine.gottschalk@uni-

bonn.de Institutes of Molecular Medicine and Experimental Immunology (IMMEI)

Grebe, Alena [email protected] Institute of Innate Immunity Gutweiler, Sebastian [email protected] Institutes of Molecular Medicine


Hackstein, Philipp [email protected] Institutes of Molecular Medicine and Experimental Immunology (IMMEI)

Hamzeh, Hussein [email protected] center of advanced european studies and research (caesar)

Hanin, Geula [email protected] Safra Center / Institute for Biological Chemistry

Heineck, Lukas [email protected] Institutes of Molecular Medicine and Experimental Immunology (IMMEI)

Hemmerling, Inga [email protected] Institute of Clinical Chemistry and Clinical Pharmacology

Hertrich, Carola [email protected] Institute of Clinical Chemistry and Clinical Pharmacology

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Herzner, Anna-Maria [email protected] Institute of Clinical Chemistry and Clinical Pharmacology

Heuser, Christoph [email protected] Institutes of Molecular Medicine and Experimental Immunology (IMMEI)

Holland, Tristan [email protected] Institutes of Molecular Medicine and Experimental Immunology (IMMEI)

Höning, Klara [email protected] Institute of Clinical Chemistry and Clinical Pharmacology

Hückesfeld, Sebastian [email protected] Life and Medical Sciences (LIMES)

Jäger, Paulina [email protected] Life and Medical Sciences (LIMES)

Jakobs, Christopher [email protected] Institute of Clinical Chemistry and Clinical Pharmacology

Janas, Marianne [email protected] Institutes of Molecular Medicine and Experimental Immunology (IMMEI)

Jansen, Vera [email protected] center of advanced european studies and research (caesar)

Jenniches, Imke [email protected] Institute of Molecular Psychiatry Jentgens, Eva [email protected] Life and Medical Sciences

(LIMES) Jikeli, Jan [email protected] center of advanced european

studies and research (caesar) Kaczmarek, Julita [email protected] Institutes of Molecular Medicine


Keßels, Steven [email protected] Institute of Clinical Chemistry and Clinical Pharmacology

Khaminets, Maria [email protected] Institute of Clinical Chemistry and Clinical Pharmacology

Kokordelis, Pavlos [email protected]


Komander, Karl [email protected] Institutes of Molecular Medicine and Experimental Immunology (IMMEI)

König, Jessica [email protected] Life and Medical Sciences (LIMES)

Kopatz, Jens [email protected] Institute of Reconstructive Neurobiology

Krämer, Benjamin [email protected]


Krause, Torsten [email protected] Institutes of Molecular Medicine and Experimental Immunology (IMMEI)

Krebs, Wolfgang [email protected] Life and Medical Sciences (LIMES)

Kreer, Chistoph [email protected] Life and Medical Sciences (LIMES)

Kreutzberg, Thomas [email protected] Institutes of Molecular Medicine and Experimental Immunology (IMMEI)

Labzin, Larisa [email protected] Institute of Innate Immunity Lambing, Silke [email protected] Institute of Clinical Chemistry and

Clinical Pharmacology Laskowski, Claudia [email protected] German Center for


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Mass, Elvira [email protected]> Life and Medical Sciences (LIMES)

Mathews, Mona [email protected] Institute of Reconstructive Neurobiology

Maurice, Nicholas J. [email protected] Institutes of Molecular Medicine and Experimental Immunology (IMMEI)

Mertens, Christina [email protected] Institute of Clinical Chemistry and Clinical Pharmacology

Mettke, Elisabeth [email protected] Institutes of Molecular Medicine and Experimental Immunology (IMMEI)

Meyer, Benjamin [email protected] Institute of Virology Meyer, Katharina [email protected] German Center for


Mitschka, Sibylle [email protected] Life and Medical Sciences (LIMES)

Mukherjee, Shatanik [email protected] center of advanced european studies and research (caesar)

Muth, Doree [email protected] Institute of Virology Nadorp, Bettina [email protected] Safra Center / Institute for

Biological Chemistry Nastaly, Maximilian maximilian.nastaly@googlemail.

com Institute of Clinical Chemistry and Clinical Pharmacology

Nent, Elisa [email protected] Institute of Molecular Psychiatry Niemeyer, Daniela [email protected] Institute of Virology Niepmann, Sven [email protected] Institute of Clinical Chemistry and

Clinical Pharmacology Nievendick, Hanna [email protected] Institutes of Molecular Medicine


Opitz, Frederike [email protected] Life and Medical Sciences (LIMES)

Papantonopoulou, Olympia

[email protected]

Life and Medical Sciences (LIMES)

Pelka, Karin [email protected] Institute of Innate Immunity Peters, Annika [email protected] Institutes of Molecular Medicine


Pohl, Judith-Mira [email protected] Institutes of Molecular Medicine and Experimental Immunology (IMMEI)

Quester, Inga [email protected] Life and Medical Sciences (LIMES)

Raju, Diana N. [email protected] center of advanced european studies and research (caesar)

Ramonet, David [email protected]


Rauen, Judith [email protected] Life and Medical Sciences (LIMES)

Reinhardt, Julia [email protected] Institute of Clinical Chemistry and Clinical Pharmacology

Rieck, Michael [email protected] Life and Medical Sciences (LIMES)

Riesenberg, Stefanie [email protected] Institute of Clinical Chemistry and Clinical Pharmacology

Ritz, Daniel [email protected] Institute of Virology Rogava, Meri [email protected] Laboratory of Experimental

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Dermatology Rohrbach, Jaqueline rohrbach.jaqueline@googlemail.

com Institutes of Molecular Medicine and Experimental Immunology (IMMEI)

Ruschel, Jörg [email protected] German Center for Neurodegenerative Diseases (DZNE)

Sander, Jil [email protected] Life and Medical Sciences (LIMES)

Schadow, Benjamin [email protected] Life and Medical Sciences (LIMES)

Schanz, Jan Oliver [email protected] Life and Medical Sciences (LIMES)

Schiffer, Christian [email protected] center of advanced european studies and research (caesar)

Schiwon, Marzena [email protected] Institutes of Molecular Medicine and Experimental Immunology (IMMEI)

Schlegel, Philipp [email protected] Life and Medical Sciences (LIMES)

Schmalenströr, Maren [email protected] Institute of Clinical Chemistry and Clinical Pharmacology

Schmid-Burgk, Jonathan

[email protected] Institute of Clinical Chemistry and Clinical Pharmacology

Schmidt, Tobias [email protected]


Schneider, Karin [email protected] Life and Medical Sciences (LIMES)

Schonauer, Sophie [email protected] center of advanced european studies and research (caesar)

Schulze, Anne V. [email protected] center of advanced european studies and research (caesar)

Schütte, Verena [email protected] Life and Medical Sciences (LIMES)

Schwickart, Anna [email protected] Institute of Clinical Chemistry and Clinical Pharmacology

Semmling, Verena [email protected] Institutes of Molecular Medicine and Experimental Immunology (IMMEI)

Seppmann, Torsten [email protected]

Life and Medical Sciences (LIMES)

Sommer, Daniel [email protected] Life and Medical Sciences (LIMES)

Stein, Axel [email protected] Institute of Biochemistry and Molecular Biology

Stein, Kathy [email protected] Department of Surgery Stunden, James [email protected] Institute of Innate Immunity Stutz, Andrea [email protected] Institute of Innate Immunity Surendran, Sandya [email protected] Life and Medical Sciences

(LIMES) Ternes, Svenja [email protected] Institute of Molecular Psychiatry Tittel, André [email protected] Institutes of Molecular Medicine


Tolksdorf, Felix [email protected] Life and Medical Sciences (LIMES)

Tuit, Sander [email protected] Life and Medical Sciences (LIMES)

Vento Asturias, [email protected] Institutes of Molecular Medicine

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Salvador and Experimental Immunology (IMMEI)

Waiskopf, Nir [email protected] Safra Center / Institute for Biological Chemistry

Wassermann, Ruth [email protected] Institute of Clinical Chemistry and Clinical Pharmacology

Welz, Meike [email protected] Institutes of Molecular Medicine and Experimental Immunology (IMMEI)

Welz, Meike [email protected] Institutes of Molecular Medicine and Experimental Immunology (IMMEI)

Wigger, Katrin [email protected] Institute of Clinical Chemistry and Clinical Pharmacology

Wijasa, Stella [email protected]


Wittlich, Michaela [email protected] Institutes of Molecular Medicine and Experimental Immunology (IMMEI)

Wollny, Robert [email protected] Institute of Virology Wolter, Franziska [email protected]


Xing, Qian [email protected] Life and Medical Sciences (LIMES)

Xue, Jia [email protected] Life and Medical Sciences (LIMES)

Yayon, Nadav [email protected] Safra Center / Institute for Biological Chemistry

Zehner, Matthias [email protected] Life and Medical Sciences (LIMES)

Zillinger, Thomas [email protected] Institute of Clinical Chemistry and Clinical Pharmacology

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