13. Minisymposium „Infektion und Immunabwehr“ Burg … 2009 - Programm... · Werner Stenzel et al., Berlin 8 Neuron and astrocyte –specific function of IKK-2 and NEMO in Toxoplasma

13. Minisymposium „Infektion und Immunabwehr“ Burg Rothenfels,

12. – 14. März 2009

13. Symposium "Infektion und Immunabwehr" Burg Rothenfels, 12. – 14. März 2009

2

SCIENTIFIC PROGRAM

13. Minisymposium „Infektion und Immunabwehr“

Burg Rothenfels,

12. – 14. März 2009

PROGRAMM

Organisation: C. Hölscher, Borstel

D. Schlüter, Magdeburg

G. van Zandbergen, Ulm

Donnerstag, 12.3.08

14.45 Abfahrt Bus-Shuttle Würzburg Hauptbahnhof (Vorplatz)

15.30 – 16.30 Ankunft, Zimmerverteilung, Kaffee und Gebäck

16.30 – 17.40 Session 1:

Signalling and effector molecules

Chair: Alexander Dalpke

1 Murine 65 kDa GBPs : important effector molecules in Toxoplasma infection.

Daniel Degrandi et al., Düsseldorf

2 Identification of a novel subcellular localization pattern of Suppressor of Cytokine Signalling – 1 (SOCS-1)

Julia Strebovsky et al., Heidelberg

3 SOCS-1 enhances Pasteurella multocida Toxin induced STAT3 activity

Katharina F. Kubatzky et al., Heidelberg


3

4 The role of Frizzled1 in M. tuberculosis infection : Modes of induction and first

insights into function

Jan Neumann et al., Kiel

5 Wnt ligands differentially regulate the inflammatory response of macrophages upon mycobacterial infection

Kolja Schaale et al., Kiel

6 Influence of optineurin on adenovirus E3-14.7K mediated TNF-resistance Wulf Schneider-Brachert et al., Regensburg

7 Downregulation of AP-1 proteins in Chlamydia pneumoniae infected host cells

Christiane Jugert et al., Luebeck

17.40-18.00 Pause

18.00 – 19.00 Session 2:

T and B cells

Chair: Andreas Limmer

1 Delayed type hypersensitivity versus humoral immune response – Influence of high and low antigen doses on the cytokine expression within spleenic compartments

Claudia Stamm et al., Luebeck

2 Injury – induced Th cell suppression after injury is restored through targeting dendritic cells in the regenerating tissue

Florian Wirsdörfer et al., Essen

3 Apoptotic Leishmania major mediate a Th2 response in BALB/c mice

Julia Barthelmann et al., Luebeck

4 Bacterial DRiPs, Yes or No

Silke Grauling – Halama et al., Heidelberg


4

5 In E.coli Nissle 1917 monocolonized Rag1 deficient mice CD4+ T cells are essential for the protection against dissemination and septic shock

Ute Eberle et al., Tuebingen

6 The regulatory T cell response during acute viral infection is locally defined and controls the magnitude and duration of the virus-specific cytotoxic T cell response

Kirsten Dietze et al., Essen

19.00 – 20.00 Abendessen

20.00 Key note lecture 1

Introduction: Dirk Schlüter

Frank Kirchof, Ulm: Mechanisms of HIV-1 immune evasion

ab ca. 21 Uhr Chill out

Freitag, 13.3.08

9.00 – 10.30 Session 3:

TLRs and innate immune responses

Chair: Christian Bogdan

1 TLR against R848 inhibits differentiation of mDCs and leads to differentiaton of tolerogenic APc from CD14+ monocytes

Sabine Woelfle et al., Heidelberg

2 Improvement of host defense in the lung

Thomas Tschernig et al., Hannover


5

3 The phosphatidylserine binding protein annexin A5 can interfere with

chlamydial infection.

Lisa Pfleiderer et al., Ulm

4 Characterization of dual production of IFN-beta and IL12/23 p40 on a single

cell basis after TLR stimulation.

Magdalena Kocur et al., Düsseldorf

5 Antiretroviral effects of Toll-like receptor ligands

Kathrin Gibbert et al., Essen

6 Functional analysis of cells from IFIT-2 knockout mice

Alexandra Siegfried et al., Tuebingen

7 The role of cytokines for NK cell activation in visceral leishmaniasis

Simone Haeberlein et al., Erlangen

8 Acute treatment against cutaneous leishmaniasis with a two component gel developing nitric oxide

Beate Lorenz et al., Mainz

9 The role of Natural killer (NK) T cells for protection against Leishmania major

infection

Klaus Griewank et al., Mainz

10:30 – 11:00 Kaffeepause

11.00 – 12.30 Session 4a:

Cytokines

Chair: Stefan Ehlers

1 Tumor-derived Prostaglandin E2 and TGF-beta synergize to inhibit PDC-derived IFN-alpha

Marijo Parcina et al., Heidelberg


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2 Role of type I IFNs in innate defence to Legionella pneumophila

Bastian Opitz Berlin

3 The visualization of IFN-beta producing cells versus their infection state in time course studies during of MCMV infection

Stephanie Borkens et al., Duesseldorf

4 Induction of pro-inflammatory IL-!2 in Plasmodium berghei infected BALB/c mice breaks blood-brain-barrier and leads to cerebral malaria

Kim Ellen Schmidt et al., Bonn

5 The role of interleukin 22 in Toxoplasma gondii induced ileitis

Melba Munoz et al., Berlin

6 The interleukin-13/ interleukin-4 receptor –alpha axis is involved in

tuberculosis associated pathology

Lisa Heitmann et al., Borstel

7 Blockade of IL-6 trans-signalling does not reactivate experimental tuberculosis

Jan Christian Sodenkamp et al., Borstel

8 IL-17 maintains protective immunity during Mycobacterium tuberculosis infection

Jochen Behrends et al., Borstel

9 The IL-23 / IL-17 axis is required for protective immune responses against

Trypanosoma cruzi infection

Hanna Erdmann et al., Borstel

12.30 – 13.30 Mittagessen

anschließend Spaziergang und Kaffee


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15.30 – 17.00 Session 4b:

Dendritic cells and macrophages

Chair: Christoph Hölscher

1 Molecular mechanisms involved in aberrant type 1 interferon-induction by

S.aureus protein A.

Sibel Durlanik et al., Heidelberg

2 Detrimental role of dendritic cells during Y.enterocolitica infection in vivo

Philipp Warnke et al., Tübingen

3 Leishmania amastigote propagation in human host cells

Elena Bank et al., Ulm

4 Role of Langerin+ skin-derived DC in Leishmania major infection

Kordula Kautz-Neu et al., Mainz

5 Opsonization of L.major with cross-reactive anti-phospholipid antibodies promotes phagocytosis by dendritic cells (DC) and induction of protective immunity

Susanna Lopez Kostka et al., Mainz

6 Role of hypoxia and HIF-1 alpa in dendritic cell immunobiology.

Jonathan Jantsch et al., Erlangen

7 Conventional and plasmacytoid bone marrow-derived dendritic cells contribute to Toll like receptor-independent IFN-alpha/ beta production in response to inactivated parapoxvirus ovis

Gottfried Alber et al., Leipzig

8 Activation of macrophages by the mycobacterial cord factor (TDM) and its synthetic analogue (TDB)

Hanne Schoenen et al., Erlangen


8

9 Interaction of Mycobacterium tuberculosis and human macrophages under hypoxic conditions.

Daniel Nickel et al., Ulm

17.00–17.30 Fachgruppenaktivitäten

Ger van Zandbergen, Dirk Schlüter Vorsitzende der Fachgruppe/des Arbeitskreises

17.30 Keynote lecture 2

Introduction: Ger van Zandbergen

Freddy Frischknecht, Heidelberg: Imaging motile pathogens

ca. 18.30 Uhr Buffet

Samstag, 14.3.08

9.00 – 10.10 Session 5a:

Immunomodulation

Chair: Norbert Reiling

1 Split tolerance after oral vaccination of mice with recombinant Escherichia coli Nissle 1917 expressing fimbrial adhesion K88

Katharina A. Remer et al., Würzburg

2 Acute graft-versus –host-disease after reduced intensity conditioning is

mediated by MyD88 mediated TLR9 sensing of bacterial DNA and can be

modulated by administration of TLR antagonist.

Rita Plickert et al., Berlin


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3 Long –term effect of sepsis –The influence of bacteremia and bacterial translocation on systemic adaptive immune responses

Timo Schwandt et al., Bonn

4 Systemic bacterial infection alters differentiation of dendritic cells in the bone marrow and mediates chronic dendritic cell dysfunction.

Eva Pastille et al., Essen

5 Increased susceptibility to infection with Aspergillus fumigatus in graft-versus-host disease is not due to impaired pathogen clearance

Bernd Echtenacher et al., Regensburg

6 Modulation of host macrophage apoptosis by Leishmania infection

Stefanie Enzenmüller et al., Ulm

7 Protective effect of filarial infection inhibiting malaria outcome in mice

Susanne Deininger et al., Bonn

10.10 – 10.30 Kaffepause

10.30 – 12.00 Session 5b:

Organ-specific regulation of immune responses

Chair: Gottfried Alber

1 Immune regulatory functions of alveolar type II epithelial cells

Dunja Bruder et al Braunschweig

2 Airway epithelial cells modify immune responses

Lotte M. Schmidt et al., Heidelberg

3 Corneal inflammation in response to filarial antigens

Katrin Gentil et al., Bonn


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4 The contribution of the innate, placental immune system to defend the fetus from infections

Diana Friedrich et al., Erlangen

5 A crucial role of the spleen in the induction of pathogenic host responses towards P. berghei ANKA infection

Beatrix Schumak et al., Bonn

6 The role of the intestinal microflora in infection with Citrobacter rodentium

Ulrich Steinhoff et al., Berlin

7 IL-4/ IL-13-dependent alternative activation of macrophages but not microglial cells is associated with uncontrolled cerebral cryptococcosis

Werner Stenzel et al., Berlin

8 Neuron and astrocyte –specific function of IKK-2 and NEMO in Toxoplasma encephalitis

Ulrike Händel et al., Magdeburg

9 Toxoplasma gondii induces behavioural changes in infected mice

M. Fahad Haroon et al., Magdeburg

12:00 Preisverleihung

12:05 Ende der Tagung

Lunchpaket / Mittagessen

13.00 Abfahrt Bus (pünktlichst!)


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ABSTRACTS (in alphabetical order of presenting authors)

Conventional and plasmacytoid bone marrow-derived dendritic cells contribute to

Toll-like receptor-independent IFN-alpha/beta production in response to inactivated

parapoxvirus ovis

Sabine Siegemund1, Andrea Hartl2, Franziska Dautel1, Ruediger Raue3, Marina A.

Freudenberg4, Mathias Buettner5, Gabriele Koehler6, Carsten J. Kirschning2, Tim

Sparwasser2, Gottfried Alber1

1Institute of Immunology, College of Veterinary Medicine, University of Leipzig, Germany; 2Institute of Microbiology, Technische Universitaet Muenchen, Germany; 3Pfizer Animal

Health, Kent, United Kingdom; 4Max Planck Institute for Immunobiology, Freiburg, Germany; 5Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Oberschleissheim,

Germany; 6Gerhard-Domagk-Institute for Pathology, Universitaetsklinikum Muenster,

Germany

Parapoxvirus ovis (PPVO) is a member of the Poxviridae family and belongs to the genus

Parapoxvirus. It displays only limited homology with orthopoxviruses and has some

molecular features such as an unusual high GC content distinct from orthopoxviruses.

Inactivated parapoxvirus ovis (iPPVO) has strong immunostimulatory capacities mediating

antiviral activity in vivo. The role of dendritic cells and the pattern recognition receptors

responsible for recognition of iPPVO are unknown. We demonstrate that bone marrow-

derived plasmacytoid dendritic cells (BM-pDC) and bone marrow-derived conventional

dendritic cells (BM-cDC) secrete IFN-alpha/beta in response to iPPVO. Furthermore, iPPVO

induces TNF-alpha and IL-12/23p40 release and MHC-II, MHC-I and CD86 up-regulation by

bone marrow-derived dendritic cells (BMDC). After engulfment, iPPVO was located in

endosomal compartments and in the cytosol of BMDC. Although iPPVO is a double-stranded

DNA virus, the DNA-recognizing toll-like receptor (TLR) 9 is not involved in iPPVO-induced

release of IFN-alpha/beta by BMDC. Moreover, we demonstrate that iPPVO elicits IFN-

alpha/beta by TLR-independent pathways, since IFN-alpha/beta release does not require

myeloid differentiation primary response gene 88 (MyD88) or TIR-domain containing adaptor

protein inducing interferon (TRIF). In contrast, iPPVO-induced TNF-alpha and IL-12/23p40

release and enhanced expression of MHC-I and CD86 but not MHC-II by BMDC partially

depends on MyD88 but not on TLR2, TLR4 or TLR9. These results provide first evidence

that iPPVO mediates its immunostimulatory properties by TLR-independent and -dependent

pathways and demonstrate an important role of cDC for IFN-alpha/beta production.


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Leishmania amastigote propagation in human host cells

Elena Bank1, Alexander Wenzel1 and Ger van Zandbergen1.

1Instiute for Medical Microbiology and Hygiene, University Clinic of Ulm

Leishmania major (L. major) is an obligate intracellular parasite elegantly misusing the

apoptotic cell clearance system for disease development. This parasite enters

polymorphonuclear neutrophil granulocytes (PMN) in its promastigote form. Hiding inside

Annexin A5 (AnxA5)-positive PMN the parasite transfers into macrophages (MF), where it

multiplies in its amastigote form. We found that the presence of AnxA5-binding population of

promastigotes is responsible for the survival and infectivity of viable promastigotes inside

PMN. Still relatively little is known about macrophage infection by the amastigote form of L.

major propagating the disease.

We developed a novel in vitro method to culture the amastigote form of L. major and

compared these amastigotes with promastigotes We found them to be higly infectious for

different types of human MF. To study amastigote development and propagation in more

detail, we generated eGFP expressing L. major. The eGFP marker is upregulated in the

amastigotes stage. Using these transfectants we infected type I (inflammatory) and typ II

(anti-inflammatory) human MF. We found that type II MF take up more parasites as

compared to type I MF. Using FACS analyses we could detect the development of

amastigotes by a higher expression level of eGFP as compared to the eGFP expression of

intraphagocytic promastigotes. Focussing on amastigote uptake using timelapse imaging we

found that dying eGFP-negative and AnxA5-positive amastigotes enter MF first followed by

viable eGFP-positive parasites. These new tools will enable us to examine L. major

amastigote propagation in human host cells.


13

Apoptotic Leishmania major mediate a Th2 response in BALB/c mice

Julia Barthelmann, Juergen Westermann, Kathrin Kalies

Institute of Anatomy, University of Luebeck

It has recently been reported that the virulent inoculum of Leishmania major (L. major)

promastigotes conventionally used for infection experiments contains about 50% of apoptotic

parasites. The depletion of apoptotic parasites lead to a reduced infectivity in BALB/c mice in

vivo.

In order to analyse the detrimental effect of apoptotic leishmania on the immune response in

the lymph node, we separated viable and apoptotic L. major by magnetic cell separation. We

studied T-cell proliferation and cytokine mRNA expression in the draining lymph node after

infection of susceptible BALB/c mice with viable L. major.

We found that the lack of apoptotic parasites in the inoculum leads to a delay of disease

progression and decreased IL-4 and IL-10 mRNA levels 6 weeks after infection, while

IFN-gamma levels remain unchanged. Furthermore, Th2-associated total serum IgG1 levels

are reduced. Disease, cytokine mRNA production and total serum IgG1 after 6 weeks could

be restored by adding apoptotic leishmania to the inoculum, thereby indicating that apoptotic

L. major support the establishment of a Th2 immune response.

Although the initial composition of the inoculum determines the disease development, the

response in the lymph node is not defined 3 days after infection: Time point and magnitude

of initial T-cell proliferation do not differ compared to conventional L. major infections and the

cytokine pattern does not correlate with the final disease outcome.

Overall, the removal of apoptotic parasites in the inoculum decreases the Th2 response, but

does not explicitly support the establishment of a Th1 phenotype in susceptible BALB/c.


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IL-17 maintains protective immunity during Mycobacterium tuberculosis infection

Jochen Behrends,1 Dominik Rueckerl,1 Manuela Heßmann,1 Uwe Mueller,2 Gottfried Alber,2

Yoichiro Iwakura,3 Stefan Ehlers4,5 & Christoph Hoelscher1

1Infection Immunology, Research Center Borstel, Germany; 2University of Leipzig, Germany; 3University of Tokyo, Japan; 4Microbial Inflammation Research, Research Center Borstel,

Germany; 5Molecular Inflammation Medicine, Christian-Albrechts-University, Kiel, Germany

Because a variety of autoimmune disorders have now been shown to depend on interleukin

(IL)-17-producing T helper (TH)17 cells, therapeutic blockade of TH17 development may

provide a novel approach to avoid adverse consequences of anti-inflammatory strategies

such as reactivation of latent tuberculosis (TB). To evaluate the potential risk of interfering

with IL-17-dependent inflammation, we analyzed the outcome of experimental TB in IL-17-

deficient (-/-) mice after infection with Mycobacterium tuberculosis (Mtb). IL-17 was important

for the induction of neutrophil chemokines after Mtb infection, but was not involved in

granuloma formation and protection during the first three months of Mtb infection. Mtb-

infected IL-17-/- mice efficiently generated interferon-gamma (IFN )-producing T cells and

IFNgamma-dependent effector responses. However, IL-17-/- mice were not able to control

mycobacterial replication during the chronic phase of experimental TB and died significantly

earlier than corresponding wildtype mice. This breakdown of immune protection in IL-17-/-

mice was associated with a drop in the frequency of IFN -producing CD4+ T cells. Our

findings reveal that IL-17 is essential for maintaining CD4+ T cell-dependent protection during

chronic stages of TB. Hence, interfering with IL-17-dependent pathways as an anti-

inflammatory therapeutic approach will possibly incur the danger of reactivating latent TB.

(Supported by the Inflammation Research Excellence Cluster)


15

The visualization of IFN-beta producing cells versus their infection state in time

course studies during of MCMV infection

Stephanie Borkens1, Vu Thuy Khanh Le2, Stefanie Scheu1

1Institute of Medical Microbiology and Hospital Hygiene, University of Duesseldorf 2Institute of Virology, University of Duesseldorf

Type I interferons are a family of multiple IFN-alphas and a single IFN-beta which were

initially identified on the basis of their antiviral activities. Previous findings identified pDCs as

the major IFN-beta producing cells in the spleen. However, which cells are actually infected

with MCMV remains unclear, although it has been suggested that at certain stages of

infection MCMV can actively replicate in macrophages and conventional DCs.

For a detailed analysis of the initial phase of MCMV infection, we used a MCMV strain which

expresses EGFP under the immediate-early promoter (MIEP) to infect IFN-beta/YFP reporter

knockin mice. This experimental approach allows for the simultaneous visualization of the

IFN-beta response and the infection status of MCMV in vivo.

In initial analyses at 24h post infection. we identified splenic pDCs as well as few CD11b+

cells expressing IFN-beta. Furthermore, we could show that the IFN-beta producing pDCs

are not directly infected with MCMV and that mainly CD11b+/c+ cells expressed EGFP as a

marker for MCMV infection.

Current studies aim at the clarification of IFN-beta-production versus the infection status of

individual cells in this simultaneous analysis during several time points of infection.


16

Immune regulatory functions of alveolar type II epithelial cells

Marcus Gereke, Harro Frauendorf, Dunja Bruder

Immune regulation group, Helmholtz Center for Infection Research, Braunschweig

Whereas the lung epithelium was long time related primarily with physical barrier and gas

exchange functions, the contribution of alveolar type II epithelial cells (AECII) in respiratory

immune regulation has become increasingly appreciated. However, their precise function in

the induction and regulation of T cell reactivity to self antigen remains poorly understood.

Utilizing a transgenic mouse model for CD4+ T cell mediated pulmonary inflammation we

found that MHC class II expressing AECII present self-antigen to CD4+ T cells resulting in

functional activation of lung-reactive T cells and finally autoimmunity. Importantly, we

unravelled a previously unknown immunological attribute of AECII in re-establishment of

peripheral T cell tolerance in the lung. Upon inflammation, AECII secrete a broad variety of

soluble factors including transforming growth factor-beta (TGF-beta), Platelet Factor-4 and

Surfactant Protein-A and -D, which suppress T cell proliferation and induce in a partially

TGF-beta dependent mechanism Foxp3 expression in lung-reactive CD4+ T cells. As a part

of the innate immune system AECII thus exhibit so far underestimated immune regulatory

function and synergize with adaptive immune mechanisms to re-establish tolerance to self-

antigen in the lung. Preliminary data suggest that upon influenza infection alveolar self

antigen recognition by CD4+ T cells does not result in increased abundance of Foxp3

expressing regulatory T cells thus indicating that the capability to respond to and to eradicate

a pathogen dominates about the necessity to protect against potentially harmful autoimmune

reactions. Currently we are investigating the contribution of AECII in balancing tolerogenic

and pathogen-specific immunity in the lung.


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Murine 65 kDa GBPs: important effector molecules in Toxoplasma infection.

Daniel Degrandi, Carolin Konermann, Cornelia Beuter-Gunia, Sarah Lahme, Anna Fischer,

Elisabeth Kravets, Klaus Pfeffer

Medizinische Mikrobiologie und Krankenhaushygiene. Heinrich-Heine-Universität Düsseldorf

The 65 kDa guanylate-binding proteins comprise a growing family of highly conserved

GTPases, found in mice, humans, and many other species. In mice, 11 members of the GBP

family have been described. In recent studies, we could show that several members of the

mGBP family colocalize with the parasitophorous vacuole (PV) of avirulent Toxoplasma

gondii (Tg) strains, suggesting a direct antiparasitic function of the mGBP proteins.

Interestingly, highly virulent Tg strains are able to modulate the cellular response to the

infection and suppress the recruitment of mGBP proteins to the PV. The mechanisms

involved in the recruitment of mGBP proteins to the PV and their molecular and biochemical

activity are the major focus of our current studies. First results indicate, that GTP-binding and

hydrolysis are needed for normal localization of the proteins in infected and uninfected cells.

Furthermore, the recruitment of mGBP proteins towards the Toxoplasma PV is dependent on

the microtubular network, since cells treated with Paclitaxel and other microtubule interacting

drugs showed no colocalization of mGBPs with the PV.

Studies in mGBP2-/- mice showed an increased susceptibility towards infections with Tg.

Also, mGBP2 deficient cells show a significantly reduced capability to control Tg growth in

vitro. These data indicate, that the family of 65 kDa mGBPs play crucial roles in the cell

autonomous defense against intracellular pathogens.


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Protective effect of filarial infection inhibiting malaria outcome in mice

Susanne Deininger1, Daniel Fernández-Ruiz1, Gwydion Brennan1, Bettina Dubben1, Achim

Hoerauf1, Sabine Specht1

1Institute for Medical Microbiology, Immunology and Parasitology, University Hospital Bonn,

Bonn, Germany

Malaria is a serious tropical disease with a high risk of mortality to date. Chronic helminth

infections and often co-endemic with malaria and both might alter the immune response

against another. By use of a co-infection model of the filarial nematode Litomosoides

sigmodontis and the malaria pathogen Plasmodium berghei ANKA in Balb/c mice we

discovered that a preceding filarial infection inhibited a subsequent infection with P. berghei

sporozoites in one-third of mice. Interestingly, this sterile protection was correlated with

presence of microfilariae, which are the first stage larvae of the filarial parasite. We observed

increased levels of activated T cells in spleen and liver of microfilaremic mice. Furthermore,

phagocytotic cells were increased in the spleens of these mice. In order to investigate the

underlying mechanism, we analysed cytokine profiles in these mice. We found that worm-

infected mice produced higher levels of IL-10, IFN-gamma and granzyme B and their CD4+

and CD8+ T cells were found to be in an activated status (CD69+) secreting IFN-gamma,

perforin and granzyme B.

Both candidates the cytotoxic T cells and the phagocytotic cells may contribute to the

mechanisms responsible for blocking parasitaemia of malaria pathogens in a part of filarial-

infected mice. A better knowledge about the protective mode of a patent filarial infection on

the outcome on malaria may provide some tools to influence the course of malaria in favour

for the host.


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The regulatory T cell response during acute viral infection is locally defined and

controls the magnitude and duration of the virus-specific cytotoxic T cell response

Kirsten Dietze1, Gennadiy Zelinskyy1, Ulf Dittmer1

1Institut fuer Virologie, Universitaetsklinikum Essen

Cytotoxic T-cells (CTL) facilitate control of acute viremia in many viral infections, including

retroviruses like HIV or HTLV. However, viruses that establish chronic infections have

developed mechanisms to evade destruction by CTL. We have used the Friend Virus (FV)

model to investigate these mechanisms. In the acute infection FV induces a strong CTL

response but the mice become persistently infected. However, regulatory CD4+ T cells (Treg)

that expand in the spleen of infected mice suppress the production of cytotoxic molecules in

CD8+ T cells and the cytotoxic function of CTL. The aim of our current work was to analyse

the compartmentalisation of the Treg response and the subsequent local suppression of

CD8+ T cells by Tregs during an ongoing retroviral infection. We found, that expansion of

effector CD8+ T cells, production of cytotoxic molecules and degranulation was directly linked

to viral loads in lymphatic organs. Consequently the expansion of induced Treg correlated

with the number and function of virus-specific CD8+ T cells. For the expansion of Treg the

presence of CTL was obligatory, what was shown by CD8 depletion experiments.

Furthermore, depleting Treg in DEREG mice resulted in enhanced expansion of effector

CD8+ T cells and improved the production of cytotoxic molecules leading to reduced viral

loads in lymphatic organs. In summary, during acute retroviral infection Treg downregulated

the expansion and function of virus-specific CTL. The immunosuppressive activity of Tregs

was locally defined to the organs in which efficient viral replication followed by a strong CD8+

effector cell response took place.


20

Molecular mechanisms involved in aberrant type I interferon-induction by S. aureus

protein A

Isabelle Bekeredjian-Ding1, Sibel Durlanik1, Marijo Parcina1, Sandra Ammann1, Wulf

Schneider-Brachert2 Klaus Heeg1

1Hygiene-Institute, Dept. of Med. Microbiology and Hygiene, UniversityHospital Heidelberg 2Institute for Med. Microbiology und Hygiene, University of Regensburg

Protein A (SpA), an immunoglobulin (Ig)-binding protein, is a major virulence factor of

Staphylococcus aureus and well-known as an immunostimulatory protein. We previously

observed that certain strains of S. aureus induce IFN-alpha secretion from plasmacytoid

dendritic cells (pDC). The aim of the project presented was therefore, to investigate the role

of SpA in type I interferon induction from pDC. Remarkably, IFN-a induction correlated with

bacterial levels of SpA expression, and subsequent experiments showed that recombinant

SpA induces IFN-alpha secretion from pDC. Moreover, IFN-I induction could be blocked by

saturating Ig or Fc fragments, indicating that the Ig-binding domain may be essential for IFN-I

induction. Most importantly, DNAse and RNAse digestion of recombinant SpA did not alter its

ability to induce IFN-I. Recently, SpA has been shown to bind TNFR1 on respiratory

epithelial cells. Here we show that anti-TNF-R mAb neutralisation can inhibit the SpA-

induced type I interferon secretion. We further show that SpA colocalizes with the TNF-R2 in

transfected HEK293 cells. Thus, SpA may represent a novel TLR-independent stimulus for

pDC activation.


21

In E. coli Nissle 1917 monocolonized Rag1 deficient mice CD4+ T cells are essential for

the protection against dissemination and septic shock.

Ute Eberle*1, Kerstin Fink*1, Martina Müller1, Frank Leithäuser2, Thomas A. Ölschläger3, Ingo

B. Autenrieth1, Julia-Stefanie Frick1

1Institute of Medical Microbiology and Hygiene, University of Tuebingen, Germany 2Institute of Pathology University of Ulm, Germany 3Institute of Molecular Biology of Infection, University of Wuerzburg, Germany *equal contribution

E. coli Nissle 1917 (EcN) is a well defined probiotic E. coli strain, which is effective in

maintaining remission in ulcerative colitis. The aim of this study was to explore safety

aspects of this frequently used probiotic strain. Therefore, germfree Rag1-/- mice were

monocolonized with EcN. Upon CD4+ T cell transfer germfree Rag1-/- mice showed a high

mortality rate due to dissemination of EcN. In peripheral organs high bacterial loads of EcN

were detected correlated with high levels of TNF in the serum. In contrast, SPF Rag1-/- mice

colonized with EcN showed no dissemination of EcN in peripheral organs even though the

numbers of bacteria in the intestine did not differ. Furthermore, EcN monocolonized C57Bl/6

mice showed no increased mortality rate, indicating that CD4+ T cells were essential either

for the inhibition of translocation or for the clearance of EcN from the peripheral organs. The

translocation and dissemination are an EcN specific effect, as it was not observed in

germfree Rag1-/- mice monocolonized with E. coli mpk, a commensal E. coli stain isolated

from the murine intestine. Additionally, the dissemination of EcN in monocolonized Rag1-/-

mice was not flagella dependent. As the translocation of EcN Dflic and DflgE to peripheral

organs was comparable to the translocation of wildtype EcN. Therefore, an unknown

bacterial factor seems to account for the dissemination of EcN in monocolonized Rag1-/-

mice, and both the immature intestinal barrier and the immature mucosa associated immune

system of germfree animals seem to be essential for the translocation of EcN.


22

Increased susceptibility to infection with Aspergillus fumigatus in graft-versus-host

disease is not due to impaired pathogen clearance

Bernd Echtenacher1, Kristina Doser 2, Matthias Edinger2 and Petra Hoffmann2

1Institute of Immunology, University Regensburg and 2Dept. of Hematology and Oncology,

University Hospital Regensburg, Regensburg, Germany

Graft-versus-host disease (GvHD) is a frequent and life-threatening complication after

allogeneic bone marrow transplantation (alloBMT) that is initiated by mature alloreactive T

cells within the graft. These show excessive proliferation and pro-inflammatory cytokine

secretion upon interaction with host antigen-presenting cells, which leads to a dysregulated

cytokine milieu and finally results in tissue damage and target organ destruction (gut, liver

and skin). Patients after alloBMT are severely immunocompromised and therefore

particularly prone to opportunistic bacterial and fungal infections. While immunosuppressive

medication for GVHD prophylaxis clearly raises the risk of infection, the contribution of GVHD

itself to this increased susceptibility is much less understood. To evaluate the impact of

GVHD on the host's defence against fungal infections, allo-transplanted BALB/c mice with or

without GVHD were infected intratracheally or intravenously with the clinically relevant

pathogen Aspergillus fumigatus. Mortality was significantly higher in GVHD animals (only

20% survival after 2wks) as compared to the non-GVHD group (75% survival for more than

5wks). Interestingly, clearance of the fungus from the lung after i.t. infection, or from spleen

and liver after i.v. infection, was rapid and comparable in both groups and no live fungus was

detectable in moribund animals. However, when lymphocytes isolated from spleen and liver

of infected animals were restimulated in vitro with germinating conidia, cells from GVHD

animals secreted significantly more pro-inflammatory TNF and IL-6 than those from control

mice. This suggests that an uncontrolled inflammatory immune response contributes to the

high morbidity and mortality of opportunistic infections in GVHD.


23

Modulation of host macrophage apoptosis by leishmania infection

Stefanie Enzenmüller1, Cordula Schropp2, Simone Fulda1, Ger van Zandbergen2 and Silke

Fischer1,2

1University Children's Hospital, Ulm. 2Institute for Medical Microbiology and Hygiene,

University Clinic of Ulm

Leishmania are obligate intracellular parasites which undergo a promastigote flagellated

stage in the sandfly and an aflagellated amastigote stage in vertebrate hosts. They cause a

spectrum of human diseases, ranging from self-limiting cutaneous infections to visceral

leishmaniasis. The infection of mammalian hosts is initiated by promastigotes that are

phagocytosed by macrophages either directly or after infection of neutrophils initially

recruited to the sandfly bite. In host cells Leishmania replicate and differentiate to

amastigotes. Like all intracellular parasites, Leishmania have evolved specialized strategies

to evade immune destruction and to interact with multiple apoptotic systems to complete their

life cycle. The repression of host cell apoptosis is one type of strategy to protect the host

macrophage in which the intracellular parasites replicate and persist.

Our main research interest is focused on how Leishmania major interferes with the host cell

apoptotic machinery in infected cells. Therefore we investigate how Leishmania modulate the

different signalling pathways of apoptosis. We used two different cell lines (monocyte-derived

macrophages THP1 and murine macrophage-like Raw 264.7). Cells were infected with L.

major in vitro and subsequently treated with external apoptosis-inducing stimuli such as UV-

light or staurosporine. First results are indicating that L. major inhibits caspase activation,

cytochrome c release from mitochondria and modulates the level of BH3-only proteins within

the infected macrophage cells.


24

The IL-23/IL-17 axis is required for protective immune responses against

Trypanosoma cruzi infection

Hanna Erdmann,1 Caroline Rossnagel,1 Nico Ghilardi,2 Yoichiro Iwakura,3 Thomas Jacobs,4

Christoph Hoelscher1

1Infection Immunology, Research Center Borstel, Germany; 2Genentech, South San

Francisco CA USA; 3 Center for Experimental Medicine, Institute of Medical Science,

University of Tokyo, Japan; 4Bernhard-Nocht-Institute for Tropical Medicine and Hygiene,

Hamburg, Germany

Interleukin (IL)-12 is a potent inducer of interferon-gamma (IFN )-producing T helper (TH)1

cells and promotes a protective cell-mediated immune response after infection with the

protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease. IL-23 is

structurally closely related to IL-12 and shares the IL-12/23p40 subunit and the

corresponding IL-12 receptor 1 subunit. However, IL-23 is not as potent as an inducer of

IFN -production than IL-12. Instead IL-23, but not IL-12, promotes the proliferation of IL-17-

producing TH17 cells. To analyze the role of IL-23 for protective immune responses during

experimental Chagas disease, IL-23p19 deficient (-/-) mice were infected with T. cruzi.

Compared to wild-type mice, IL-23p19-/- mice developed a higher parasitemia and an

increased mortality. However, this susceptibility was not due to an impaired TH1 immune

response. Because IL-23 supports the development of IL-17-secreting TH17 cells, we

infected IL-17-/- mice with T. cruzi to study the relevance of IL-17 for protective immune

responses. Like IL-23-/- mice, IL-17-/- mice exhibited a higher parasitemia, an elevated

mortality and an altered liver pathology. Moreover, TH1 immune responses were not affected

by the absence of endogenous IL-17. Together, we suggest here that in addition to TH1

cells, IL-23-dependent TH17 cells are required for a successful resolution of T. cruzi

infection. One important effector cytokine that mediates this IFN -independent arm of the

protective immune response during experimental Chagas disease appears to be IL-17.

Downstream effector mechanisms induced by IL-17 are currently under investigation.

(Supported by the Inflammation Research Excellence Cluster).


25

The contribution of the innate, placental immune system to defend the fetus from

infections

Diana Friedrich, André Gessner, Daniela Klaffenbach, Jörg Dötsch, and Markus Schnare

Institut für Mikrobiologie - Klinische Mikrobiologie, Immunologie und Hygiene des

Universitätsklinikums Erlangen, Wasserturmstr. 3-5, 91054 Erlangen

The placenta establishes an anatomical barrier between the maternal side and the

developing fetus. Nevertheless, it is a potential origin where pathogens coming from the

maternal tissue might traverse to the fetal itssue. Therefore, we hypothesized that the

placenta upon pathogen encounter is able to orchestrate an immune response by releasing

inflammatory chemokines and cytokines. Furthermore the placenta might be able to express

a broad repertoire of antimicrobial effector proteins to directly fight against the incoming

bacterial threat.

The stimulation of isolated placental cells with TLR-ligands or complete bacteria enhanced

the secretion of the chemokine IL-8, the proinflammatory cytokines IL-6 and TNF as well as

the anti-inflammatory cytokine IL-10 in a stimulus dependent manner. In addition purified

placental cells expressed the antimicrobial effector proteins bactericidal/permeability-

increasing (BPI), beta-defensin 2, secretory leucocyte protease inhibitor and acyloxyacyl

hydrolase. Group B streptococci were potent stimulators of these products.

Because the synthesized spectrum of AMPs and cytokines identified from isolated placenta

cells is similar to what can be observed from neutrophilic granulocytes, we separated

trophoblasts from lymphocytes by MACS. Indeed by flow cytometry we could demonstrate

that granulocytes were present in the isolated trophoblast fraction. After the separation no

lymphocytes could be detected in the purified cell fraction. Via confocal scanning laser

microscopy we were able to detect intracellular expression of BPI in purified trophoblasts.

This separation step will enable us to determine the specific contribution of the purified

trophoblasts and the associated hematopoietic cells for the expression of the cytokines or

antimicrobial effector proteins.


26

Corneal inflammation in response to filarial antigens

Katrin Gentil1, Eric Pearlman2, Achim Hoerauf1

1 Institute for Medical Microbiology, Immunology and Parasitology, University Clinic Bonn 2 Department of Ophthalmology and Center for Global Health and Diseases, Case Western

Reserve University, Cleveland, OH, USA

River blindness caused by the filarial nematode Onchocerca volvulus is the second leading

cause of blindness in the developing world. Infections with O. volvulus cause corneal

inflammation eventually leading to sclerosing keratitis and blindness.

Our group has been using a mouse model of filarial infection to investigate the mechanisms

of neutrophil migration to the cornea. We were investigating the role of Toll-like receptors

(TLRs) in the generation of adaptive immune responses and migration of granulocytes to the

corneal stroma. We found decreased IFNgamma production by splenocytes of immunized

TLR2-/- mice when compared with immunized C57BL/6 mice. Similarly, we found decreased

CXC chemokine production in the cornea and decreased neutrophil infiltration into the

corneal stroma in TLR2-/- mice. In contrast, IL-5 production and eosinophil migration were

unaffected in TLR2-/- mice.

Further analysis revealed decreased neutrophil migration in IFNgamma-/- mice. We could

demonstrate that IFNgamma primes macrophages for activation by filarial extracts by

upregulation of TLR2 expression on the cell surface leading to increased production of pro-

inflammatory cytokines. These in turn induce CXC chemokine production by corneal

fibroblasts that attract neutrophil migration to the cornea.

These findings demonstrate that although parasites typically induce strong Th2 responses,

the low levels of Th1 responses present during filarial infection are potent inducers of

neutrophil migration and corneal inflammation.


27

Antiretroviral effects of Toll-like receptor ligands

Kathrin Gibbert1, Ulf Dittmer1

1Institut für Virologie; Universitaetsklinikum Essen

The Toll-like receptor (TLR) system plays an important role in the recognition of infectious

pathogens and their signals induce the coordinated activation of innate and adaptive immune

response. Nothing was known about the simultaneous activation of the TLR 3, 7 and 9

ligands which induce various cytokines like Type I Interferons or Interleukin-12 in acute

retroviral infection. In the current study, we analyzed the expression of inflammatory

cytokines by BM-derived DCs stimulated with different combinations of TLR ligands. A

simultaneous incubation with the TLR 3 ligand Poly I:C and the TLR 7 ligand Resiquimod

(R848) led to a stronger induction of IL-12 compared to the stimulation with just one of those

ligands. We used the Friend retrovirus model to get more insight into the role of the

combined TLR ligand stimulation in an acute retroviral infection. Treatment of FV-infected

mice with Poly I:C and R848 alone or in combination led to a strong reduction in viral loads in

the blood and the spleen. An expansion of NK cells and a strong activation of B cells was

found in the TLR ligand treated animals. We could show that the depletion of CD8+ T cells in

Poly I:C-treated, FV-infected animals led to an increase of viral loads in the spleen and the

bone marrow whereas the depletion of NK cells resulted in higher viral loads in the bone

marrow but not in the spleen. In summary Poly I:C and R848 showed a very strong antiviral

activity in vivo. Thus, the use of TLR ligands with strong antiviral activity might be a good

feature for the treatment of retroviral infections.


28

Bacterial DRiPs, Yes or No?

Silke Grauling-Halama and Gernot Geginat

Institut für Medizinische Mikrobiologie und Hygiene, Fakultät für Medizin Mannheim der

Universität Heidelberg, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim

The rapid recognition of infected cells by CD8 T cells requires fast processing of pathogen-

derived antigens in the cytoplasm of the host cell followed by subsequent presentation of

antigenic peptides in the context of MHC class I molecules on the surface of infected cells. A

still unsolved paradox is the discrepancy between a rather long half-live of antigens in the

presence of rapid presentation of pathogen-derived antigenic peptides. Studies with virally

infected cells suggest that instable structurally altered, so-called defective ribosomal

products “DRiPs” are the main source of antigen in infected cells. As viral DRiPs can’t be

studied independent of host cells we took advantage of the facultative intracellular bacterium

Listeria monocytogenes which enabled us to characterize T cell antigens in infected cells as

well as independent of host cells. Our data indicate that similar to virally infected cells the

rapid processing of L. monocytogenes derived T cell antigens also depends on an instable

form of antigen. In contrast to viral DRiPs, however, the instable form of antigen is not

defined by structural alterations of antigens but is a temporary state that antigens are

exposed to in their statu nascendi. In summary, our studies suggest a general antigen

processing model that does not necessitate the assumption of defective protein synthesis as

basis of rapid antigen processing and presentation of stable T cell antigens.


29

The role of Natural killer (NK) T cells for protection against Leishmania major infection

Klaus Griewank, Beate Lorenz, Michael Fischer, Susanna Lopez Kostka, Esther von Stebut-

Borschitz

Dept of Dermatology, Johannes Gutenberg-University, Mainz

Leishmaniasis is a serious disease affecting 12 million people worldwide and causing tens of

thousands of deaths every year. Infection with Leishmania is also a classic immunological

model with resistant mouse strains developing Th1 immunity and susceptible strains initiating

a Th2 response leading to failure to contain the infection and eventually death. Here we

studied the influence of NKT cells on the development of immunity against L. major using a

physiological low dose infection model. Our initial results showed that NKT cell-deficient

(CD1d-/- or Ja18-/-) C57BL/6 mice were better able to contain Leishmania infections than

their wildtype counterparts. Lesions and parasite burdens in CD1d-/- mice were at least 2-

fold smaller than in C57BL/6 mice (lesions ~2-fold difference wk3, 5 and 8, p<0.05 to 0.0001,

parasite burden ear wk5 p<0.05). Application of 100 ng of the NKT cell stimulating glycolipid

alpha-Galactosyl-Ceramide (aGalCer) led to a more severe course of disease in C57BL/6

mice associated with higher parasite burdens in infected lesions and spleens (>10 fold; p<0.1

ear, p<0.05 spleen). In contrast, aGalCer administration to susceptible BALB/c mice

improved their ability to contain Leishmania infections as measured by differences in lesion

sizes and parasite burdens ( 5–fold; p< 0.05 wk5 and 8). In summary, our findings show that

in low dose infections with L. major, NKT cells can alter the immune response against

Leishmania, most likely by influencing the cytokine milieu early on. Modulating NKT cell

responses, for instance with glycolipid ligands, could be a successful approach to develop a

long awaited vaccine against this important human pathogen.


30

The role of cytokines for NK cell activation in visceral leishmaniasis

Simone Haeberlein, Heidi Sebald, Ulrike Schleicher and Christian Bogdan

Microbiology Institute - Clinical Microbiology, Immunology and Hygiene; University Clinic of

Erlangen, Wasserturmstraße 3-5, 91054 Erlangen

The innate immune response in C57BL/6 mice infected with the intracellular protozoan

parasite Leishmania (L.) infantum, an agent of visceral leishmaniasis, is characterized by the

stimulation of natural killer (NK) cells. NK cells activated in vivo show enhanced cytotoxic

activity and secrete interferon (IFN)-gamma which is crucial to induce production of

Ieishmanicidal nitric oxide by infected macrophages.

We previously demonstrated that the activation of NK cells in L. infantum-infected mice

requires the presence of myeloid dendritic cells that are able to phagocytose the parasites

and sense them by toll-like receptor 9. This stimulates the production of interleukin (IL)-12,

which was shown to be essential for the induction of NK cell effector functions, whereas type

I interferons were dispensable. In our ongoing studies we now identified IL-18 as an

additional signal for NK cell activation during L. infantum infection. The IFN-gamma secretion

and the anti-tumor cytotoxic activity of splenic NK cells from infected C57BL/6 IL-18-/- mice

was significantly reduced compared to wild-type controls. Surprisingly, the degranulation of

NK cells measured by CD107a surface expression, which is assumed to correlate positively

with cytotoxicity, was in fact increased in IL18-/- mice, despite the decreased cytolytic activity

in the chromium-release assay.

In conclusion, priming of NK cells by IL-18 is crucial to achieve full NK cell activation in L.

infantum-infected mice. Subsequent work will further investigate the differential effects of IL-

18 and analyse whether additional cytokines such as IL-15 are also involved in the process

of NK cell activation in experimental leishmaniasis.


31

Neuron- and astrocyte-specific function of IKK-2 and NEMO in Toxoplasma

encephalitis

Ulrike Händel,1 Martina Deckert,2 Dirk Schlüter 1

Institut für Medizinische Mikrobiologie, OvG Universität Magdeburg, 39120 Magdeburg,

Germany; 2 Abteilung für Neuropathologie, Universität zu Köln, 50937 Köln, Germany;

The obligate intracellular parasite Toxoplasma gondii induces a chronic persisting

encephalitis and is controlled by T and B cells. Although T. gondii infects brain resident cells

including astrocytes, neurons and microglia, the function of these cells in Toxoplasma

encephalitis (TE) is much less well defined compared to T and B cells. Since the transcription

factor NF- B is a major regulator of immune responses and intracellular survival of T. gondii

may be dependent on a manipultation of the NF-kB pathway, we studied the role of neuronal

and astrocytic IKK-2 and NEMO, two members of the NF-kB signalling cascade, in TE.

After infection of mice deficient in neuronal or astrocytic expression of IKK-2 and NEMO,

respectively, with T. gondii, all mouse strains developed a TE with only slight differences in

survival and parasite control as compared to control mice. However, neuronal loss was more

pronounced in inflammatory lesions of mice with neuronal deletion of IKK2. In addition,

cultured primary IKK2-deficient neurons exhibited an increased cell death after infection with

T. gondii as compared to neurons from control mice.

These findings suggest that IKK-2 is important for an optimal survival of neurons in TE but

that a topographally restricted loss of neurons close to inflammatory infiltrates does not result

in an increased mortality.


32

Toxoplasma gondii induces behavioural changes in infected mice.

M. Fahad Haroon1, F. Angenstein4, H. Lison2, W. Wetzel2, J. Goldschmidt3, H. Scheich2, E.

Budinger2, D. Schlüter1

1 Institute of Medical Microbiology, Universitätsklinikum Magdeburg, Leipziger Straße 44,

39120 Magdeburg 2 Department Auditory Learning and Speech, Leibniz Institute for Neurobiology (IfN),

Brenneckestraße 6, 39118 Magdeburg 3 Special Laboratory Non-invasive Brain Imaging, Leibniz Institute for Neurobiology (IfN),

Brenneckestraße 6, 39118 Magdeburg 4 Special Laboratory Behavioural Pharmacology, Leibniz Institute for Neurobiology (IfN),

Brenneckestraße 6, 39118 Magdeburg

It has been previously reported that Toxoplasma gondii-infected mice exhibit an altered

behaviour and loose their natural fear of cat urine. However, it is unclear whether these

behavioural changes are due to (i) infection of brain areas important for behaviour, e.g.

amygdala, (ii) active alteration of neuronal functions by the parasite or (iii) the ensuing

immune response. To address these questions we infected BALB/c mice with T. gondii cysts

(DX strain) and analysed these animals at days 30 and 60 pi. MRI and histology depicted

large, randomly distributed inflammatory lesions in the infected mice without any predilection

for brain areas modulating behaviour. These findings suggest that a preferential anatomical

location of parasite-induced inflammation is not responsible for behavioural changes of mice.

Immunohistochemistry showed that T. gondii infected mainly glial cells and neurons and that

T. gondii cysts persisted in neurons. Interestingly, cyst harbouring neurons had frequently

axonal contact to non-infected neurons and both, the axon and the non-infected neuron,

stained positive for amorphous T. gondii antigen. This may suggest that the persisting

obligate intracellular parasite might actively influence the neuronal activity of non-infected

neurons. Furthermore, thallium uptake experiment revealed that cyst harbouring neurons

showed no dynamic influx of potassium, which indicates that they were electrically silent and

functionally inactive. This might additionally contribute to an altered neuronal activity and

behavioural changes of mice.


33

The interleukin-13/interleukin-4 receptor-alpha axis is involved in tuberculosis-

associated pathology

Lisa Heitmann,1 Tanja Schreiber,1 Thorsten Thye,2 Andrew N. J. Mckenzie,3 Frank

Brombacher,4 Rolf D. Horstmann,2 Christian G. Meyer,2 Stefan Ehlers,5,6 & Christoph

Hoelscher1

1Infection Immunology, Research Center Borstel, Germany; 2Bernhard Nocht Institute for

Tropical Medicine, Hamburg, Germany; 3MRC Laboratory of Molecular Biology, Cambridge,

UK; 4University of Cape Town, South Africa; 5Molecular Inflammation Medicine, Research

Center Borstel, Germany; 6Molecular Inflammation Medicine, Christian-Albrechts-University,

Kiel, Germany.

Reactivation and granuloma necrosis (GN) are pivotal events of post-primary tuberculosis

(TB). To date, there is no clear evidence whether interleukin (IL)-4 and IL-13 are involved in

the pathogenesis of the disease. Therefore, we genotyped polymorphisms in IL-4, IL-13 and

the IL-4 receptor alpha-chain (IL-4R ) in patients with pulmonary TB from Ghana. A

structural variant of the IL-4R , which has been shown to be associated with an increased

transcription of the gene, was found to be significantly associated with increased cavity size

indicating a prominent role of the IL-4R in human GN. In Mtb-infected mice, however, IL-4

and IL-13 are only moderately induced and necrotizing granulomas are not apparent. To

address the question how IL-4R -mediated mechanisms affect the outcome of murine TB,

IL-13-overexpressing (IL-13tg) mice were infected with Mycobacterium tuberculosis (Mtb).

Aerosol infection of IL-13tg mice resulted in a profound induction of arginases concomitant

with increased bacterial loads at chronic stages of the disease very much resembling

reactivation TB. Importantly, in IL-13tg mice Mtb infection resulted in tissue pathology similar

to GN in human TB. From our data we conclude that IL-4R -dependent mechanisms are

involved in the pathogenesis of post-primary TB. (Supported by a BMBF Collaborative Grant

and the Inflammation Research Excellence Cluster).


34

Role of hypoxia and HIF-1alpha in dendritic cell immunobiology

Jonathan Jantsch1, Johannes Schödel2, Melanie Volke1,2 , Nadine Turza3, Ulrike Schleicher1,

Alexander Steinkasserer3, Christian Bogdan1, Markus Schnare 1, Carsten Willam 2

1Microbiology Institute – Clinical Microbiology, Immunology and Hygiene, 2Department of

Nephrology and Hypertension, and 3 Department of Dermatology, University Hospital

Erlangen, Friedrich-Alexander University of Erlangen-Nuremberg, Erlangen, Germany

Dendritic cells play a key role in linking innate and adaptive immunity. In inflamed tissues

oxygen tensions are thought to be low. The role of hypoxia and of the key player of hypoxic

gene regulation, the transcription factor hypoxia inducible factor 1alpha (HIF-1 alpha), are

largely unkown. Therefore, we investigated the effects of hypoxia and HIF-1alpha on murine

DC activation and function in the presence or absence of lipopolysaccharide (LPS).

Hypoxia alone did not activate murine DC, whereas hypoxia combined with LPS led to

augmented DC activation compared with LPS alone. Interestingly even under normoxic

conditions, DC activation was accompanied by accumulation of HIF-1alphaprotein levels,

induction of glycolytic HIF target genes, and enhanced glycolytic activity. Predominantly, this

LPS induced stabilisation of HIF-1alpha under normoxic conditions relies on a MyD88-

dependent NF-kappaB activity. Using RNA interference techniques, knockdown of

HIF-1alpha significantly reduced glucose use in DC, inhibited their maturation, and led to an

impaired capacity of DC to stimulate allogeneic T cells.

Altogether, our data indicate that HIF-1 alpha and hypoxia play a crucial role for DC

activation in inflammatory states.


35

Downregulation of AP-1 proteins in Chlamydia pneumoniae infected host cells

Christiane Jugert1, Matthias Klinger2, Werner Solbach1, Ger van Zandbergen3,

Jan Rupp1

1Institut für Med. Mikrobiologie und Hygiene, Ratzeburger Allee 160, 23538 Luebeck 2Institut für Anatomie, Ratzeburger Allee 160, 23538 Luebeck 3Institut für Med. Mikrobiologie und Hygiene, Albert-Einstein-Allee 11, 89081 Ulm

The development of Chlamydia pneumoniae (Cp) depends on the regulation of cell type

specific transcription factors of the host cells. The protein families of the transcription factor

AP-1 are known to determine cell life and death in response to extracellular stimuli.

Chlamydia-infected host cells are resistant to apoptosis induced by a wide spectrum of

proapoptotic stimuli. Our work focuses on the regulation of AP-1 proteins in Chlamydia

infected host cells.

HEp2-cells were infected with Cp for up to 72 hours. Nuclear proteins were extracted and the

DNA binding activity of AP-1 was measured by EMSA. To analyse the expression of different

AP-1 proteins, Western blot analysis were performed.

Whereas AP-1 activity was upregulated in Cp infected HEp2-cells in the early phase of the

developmental cycle, AP-1 proteins were downregulated in the late phase. Degradation of

c-Jun protein could also be shown in a degradation assay with cell extracts.

The overexpression of c-Jun through transient transfection resulted in smaller inclusion

bodies and lower recovery indicating that down regulation of c-Jun is necessary for rapid

development of Cp.

Summing up, our data indicate that AP-1 proteins are differentially regulated in early and late

stages of Cp infected HEp2-cells. We assume that Cp downregulates the expression of AP-1

proteins through degradation to inhibit host cell apoptosis and to fulfil its developmental

cycle.


36

Role of Langerin+ skin-derived DC in Leishmania major infections

Kordula Kautz-Neu1, Madelon Noordegraaf2,3, Stephanie Dinges1, Clare L. Bennett2,4, Björn

E. Clausen2,3, Esther von Stebut1

1Dept. of Dermatology, Johannes-Gutenberg University, 55131 Mainz, Germany; 2Dept. of

Cell Biology and Histology, Academic Medical Center, University of Amsterdam, 1105 AZ

Amsterdam, The Netherlands; 3Dept. of Immunology, Erasmus University Medical Center,

3015 GE Rotterdam, The Netherlands; 4Dept. of Haematology, University College London,

Royal Free Hospital, London, NW3 2PF, United Kingdom

Langerhans cells (LC) constitute a unique subset of dendritic cells (DC) in the epidermis.

These DC are characterized by the expression of MHCII, CD11c and Langerin. In addition to

LC, the skin contains a second type of Langerin+ DC, the Langerin+ dermal DC (dDC).

Epidermal LC and Langerin+ dDC migrate to skin draining lymph nodes under both steady

state and inflammatory conditions. The exact DC subtype responsible for the induction of

protective immunity against L. major has not been identified so far. In this study we analyzed

the role of Langerin+ skin-derived DC in the physiologically relevant low dose Leishmania

infection model. We utilized a diphtheria toxin (DT)-based system that allows the inducible in

vivo ablation of Langerin+ cells. In knock-in mice expressing a DT receptor (DTR) cDNA

under the control of the Langerin gene (Langerin-DTR mice) the entire population of

epidermal LC was eliminated 48 h after a single injection of DT. Interestingly, in low-dose

infections with L. major (1x103 parasites), DT-treated Langerin-DTR mice developed

significantly smaller lesions, increased IFNgamma/IL-4 ratios and decreased parasite loads

compared to control mice. Since repopulation of skin with dDC is much faster than with LC,

selective depletion of LC only showed that LC, not dDC, were responsible for this effect. In

re-infection experiments, no difference was observed between DT- and PBS treatment

suggesting that Langerin+ skin-derived DC are not required for development of an intact

memory response. Interestingly, the number of CD4+ Foxp3+ regulatory T cells (Treg) in

infected ears was reduced in DT-treated Langerin-DTR mice in comparison to control mice.

In contrast to prior studies, our data clearly reveal a suppressive role of epidermal LC in the

course of L. major infection via induction of Treg, as the depletion of these cells leads to a

better disease outcome.


37

Characterization of dual production of IFN-beta and IL12/23 p40 on a single cell basis

after TLR stimulation

Magdalena Kocur1, Stefanie Scheu1

1Institute of Medical Microbiology and Hospital Hygiene, Heinrich-Heine-University

Düsseldorf

Type I Interferons and IL-12/23 p40 play important roles in the response to bacterial and viral

infections and link innate and adaptive immunity. Their expression is activated in response to

TLR ligand binding mainly on antigen presenting cells. It has been shown that IFN-beta

contributes to the regulation of p40 expression, but data concerning its possible negative or

positive effect remain controversial. To determine the localization and kinetics of the

production of these cytokines in vivo, a double reporter mouse system for IFN-beta/YFP and

IL12/23 p40/GFP was established. Previous findings showed that after injection of the TLR9

ligand CpG in vivo IFN-beta is mainly expressed in plasmacytoid dendritic cells, but to a very

low extent additionally by conventional dendritic cells. For the expression of IL12/23 p40 only

the in vivo expression after injection of the TLR4 ligand LPS was determined to be restricted

to conventional dendritic cells until now. Here we characterize phenotypically cells, which

show dual versus single expression of IFN-beta and IL12/23 p40 after systemic stimulation

with several TLR-ligands. In vitro analysis of GM-CSF DCs will determine a possible co- or

counterregulation of both cytokines after TLR stimulation in individual cells. The

characterization of the effect of combined versus single TLR stimulation is facilitated by the

analysis of the frequency of secreted cytokine amounts by ELISA versus quantification of

cytokine producing cells as identified by the respective reporter allele.


38

SOCS-1 enhances PASTEURELLA MULTOCIDA Toxin induced STAT3 activity

Dagmar Hildebrand, Klaus Heeg and Katharina F. Kubatzky

Hygiene Institute, Dept. of Hygiene and Medical Microbiology, Im Neuenheimer Feld 324,

69120 Heidelberg

Pasteurella multocida Toxin (PMT) is a mitogenic protein toxin that causes respiratory

diseases in wild and domestic animals. PMT is able to perturb mammalian signalling

cascades through activation of heterotrimeric G proteins and downstream signalling events.

Recently we showed that PMT induces signal transducers and activators of transcription

(STAT) activity through Galphaq mediated activation of Janus kinases (JAK). Activation of

the JAK-STAT pathway is persistent, as PMT does not induce expression of suppressor of

cytokine signalling (SOCS) proteins.

Overexpression of SOCS-1 in HEK293 cells, however, did not decrease STAT3 activity, but

enhanced it significantly. SOCS proteins also act as E3 ubiquitin ligases that target proteins,

such as JAKs, to proteasomal degradation. Function as an E3 ligase, however, is dependent

on the phosphorylation status of SOCS-1 and serine phosphorylation of SOCS-1 was shown

to abrogate proteasomal degradation. We therefore determined the expression levels of the

tyrosine kinase JAK2 and found them to be elevated in the presence of SOCS-1, eventually

causing hyperphosphorylation of JAK2 and STAT3. The serine/threonine kinases of the Pim

family are STAT dependent genes and the protein was shown to interact with SOCS-1

previously. Stimulation of cells with PMT induced expression of Pim-1 that accumulated over

time. In addition, we found that SOCS-1 is heavily threonine phosphorylated after PMT-

stimulation but not after stimulation with IL-6. We hypothesise that persistent expression of

Pim-1 leads to phosphorylation of SOCS-1 which protects JAK2 from proteasomal

degradation. JAK2 accumulates, leading to hyperactivation of STAT3 and enhanced

transforming potential.


39

Opsonization of L. major with cross-reactive anti-phospholipid antibodies promotes

phagocytosis by dendritic cells (DC) and induction of protective immunity

Susanna Lopez Kostka, J. Hurst1, Filiz Demircik2, Philipp von Landenberg1, Mark C. Udey3,

Ari Waisman2, and Esther von Stebut.

Department of Dermatology, 1Institute of Clinical Chemistry and Laboratory Medicine, 21st

Medical Department, Johannes Gutenberg-University Mainz, Germany, and 3Dermatology

Branch, NCI, NIH, Bethesda, USA.

DC acquire L. major through FcgR/IgG-mediated phagocytosis. Thus, both B cell- and Fcg-

deficient mice were more susceptible to leishmaniasis which was directly attributable to a

failure of DC to prime T cells efficiently and to reduced production of IFNg. We now

investigated how initial B cell responses to the parasite itself develop. L. major parasites

display phospholipids on their surface. Here, parasites were opsonised by incubation with

normal mouse serum (NMS), immune serum (IS) from infected mice or serum containing

anti-phospholipid IgG (PhAk-S). Binding of Ab to Leishmania was detected by FACS.

Second, both IS as well as PhAk-S significantly enhanced phagocytosis of L. major by DC as

compared to unopsonized controls or NMS (35±5 and 27±5% vs. 13±1 and 18±3% infected

DC, n 5). Next, mice were infected with parasites opsonised with NMS, IS or PhAk-S. Both

Leishmania-specific IgG (IS) as well as cross-reactive PhAk-S significantly improved disease

outcome. Finally, genetically modified C57BL/6 mice which produce membrane-only IgM+ B

cells and have no immunoglobulins (“IgMi”) displayed increased susceptibility as compared

to wild type mice. Interestingly, the IgMi phenoptye was normalized upon reconstitution with

NMS, but if IS was used for adoptive transfer, the mice showed a significantly improved

disease outcome (smaller lesions/faster resolution). In conclusion, our findings suggest that

cross-reactive antibodies are found in NMS which bind to pathogens to facilitate

phagocytosis of L. major leading to DC-/IL-12-mediated induction of protective immunity.

Prior B cell-priming does not seem to be absolutely required to facilitate clearance of this

important human pathogen in vivo.


40

Treatment against cutaneous leishmaniasis with a two component gel developing

nitric oxide

Beate Lorenz, Alfred Goldinger1, and Esther von Stebut.

Department of Dermatology, and 1Central Pharmacy, Johannes Gutenberg-University Mainz,

Germany.

Nitric oxide (NO) released by e.g. activated MF exerts powerful effects against L. major. We

have now investigated the therapeutic potential of a two-component gel containing ascorbic

acid 5% and sodium nitrite 5%; once mixed the gel releases exogeneous NO. Next,

Leishmania-resistant C57BL/6 and -susceptible BALB/c mice were infected intradermally

with physiological doses of 103 L. major. Mice were treated early on, or – similar to the

clinical situation – on fully developed lesions. Treatment was performed 2x/week (200 mg

gel). Lesions were significantly smaller in all treatment groups reaching maximal differences

starting 2 weeks post treatment (e.g. C57BL/6 4.5±0.8 early treatment groups vs. 10.3±1.2

mm3 in controls, n 16). In addition, we observed a significant reduction of the parasite load

both after early and delayed treatment as compared to untreated controls (C57BL/6 early:

0.8±0.2 versus 9±2x104 parasites/wk6; late: 1±1 versus 10±3 x104 parasites/wk9, n 8). In

addition, visceralisation of parasites into spleen was significantly inhibited in all treatment

groups. Finally, antigen-specific cytokine release in draining lymph node cells revealed

strongly decreased IL-4 and increased amounts of IFNg associated with higher IL-12p40

release in all treatment groups as compared to controls, whereas IL-10 levels remained

unaltered. In conclusion, our data indicate that local application of NO donors may be useful

in treating newly developed as well as established lesions of L. major infection with no

severe side effects. Together with a direct parasite killing effect by exogeneous NO, the

mechanism of action is an alteration of the cytokine profile towards Th1-associated protective

immunity.


41

The role of interleukin 22 in Toxoplasma gondii induced ileitis.

Melba Muñoz1, Markus Heimesaat1, Robert Sabat2, Christoph Hoelscher3, Oliver Frey4,

Thomas Kamradt4, Wenjun Ouyang5, Oliver Liesenfeld1

1Institute of Microbiology and Hygiene, Campus Benjamin Franklin, University Hospital

Charité Berlin, Germany. 2Interdisciplinary Group of Molecular Immunopathology,

Dermatology/Medical Immunology, Campus Mitte, University Hospital Charité Berlin,

Germany. 3Infection immunology, Research Center Borstel, Borstel, Germany, 4Institue of

Immunology, Friedrich Schiller University Jena, Germany, 5Immunology Department,

Genentec Inc., South San Francisco, CA USA.

Peroral infection in susceptible C57BL/6 mice with Toxoplasma gondii leads to the

development of small intestinal inflammation with massive necrosis (pan-ileitis) dependent on

Th1 cytokines. The role of Th17 cells in ileitis is unknown. We have previously shown that IL-

23 but not IL-17 mediates T. gondii-induced small intestinal immunopathology. Therefore, we

investigated the role of IL-22 in the development of T. gondii-induced ileitis. IL-23 dependent

upregulation of IL-22 was essential for the development of T. gondii-induced small intestinal

immunopathology whereas IL-17 was downregulated and dispensable. IL-22-/- mice did not

develop small intestinal necrosis although they harbored the same number of parasites as

both Wild-type and IL-17-/- mice. Commensal gut flora was also important in the upregulation

of IL-22 in the ileum of infected mice. Interestingly, IL-22 was not exclusively produced by

CD4+ T cells but also by a non-T non-B cell population in the small intestinal lamina propria.

In conclusion, IL-22 induced by IL-23 but not IL-17 is a key mediator of immunopathology in

the small intestine.


42

The role of Frizzled1 in M. tuberculosis infection: Modes of induction and first insights

into function

Jan Neumann1, Stefan Ehlers2,3, Norbert Reiling1

1Microbial Interface Biology, 2Microbial Inflammation Research, Research Center Borstel,

23845 Borstel, 3Molecular Inflammation Medicine, Christian-Albrechts University, 24118 Kiel,

Germany.

Frizzleds represent seven-pass transmembrane receptors for a family of secreted

glycoprotein ligands termed "Wnts". Multiple Wnt and Frizzled (Fzd) homologs conduct

Wnt/Fzd signaling events through three known pathways: the beta-catenin pathway, the

planar cell polarity (PCP) pathway and the Ca2+-dependent pathway. These signaling events

assign key functions in embryonic development, stem cell homeostasis and very recently

also in inflammation. In the current study we have analysed the regulation of the Wnt/beta-

catenin pathway in experimental M.tb infection. We observed that the Wnt/beta-catenin

signaling is drastically down-regulated in lung homogenates of M.tb-infected mice. In contrast

Frizzled1 (Fzd1), a Frizzled homolog associated with beta-catenin signaling, was identified to

be up-regulated during M.tb infection. In macrophages Fzd1 mRNA expression is triggered

by mycobacteria via TLR2 and MyD88. To gain insights into Fzd1 function in the context of

M.tb infection, we analysed the cellular response of macrophages to its ligand Wnt3a, which

we find constitutively expressed in lung homogenates of M.tb infected mice. Wnt3a is able to

induce beta-catenin signaling in macrophages in a Fzd1-dependent manner. Currently we

investigate the functional relevance of Wnt3a and Fzd1 in mycobacteria induced immune

responses.


43

Interaction of Mycobacterium tuberculosis and human macrophages under hypoxic

conditions

Daniel Nickel1, Steffen Stenger1

1Institute for Medical Microbiology and Hygiene, University Hospital of Ulm, Germany

Mycobacterium tuberculosis (MTb) is an intracellular pathogen that has adapted to the milieu

of human lung tissue. Most strikingly mycobacteria manage to survive within a defined

cellular conglomerate termed granuloma. Recent studies indicate that oxygen tension in

granulomatous areas is far lower as compared to the environmental atmosphere even

though exact figures are not available. Due to technical obstacles this physiological situation

has not been sufficiently considered in the past when immunological analyses were

performed. Therefore we established a model to co-culture human macrophages and virulent

MTb under hypoxic conditions. Using a hypoxia chamber integrated in our high safety air flow

system we demonstrated that bacterial uptake, cytokine release (IL-10, CCL5, TNF),

intracellular growth and cellular viability were virtually identical at 20% O2 and 3% O2.

Therefore the interaction of human macrophages and MTb appears to be stable across a

broad range of oxygen tension. Western Blotting of macrophages for HIF-1alpha, a surrogate

marker of anaerobic metabolism, revealed that this molecule is not yet stabilized at 3%, the

condition used in our experiments. However, an oxygen tension of 1% induces anaerobic

metabolism, i.e. expression of HIF-1 alpha, without disturbing cellular viability. Ongoing

studies are investigating whether further restriction of oxygen availability to 1% or lower will

alter the immunological features of the host pathogen interaction in human tuberculosis.


44

Role of type I IFNs in innate defence to Legionella pneumophila

Bastian Opitz

Department of Internal Medicine/Infectious Diseases and Pulmonary Medicine, Charité

Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany

Legionella pneumophila, a Gram-negative facultative intracellular bacterium, is the causative

agent of a severe pneumonia known as Legionnaires´ disease. Classically, type I IFNs (IFN-

alpha/beta) have been associated with antiviral immunity. IFN-alpha/beta signal through the

IFN-alpha/beta receptor (IFNAR) leading to induction of hundreds of IFN-stimulated genes

(ISGs), many of which have anti-microbial activities. Recently, it was demonstrated that type

I IFNs are also produced in host cells infected with (intracellular) bacteria or stimulated with

cytosolic DNA. These responses were mediated by a so far unidentified cytosolic pattern

recognition receptor, and independent of the Toll-like receptors, NOD-like receptors as well

as RIG-like receptors.

Here we show by RNAi and overexpression assays that L. pneumophila-infected host cells

produced IFN-beta dependent on IRF3, but independent of the recently suggested pattern

recognition receptor ZBP1 (DLM-1/DAI). We observed enhanced L. pneumophila replication

in mouse macrophages lacking IFNAR and human cells after IRF3 knock-down, suggesting

that endogenously produced IFN-beta activates a cell-autonomous defence against

Legionella. Moreover, IFN-beta treatment restricts Legionella replication in human and

murine host cells. Type I IFNs appear not to influence the establishment of the Legionella-

containing vacuole (LCV) or fusion of the LCV with the lysosome. Moreover, type I IFN-

mediated cell-autonomous defence was independent of autophagy and pyroptosis. We thus

hypothesize a crucial involvement of antibacterially acting ISGs. Ongoing studies focus on

the role of IFN-induced immunity-related GTPases (IRGs).


45

Tumor-derived Prostaglandin E2 and TGF-beta synergize to inhibit PDC-derived IFN-

alpha

Isabelle Bekeredjian-Ding1, Meike Schäfer2, Evelyn Hartmann3, Marijo Parcina1, Stefan

Endres2, Barbara Wollenberg4, Gunther Hartmann2,3

1Hygiene-Institute, Dept. of Medical Microbiology and Hygiene, UniversityHospital Heidelberg 2Dept. of Internal Medicine, Division of Clinical Pharmacology, University of Munich 3Institute for Clinical Biochemistry and Pharmacology, University Hospital Bonn 4Department of Otorhinolaryngology, University of Schleswig-Holstein

Due to their capacity to produce high levels of type I interferons (IFN-I) plasmacytoid

dendritic cells (PDC) are the major targets for adjuvant therapies with CpG ODN. However,

PDC infiltrating tumor tissues often lose their IFN-secreting capacity. The tumor environment

is characterized by the presence of high concentrations of immunosuppressory mediators

such as PGE2, IL-10 and TGF-b. Here we show that prostaglandin E2 (PGE2) and TGF-b

increase IL-6 and IL-8 and synergistically inhibit IFN-a and TNF production upon TLR7- or

TLR9-mediated activation of human PDC. Receptor expression studies showed that the

effects of PGE2 are most likely mediated by EP2 receptor engagement. Further phenotypical

characterization of PGE2 and TGFb-treated PDC revealed a down-regulation of CD40

paralleled by an up-regulation of CD86. PDC interaction with other infiltrating immune cells

may thus be skewed to a tolerogeneic action at the tumor site. Moreover, PGE2 and TGF-b

increase CXCR4 and decrease CCR7 in TLR-stimulated PDC suggesting that PDC lose their

ability to migrate to the lymph node and may be retained in the periphery. We therefore

speculate that cyclooxygenase inhibitors and TGF-b antagonists may improve TLR7 and

TLR9-based immunotherapy in diseases associated with immunosupression.


46

Systemic bacterial infection alters differentiation of dendritic cells in the bone marrow

and mediates chronic dendritic cell dysfunction

Eva Pastille1, Yang Zhang2, Nadine Kroell1, F. Ulrich Schade1, Stefanie B. Flohé1,

1Surgical Research, Department of Trauma Surgery, University Hospital Essen, Essen,

Germany; 2Department of Molecular Biology, University Hospital Essen, Essen, Germany

Polymicrobial sepsis rapidly causes a dysfunction of dendritic cells (DC) that is maintained

even after resolution of the infection. This dysfunction is characterized by an impaired

capacity of DC to secrete the Th1-polarizing cytokine IL-12 and to induce a protective Th1

cell response, while the production of IL-10 is enhanced. The long-lasting DC dysfunction

causes an enhanced susceptibility to secondary infections that represent life-threatening

complications for sepsis patients. We investigated whether DC dysfunction is mediated by a

defective differentiation of DC from bone marrow. Sepsis was induced in mice by cecal

legation and puncture (CLP) and 4 d after CLP or sham operation, when mice recovered

from sepsis, DC differentiation from bone marrow was analyzed in vitro. Upon stimulation

with CpG and CD40 ligand, bone marrow-derived dendritic cells (BMDC) from septic mice

released extended levels of IL-10 but similar levels of IL-6 compared to sham-BMDC. When

injected subcutaneously, BMDC from septic mice showed a reduced capacity to promote

Th1-cell polarization. Intranasal application of BMDC from septic mice along with

Pseudomonas aeruginosa into naïve mice impaired bacteria clearance in the lung. Lungs of

mice treated with BMDC from septic mice contained less IL-12 and IFN-gamma in

comparison to lungs of mice injected with sham-BMDC. Co-cultures of BMDC and naive

peritoneal macrophages showed an enhanced level of IL-10 upon Pseudomonas aeruginosa

stimulation when BMDC derived from septic mice. Thus, during systemic bacterial infection

DC follow an altered developmental pathway and mediate chronic immunosuppression

through interference of the innate and adaptive immune system.


47

The phosphatidylserine binding protein annexin A5 can interfere with chlamydial

infection

Lisa Pfleiderer1, Jan Rupp2, Martin Herrmann3 and Ger van Zandbergen1

1Institute for Medical Microbiology and Hygiene, University Clinic of Ulm. 2Institute for Medical

Microbiology and Hygiene, Center for Structural and Cell Biology in Medicine, University of

Lübeck. 3Department for Internal Medicine 3 University Hospital Erlangen

Chlamydophila pneumoniae (Cp) is an obligate intracellular pathogen causing respiratory

disease. Cp can transfer itself between host cells without being detected by the immune

system. It has been suggested that Cp can be released from infected cells hiding in apoptotic

bodies. These dying cell parts, present phosphatidylserine (PS) as an apoptotic eat me

signal for phagocytes. Recognition of PS induces a non-inflammatory uptake-mechanism

and can be blocked by the PS-binding protein annexin-A5 (AnxA5).

In this study we focus on chlamydial release of dying infected epithelial cells. In addition we

investigated the role of PS on infected apoptotic granulocytes for chlamydial transfer to

macrophages.

First we used HeLa cells transgenic for GFP on the endoplasmatic reticulum (ER), since it

was suggested that apoptotic bodies consist of ER-membrane. Chlamydia infected

transgenic HeLa cells were monitored using timelapse imaging. We found that 4 days after

infection chlamydia.was present outside the HeLa cells in ER-GFP apoptotic bodies. To

study the effect of AnxA5 on chlamydial infection we used 3 day old Cp infected polymorph

nuclear neutrophil granulocytes (PMN), one of the first host cells in the lung. We transferred

chlamydia from these apoptotic PS-positive cells to either macrophages or alveolar

macrophages, as other potential lung host cells. Co-incubation with AnxA5 resulted in a

lower infection rate and the presence of smaller Cp. inclusions.

Our data suggest that chlamydia can hide in PS-positive apoptotic bodies, in part consisting

of ER-membrane. Recognition of PS is an important step in the silent propagation of

chlamydia.


48

Acute graft-versus-host-disease after reduced intensity conditioning is mediated by

MyD88 mediated TLR9 sensing of bacterial DNA and can be modulated by

administration of TLR9 antagonist

Rita Plickert,1 Markus M. Heimesaat,1 Axel Nogai,2 Stefan Bereswill,1 André Fischer,1

Christoph Loddenkemper,3 Ulrich Steinhoff,4 Eckhard Thiel,2 Marina Freudenberg,5 Ulf B.

Göbel1 und Lutz Uharek2

1Institut für Mikrobiologie und Hygiene, Charité - Universitätsmedizin Berlin, Charité Centrum

5, Campus Benjamin Franklin, Hindenburgdamm 27, D-12203 Berlin, Germany 2Medizinische Klinik III (Hämatologie, Onkologie und Transfusionsmedizin), Charité -

Universitätsmedizin Berlin, Charité Centrum 14, Campus Benjamin Franklin,

Hindenburgdamm 30, D-12200 Berlin, Germany 3Institut für Pathologie, Charité - Universitätsmedizin Berlin, Campus Benjamin Franklin,

Hindenburgdamm 30, D-12200 Berlin, Germany 4Max Planck Institute of Infection Biology, Schumannstrasse 20-21, 10117 Berlin, Germany 5Max Planck Institut für Immunbiologie, Stübeweg 51, D-79108 Freiburg, Germany

Graft-versus-host-disease (GvHD) is a major cause for morbidity and mortality after

allogeneic stem cell transplantation. Acute intestinal GvHD is mediated by bacterial sensing

via toll-like-receptors (TLR), but the immunobiology in situ is poorly understood. In a murine

stem cell transplantation model based on reduced intensity conditioning, we investigated the

impact of bacterial sensing via TLRs on intestinal GvHD. Following transplantation, wild type

mice displayed intestinal tissue damage, accompanied by elevated numbers of T-cells,

neutrophils, apoptotic and proliferating cells in the colon. In TLR9-/- mice the numbers of T-

cells, neutrophils and apoptotic cells after transplantation in the colon were significantly

reduced, resulting in amelioration of the tissue damage and in improved survival of TLR9

deficient mice. Similar changes were observed in MyD88-/- mice, supporting our results in

TLR9-/- mice.

The administration of a TLR9 antagonist in wild type mice after transplantation showed

similar results as demonstrated in TLR9-/- and MyD88-/- mice, indicating that intestinal GvHD

can be influenced by the modulation of TLR9 sensing.

These results indicate an important role of TLR9 in the pathomechanism of GvHD and may

lead to a therapeutic approach in the future.


49

Split tolerance after oral vaccination of mice with recombinant Escherichia coli Nissle

1917 expressing fimbrial adhesin K88

Katharina A. Remer, Marita Bartrow, Bianca Roeger, Heidrun Moll,

Tobias A. Oelschlaeger

University of Würzburg, Institute for Molecular Infection Biology, Würzburg, Germany

Probiotic bacteria are live, non-pathogenic micro-organisms which are beneficial for the

health of the host. One of these, the Escherichia coli strain Nissle 1917 (EcN) is used as a

probiotic agent for the treatment of infectious and chronic inflammatory diseases of the

intestine. Since it has been shown that EcN can serve as a safe carrier for targeted delivery

of recombinant molecules to the intestinal mucosa, we constructed the recombinant strain

EcN pMut2-kanK88 (EcN-K88) stably expressing the determinant for the K88 fimbrial

adhesin on the bacterial surface. K88 is a key virulence factor of enterotoxigenic E. coli

(ETEC), which are the cause of one of the economically most important diseases in swine

husbandry. Massive efforts have therefore been made to develop a vaccine for the induction

of protective mucosal immunity against ETEC.

After oral application of EcN-K88 to mice for one week, EcN-K88 as well as wild-type EcN

and EcN mock-transformed with the plasmid vector only could be detected in faecal samples

for a prolonged period of time after the last feeding, indicating that EcN can transiently

colonise the murine intestine. Oral application of EcN-K88 resulted in significant IgG serum

titers against K88 as early as seven days after the initial feeding with EcN-K88, but no

significant IgA titers. In contrast, we failed to detect any specific T cell responses towards the

K88 antigen both in spleen and mesentheric lymph nodes. Although dendritic cells readily

up-regulated maturation and activation markers in response to K88 stimulation, accompanied

by secretion of interleukin (IL)-12, IL-6, IL-10 and tumour necrosis factor, restimulation of T

cells from mice having received EcN-K88 with K88-loaded dendritic cells did not result in

detectable T cell proliferation and IL-2 secretion, but rather induced an IL-10 bias. While the

serum antibody responses clearly demonstrate that K88 is recognized by the humoral

immune system, our findings indicate that oral application of probiotic EcN expressing the

K88 fimbrial adhesin induces a selective T cell tolerance towards the antigen.


50

Wnt ligands differentially regulate the inflammatory response of macrophages upon

mycobacterial infection

Kolja Schaale1, Stefan Ehlers2,3 and Norbert Reiling1

1Microbial Interface Biology, 2Microbial Inflammation Research, Research Center Borstel,

23845 Borstel, 3Molecular Inflammation Medicine, Christian-Albrechts University, 24118 Kiel,

Germany.

Wnt proteins are secreted palmitoylated glycoproteins with multiple functions in cell

proliferation, migration as well as tissue organization. They are best known for their role in

embryonic development and tissue homeostasis. Deregulation of Wnt signaling has been

shown to promote carcinogenesis. Recently we identified Wnt signaling to be involved in the

regulation of inflammatory processes: Wnt5a is induced in human macrophages in response

to mycobacteria and conserved bacterial structures and contributes to the regulation of the

proinflammatory cytokines IL-12 and IFN-gamma. To gain deeper insights into Wnt mediated

modulation of inflammatory processes we now used murine bone marrow derived

macrophages and analyzed the effects of the addition of exogenous Wnt homologs. We

monitored Wnt-mediated activation of primary macrophages by measuring the activation of

signaling pathways and transcription factors, analyzed the expression of target genes by

Real-Time PCR and measured the secretion of inflammatory cytokines by ELISA.

Exogenous Wnt5a - but not Wnt3a - was able to induce cytokine expression in primary

macrophages. In infection experiments Wnt5a promoted the mycobacteria-induced

macrophage activation and enhanced the expression of inflammatory mediators in murine

macrophages. In contrast, addition of Wnt3a reduced the expression of inflammatory

mediators upon mycobacterial infection. These data corroborate our previous findings and

further support the notion that TLR/NF-kappaB and Wnt signaling, both being evolutionary

highly conserved pathways, are functionally interconnected.


51

Induction of pro-inflammatory IL-12 in Plasmodium berghei infected BALB/c mice

breaks blood-brain-barrier and leads to cerebral malaria

Kim Ellen Schmidt1, Beatrix Schumak2, Sabine Specht1, Bettina Dubben1, Frank

Juengerkes2, Volker Schmitz3, Andreas Limmer2 and Achim Hoerauf1

1Institute of Medical Microbiology, Immunology and Parasitology, University Hospital Bonn 2Institute of Molecular Medicine and Experimental Immunology, University Hospital Bonn 3Department of Internal Medicine I, University Hospital Bonn,

Infection with Plasmodium parasites causes malaria and often results in development of

severe complications like severe malaria anaemia or cerebral malaria, which are mainly

attributed to overwhelming inflammatory immune reactions of the host. Murine models that

differ in the development of experimental cerebral malaria (ECM) allow detailed studies of

host responses towards infection with the rodent malaria parasite Plasmodium berghei ANKA

(PbA). Here we show that BALB/c mice, which are known to be resistant against

experimental cerebral malaria can be driven into interferon-gamma (IFN-gamma) dependent

ECM pathology and subsequent death if they received CpG after Plasmodium infection. CpG

application resulted in production of pro-inflammatory cytokines as well as neuro-pathological

symptoms, like hemiplegia, convulsions, coma leading to increased mortality. Importantly,

CpG application caused permeabilization of the blood brain barrier, comparable to ECM-

susceptible PbA infected C57 BL/6 mice. Experiments in genetically deficient mice revealed

that ECM development in BALB/c mice after CpG application was dependent on the

presence of interleukin 12. The pathological consequences of CpG application could be

ascribed to interleukin 12 as similar results were obtained with injection of recombinant

Adenovirus expressing interleukin 12. Taken together our data suggest that the induction of

pro-inflammatory mediators provokes ECM development in BALB/c mice in an IL-12

dependent manner.


52

Airway epithelial cells modify immune responses

Lotte M. Schmidt, Magdalena Dahmen, Fabian Fey and Alexander H. Dalpke

Department of Hygiene and Medical Microbiology, University of Heidelberg, Germany

Airway epithelial cells are lining the body’s largest surface and represent the first line of

defence against invading pathogens. Moreover, they are also playing an important role in

innate mucosal immunity by secretion of antibacterial proteins. Nowadays, it turns out that

the airway epithelium also has simple immune functions, mediated in part by the expression

of TLRs. However, as upper airways are not an entirely sterile compartment the question

was raised how local immunity and homeostasis is regulated. We speculated that epithelial

cells within the airway microenvironment are regulators of professional immune cells. We

show that epithelial-cell conditioned dendritic cells display properties of alternative activation

with increased expression of arginase-1 and interleukin 10 and reduced TLR-mediated

upregulation of CD40, CD86 and pro-inflammatory cytokines. These “alternative “activated

dendritic cells also showed a reduced capacity of induction of T-cell proliferation. Modulation

of T-cell proliferation by epithelial cells was mediated in two different ways, first directly via

TGF-beta and second indirectly via the influence on antigen presenting cells. Modified T cells

showed increased differentiation towards interleukin 10 producing Tr1 cells.

Our results indicate that bronchial epithelial cells are capable of inducing a non-inflammatory

microenvironment. Furthermore we now hypothesize that during infections this inhibition is

transiently down-regulated resulting in a “release of inhibition”. First results with supernatant

showed that the infection of epithelial cells in the absence of further immune cells is not

sufficient to convert this anti-inflammatory microenvironment into an immune activated state.

This hints towards a role of migratory immune cells invading the lung during infection.


53

Influence of optineurin on adenovirus E3-14.7K mediated TNF-resistance

Laura Klingseisen; G.Knoll; M.Kurz; Wulf Schneider-Brachert

Institute for Med. Microbiology; University of Regensburg

Recently we have shown, that adenovirus E3-14.7K (14.7K) expressing cells are protected

from TNF-mediated apoptosis by inhibition of TNF-Receptor-1 (TNFR1) internalization. In the

presence of 14.7K the recruitment of essential adaptorproteins involved in both apoptosis

signalling (TRADD, FADD, and caspase-8) and clathrin-mediated endocytosis (rab5 and

dynamin2) to the activated TNFR1 is blocked. The 14.7K interacting protein optineurin

seems to link 14.7K not only to the apoptotic pathway but also to the membrane trafficking

network via its additional interacting partners rab8 and huntingtin. Therefore we investigated

wether the interaction of 14.7K with optineurin is essential to prevent TNF-mediated

apoptosis.

We generated several mutants of 14.7K and characterized their ability to protect cells from

apoptosis and to interact with optineurin. Finally 14.7K expressing cells were treated with

optineurin targeting siRNA to examine the importance of optineurin for 14.7K mediated

inhibition of apoptosis.

The 14.7K mutants defined three groups: (i) ones, which interacted with optineurin and

protect cells from cell death, (ii) others that interacted with optineurin but failed to protect,

and those (iii) that were incapable for interaction and inhibition of apoptosis. Interestingly the

suppression of optineurin by siRNA transfection did not revert the TNF-resistant phenotype.

Collectively, these data suggest that optineurin might not be essentially involved in 14.7K

mediated inhibition of TNFR1 induced apoptosis.

Additional work is under progress to address the effect of optineurin on TNFR1-

internalization.


54

Activation of macrophages by the mycobacterial cord factor (TDM) and its synthetic

analogue (TDB)

Hanne Schoenen7, Kerstin Werninghaus1,7, Anna Babiak1, Olaf Groß2, Christoph Hoelscher3,

Else Marie Agger4, Falk Nimmerjahn6, Juergen Ruland2, and Roland Lang1,7 1Institute of Medical Microbiology, Immunology and Hygiene, Technical University Munich, D-

81675 Munich, Germany; 2Department of Hematology, Technical University Munich, 80333

Munich, Germany; 3Division of Infection Immunology, Research Center Borstel, D-23845

Borstel, Germany; 4Adjuvant Research, Department of Infectious Disease Immunology,

Statens Serum Institute, 2300 Copenhagen, Denmark; 5Semmelweis University School of

Medicine, Budapest H-1089, Hungary; 6Molecular Medicine, 7Institute of Clinical

Microbiology, University Hospital Erlangen, 91054 Erlangen, Germany; 8Division of

Immunology, CLS, Institute for Infectious Diseases and Molecular Medicine, University of

Cape Town, 7925 Cape Town, South Africa

Mycobacterium tuberculosis (MTB) causes nearly two million deaths per year. The live

vaccine M. bovis (BCG) does not prevent pulmonary tuberculosis and can not be used in

immunocompromised people. Immunization with recombinant subunit vaccines is an

attractive new vaccination strategy. These subunit vaccines require adjuvants that trigger

innate immunity which then activate the adaptive immune system. In mouse vaccination

studies the mycobacterial cord factor Trehalose-6, 6-dimycolate (TDM) and its synthetic

analogue Trehalose-6,6-dibehenate (TDB) have shown to be efficient adjuvants. Injection of

the recombinant protein H1 derived from MTB in combination with TDB as adjuvant lead to a

strong Th1 and Th17 T-cell response in mice. Furthermore, macrophages and DCs respond

with cytokine and NO production in vitro. We have addressed the mechanism of APC

activation by TDB and TDM utilizing different techniques. Using knock-out mice it was shown

that the response to the glycolipids was independent on the Toll-like receptor associated

signalling molecule MyD88 in vitro. Microarray analysis revealed that the stimulation of

macrophages with TDB leads to upregulation of several C-type lectins including Dectin-1.

However the recognition of these glycolipids was independent of Dectin-1 and Dap12,

whereas the adaptor protein Fc-receptor gamma chain was essential. Furthermore, knock-

out and inhibition studies showed that the Syk-Card9-Bcl10-Malt1 pathway was required for

glycolipid recognition.

To get more detailed information about the mechanism of APC activation mediated by TDB

further investigations are required and will be focused on potential receptor candidates of the

C-type lectin family and the signalling events after glycolipid recognition.


55

A crucial role of the spleen in the induction of pathogenic host responses towards P.

berghei ANKA infection

Beatrix Schumak1, Kim Schmidt2, Sabine Specht2, Frank Juengerkes1, Günter J.

Hämmerling3, Achim Hoerauf2, Andreas Limmer1

1Institute of molecular Medicine and experimental Immunology, University Hospital Bonn; 2Institute of medical Microbiology, Immunology and Parasitology, University Hospital Bonn; 3German Cancer Research Center, Department of Tumor Immunology, Heidelberg

Cerebral malaria is a severe complication of Plasmodium infection and is generally assumed

to be an immune-mediated pathology as a result of excessive inflammation. Interferon

gamma and activated T cells are among the few factors that are known to be essential for

the development of experimental CM (ECM) in susceptible mouse strains upon infection with

P. berghei ANKA. It has been long speculated where pathogenic T cells and inflammatory

cytokines causing ECM are generated. Among direct effects on the brain, the spleen has

been taken into consideration. We show that splenectomized animals were protected against

ECM; in addition, our data demonstrate the relevance of an intact micro-architecture of the

spleen. PbA infected RAG-deficient mice developed ECM only if they received effector cells

from PbA infected wildtype donors but not after transfer of lymphocytes from naïve donors.

Furthermore, we determined a crucial role for dendritic cells in the detection of Plasmodium

parasites and subsequent induction of detrimental parasite specific immune responses. Mice

depleted of DCs or deficient in IL-12p35 were protected against ECM and lacked PbA

specific T cells, which emphasizes the importance of these antigen presenting cells in the

induction of parasite-specific T cells responses. In addition, support of T cell priming and

induction of interferon gamma belong to the most prominent biological functions of IL-12, a

cytokine which is almost exclusively produced by DCs. Taken together, our data show the

importance of the spleen in the induction of pathological host responses towards blood stage

PbA infection in susceptible mice.


56

Long-term effect of sepsis - The influence of bacteremia and bacterial translocation on

systemic adaptive immune responses

Timo Schwandt1, Frank Juengerkes1, Beatrix Schumak1, Gerrit Gielen*, Joerg Kalff2,

Percy Knolle1, Bernhard Holzmann3, Andreas Limmer1

1Institute of Molecular Medicine and Experimental Immunology, University of Bonn,

Germany; 2Department of Surgery, University of Bonn, Germany; 3Department of Surgery,

Technical University Munich, Germany

Bacterial translocation is a possible risk of abdominal surgery and could be the cause of life-

threatening consequences such as organ failure and septic shock. Patients surviving septic

shock often suffer from opportunistic infections as well as defects in adaptive immunity.

Here, we mimicked abdominal surgery by intestinal manipulation (IM) in mice. To study

septic conditions, mice received E.coli expressing luciferase either intravenously or per os

prior to subjection to CASP as well as IM. Translocation of gut-derived bacteria was

monitored by in vivo imaging (Xenogen) and additional microbiological assays. We detected

comparable amounts of bacteria in lung, liver and spleen of mice that underwent CASP or

were injected i.v. with E.coli. In contrast, bacteria were detected in lung and liver but not in

spleen of mice that were subjected to IM or received E.coli by injection into the hepatic portal

vein. Depletion experiments imply that Kupffer cells as well as soluble mediators such as

Tumor necrosis factor play an important role in trapping and clearance of translocated

bacteria in liver and lung.

Our experiments demonstrated that translocation of bacteria did not cause immune

suppression as long as they did not reach the spleen in high numbers. We suggest that liver

and lung fulfill a filter function to prevent systemic distribution of gut-derived bacteria. Failure

of or bypassing these barriers might enable bacteria to access the spleen and thus cause

systemic suppression of adaptive immunity.


57

Functional analysis of cells from IFIT-2 knockout mice

Alexandra Siegfried, Susanne Berchtold, Birgit Manncke, Ingo B. Autenrieth, Erwin Bohn

Institut für Medizinische Mikrobiologie und Hygiene, UKT Tuebingen

Interferon-induced tetratricopeptide repeat protein (IFIT)-2 is a type I interferon induced gene

which we found to be highly expressed during Y. enterocolitica infection as well as in a

mouse model for IBD. The biological functions of IFIT-2 are so far elusive. It is only known

that IFIT-2 may interact with the translation initiation factor eIF3c and may affect protein

translation. In addition, hIFIT-2 may interact with cytokeratin and may influence tumor cell

migration. In this study IFIT-2 knockout mice were generated to further assess the biological

roles of IFIT-2. The effects of mouse IFIT-2 on cell composition, T cell proliferation, B cell

maturation, LPS induced cytokine secretion and first results on its regulatory properties will

be discussed.


58

Blockade of IL-6 trans-signalling does not reactivate experimental tuberculosis

Jan Christian Sodenkamp,1 Georg H. Waetzig,2 Juergen Scheller,3 Dirk Seegert,2 Stefan

Rose-John,3 Stefan Ehlers,4,5 Christoph Hoelscher1

1Division of Infection Immunology, Research Center Borstel, Borstel, Germany; 2CONARIS

Research Institute AG, Kiel, Germany; 3Department of Biochemistry, Christian-Albrechts-

University, Kiel, Germany; 4Molecular Inflammation Medicine, Research Center Borstel,

Borstel, Germany; 5Molecular Inflammation Medicine, Christian-Albrechts-University, Kiel,

Germany

Therapeutic approaches for chronic inflammatory conditions, such as rheumatoid arthritis

and Crohn's disease, using e.g. TNF targeted biologics are associated with an increased risk

of infectious complications, especially tuberculosis (TB). The pro-inflammatory cytokine

interleukin (IL)-6 can bind to gp130 on cells lacking the IL-6 receptor (IL-6R) when it forms a

complex with the soluble IL-6R (sIL-6R) (IL-6 trans-signalling). Trans-signalling is

counteracted by soluble gp130 (sgp130). In mice, specific neutralization of the IL-6/sIL-6R

complex in vivo by sgp130Fc fusion proteins with 10- to 100-fold higher inhibitory activity

than natural sgp130 has been shown to suppress inflammatory bowel disease and

ameliorate rheumatoid arthritis. Hence, blockade of IL-6 trans-signalling by sgp130Fc

provides a novel approach for the treatment of a range of chronic inflammatory diseases.

However, the effect of sIL-6R neutralization on the outcome of experimental TB has not been

investigated so far. To evaluate the potential risk of interfering with IL-6 trans-signalling, we

analyzed the outcome of experimental TB under treatment with an optimized sgp130Fc

fusion protein. C57BL/6 mice were infected with Mycobacterium tuberculosis via the aerosol

route. At the onset of experimental TB or during an already established infection, mice were

treated with an anti-TNF antibody and with various doses of sgp130Fc for five weeks.

Whereas neutralization of TNF resulted in a strikingly impaired cell-mediated immune

response followed by pulmonary granuloma desintegration and exacerbated bacterial loads,

protective immune responses were unaffected after treatment with sgp130Fc. Our finding

that blockade of IL-6 trans-signalling does not reactivate experimental TB implies the

treatment of chronic inflammatory diseases with sgp130Fc as a superior approach to avoid

adverse consequences of anti-inflammatory strategies.

(Supported by the Inflammation Research Excellence Cluster)


59

Delayed type hypersensitivity versus humoral immune response – Influence of high

and low antigen doses on the cytokine expression within splenic compartments

Claudia Stamm, Juergen Westermann, Kathrin Kalies

Institute of Anatomy, University of Luebeck

Immunisations with different antigen doses provoke different immune responses. Thus,

injection of a low dose of sheep red blood cells (SRBC) (1x105) induces a delayed type

hypersensitivity (DTH) reaction after reexposure to the antigen, whereas the injection of a

high dose (1x109) induces a humoral immune response and suppresses a DTH. In order to

examine these mechanisms, we investigate the sequence of events occurring in the

compartments of the spleen from 1 h after immunisation up to 10 days.

After primary injection of a low dose, SRBC are hardly detectable in the spleen. Moreover,

the isolation of individual splenic compartments followed by an exponential amplification

during real-time RT-PCR is not sensitive enough to detect any changes in cytokine

expression before T-cell proliferation takes place after 2-3 days. Interestingly, IFN-gamma

expressing cells appear in the B-cell follicles between 3-6 days after immunisation.

In contrast after primary injection of a high dose, SRBC localize in the marginal zone and in

the red pulp. Increased IL12p40 transcription is found in the marginal zone 4 h after injection

and is followed by an increase of IL12p40 and IL2 in the T-cell zone. Surprisingly, compared

to the low-dose injection a twofold higher T-cell proliferation is observed after 2-3 days.

Overall, Th1-cytokines are usually associated with DTH. As the high-dose immunisation

doesn’t induce DTH but unexpectedly upregulates Th1-inducing cytokines, we hypothesize

that antigen-specific Th1 cells are induced but unable to reach the skin.


60

The role of the intestinal microflora in infection with Citrobacter rodentium

Stephen T. Reece, Anne Kohler, Petra Krienke, Marcus Heimessat, Ulrich Steinhoff

Max-Planck Institute für Infektionsbiologie and Charité Universitätsmedizin, Institut für

Mikrobiologie und Hygiene, Berlin

Citrobacter rodentium (C. rodentium) is a gram negative enteric pathogen that triggers

intestinal inflammation and hyperplasia. During infection, the bacillus attaches to the colonic

epithelium and forms attaching and effacing (A/E) lesions, analogous to the groups of human

gastrointestinal pathogens Enterohaemorraghic Escherichia coli (EHEC) and

Enteropathogenic E. coli (EPEC). After infection via the oral route, the bacterium rapidly

colonises the host colonic epithelium, induces an acute colonic inflammatory response that

peaks between day 7 and 14 post infection and susceptible mouse strains and certain

genetic KO mice succumb at this timepoint. Resistant mouse strains rapidly clear C.

rodentium from the colon with very low or absent bacterial titres present by day 28 post

infection. It has been postulated that C. rodentium utilises A/E lesion formation to out

compete normal flora to attach to the colonic epithelium and A/E lesion formation is critical

for both colonisation of the host and the local inflammatory response. However, the role of

normal colonic microflora during C. rodentium infection has not yet been addressed. In the

present study we show that despite being a carrier of C. rodentium, infection is not lethal for

germfree mice. Interestingly, the association of normal flora to previously germfree mice that

have been infected C. rodentium mice will lead to bacterial clearance in these animals. Our

data indicate that bacterial resistance is not due to antibodies but to the regulation of genes

of the fat-metabolism which are induced by the intestinal microflora.


61

IL-4/IL-13-dependent alternative activation of macrophages but not microglial cells is

associated with uncontrolled cerebral cryptococcosis

Werner Stenzel1, Uwe Müller2,4, Gabriele Köhler3, Frank L. Heppner1, Manfred

Blessing2,4, Andrew N.J. McKenzie5, Frank Brombacher6, and Gottfried Alber2

1Department of Neuropathology, Charité Universitätsmedizin, Berlin, Germany; 2Institute of

Immunology, College of Veterinary Medicine, University of Leipzig, Leipzig, Germany; 3Gerhard Domagk Institute for Pathology, University of Münster, Münster, Germany; 4Molecular Pathogenesis, College of Veterinary Medicine, Center for Biotechnology and

Biomedicine, University of Leipzig, Leipzig, Germany; 5Medical Research Council Laboratory

of Molecular Biology, Cambridge, United Kingdom; 6Institute of Infectious Disease and

Molecular Medicine and International Centre for Genetic Engineering and Biotechnology,

University of Cape Town, Cape Town, South Africa

IL-4- and IL-13-dependent Th2-mediated immune mechanisms exacerbate murine

Cryptococcus neoformans-induced bronchopulmonary disease. To study the roles of IL-4

and IL-13 in cerebral cryptococcosis, interleukin (IL)-4 receptor a-deficient (IL-4Ra-/-), IL-4-

deficient (IL-4-/-), IL-13-deficient (IL-13-/-), IL-13 transgenic (IL-13T/+), and wild-type (WT) mice

were infected intranasally. IL-13T/+ mice displayed higher fungal brain burden than WT mice,

whereas the brain burden of IL-4Ra-/-, IL-4-/-, and IL-13-/- mice was significantly lower as

compared to WT mice. Upon infection 68 % of WT mice, and 88 % of IL-13-overexpressing

IL-13T/+ mice developed cerebral lesions. In contrast, IL-4Ra-/-, IL-4-/-, and IL-13-/- were

essentially protected from CNS invasion by the fungus. Furthermore, IL-13T/+ mice harbored

large pseudocystic lesions in the CNS parenchyma, bordered by voluminous foamy

alternatively activated macrophages (aaMph) containing intracellular cryptococci. In WT

mice, also aaMph tightly bordered pseudocystic lesions. In resistant IL-4-/-, IL-13-/-, and IL-

4Ra-/- mice, no aaMph were discernible. Microglial cells of all mouse genotypes neither

internalized cryptococci nor were found to express markers of alternative activation although

they displayed similar IL-4Ra expression as macrophages. These data provide first evidence

of the development of aaMph in a CNS infectious disease model and point to distinct roles of

macrophages vs. microglial cells in the CNS immune response against C. neoformans.


62

Identification of a novel subcellular localization pattern of Suppressor of Cytokine

Signaling-1 (SOCS-1)

Julia Strebovsky, Andrea Baetz, Christian Koelsche, Alexander H. Dalpke

Dept. of Hygiene and Medical Microbiology, University Heidelberg

Suppressor of cytokine signaling (SOCS) proteins are inducible feedback inhibitors of janus

kinases and signal transducer and activator of transcription (STAT) signaling pathways.

SOCS proteins are induced by various cytokines as well as Toll-like receptor (TLR) ligands

and are supposed to act inhibitory at the level of cytokine receptor phosphorylation, janus

kinase activation or STAT recruitment.

We surprisingly observed that SOCS-1, but no other members of the SOCS family,

predominantly localizes to the nucleus which could be attributed to a novel bipartite nuclear

localization signal (NLS) located between the SH2 domain and the SOCS box. Using a

mutational approach the NLS could be narrowed down to several pivotal arginine amino

acids. Although primarily expressed within the nucleus a fraction of GFP-SOCS-1 was also

observed within the cytoplasm being ligated to the plasma membrane. GFP-SOCS-1 was

demonstrated to clearly suppress IFN-alpha mediated STAT1 phosphorylation. GFP-SOCS-1

proteins bearing mutations in the NLS evidently showed a loss of nuclear localization and

were still able to interfere with STAT activation, however were less inhibitory in STAT-

dependent reporter assays. To further analyse the role of nuclear SOCS-1 inducible cell-lines

have been established. Using photoactivable SOCS-1 constructs we could confirm that

translocation from the cytoplasm to the nucleus occurs very rapidly and that SOCS-1 is

highly mobile within the nucleus.

Thus, we claim a new role for SOCS-1 which is independent from feedback inhibition of

Jak/STAT pathways but dependent on its nuclear availability.


63

Improvement of host defense in the lung

Thomas Tschernig, Felicitas Hecke, Tanja Kerber-Momot, Ulrich Maus, Reinhard Pabst.

Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625 Hannover

MALP-2 is a synthetic lipopeptide and a specific ligand of TLR-2/6. It stimulates the host

defense by induction of chemokines and proinflammatory cytokines. In cocultures with rat

alveolar macrophages and M. bovis BCG MALP-2 led to an enhanced killing of the bacilli.

After intratracheal infection of mice with M. bovis BCG and simultaneous MALP-2 application

decreased numbers of the bacteria were found in the BAL and the lung homogenate at

different times of analyses. In an acute model of lung infection caused by P. aeruginosa

MALP-2 lead to reduced numbers of bacteria in the lung tissue and to a better clinical course

of the infection when the application was performed one day prior to the infection. Thus, local

stimulation of the pulmonary immune system via the TLR-2/6 improves host defense to

bacteria in the lung.


64

Detrimental role of dendritic cells during Y. enterocolitica infection in vivo

Philipp Warnke1, Tanja Linzer2, Sandra Schmitz1, Ingo B. Autenrieth1, Stella Autenrieth1,2

1Institute of Medical Microbiology and Hygiene, University Hospital of Tübingen, Tübingen,

Germany; 2Department of Immunology, University of Tübingen, Tübingen, Germany

CD4 T cells have been demonstrated to be required for control of Yersinia enterocolitica

infection in mice. However, virulence factors of Y. enterocolitica may inhibit T cell priming by

targeting dendritic cells (DC) in several ways: inhibition of maturation and antigen uptake as

well as by the induction of cell death as demonstrated in bone marrow-derived DC. Here we

investigated the role of DC in severe Y. enterocolitica infection in vivo using C57BL/6 wild

type and transgenic mice expressing the diphtheria toxin (DT) receptor under the control of

the CD11c promoter (CD11c.DOG mice) in order to deplete DC in vivo. Repetitive application

of DT led to significant ablation of DC. Upon Y. enterocolitica infection of wild type and DC

depleted CD11c.DOG mice we observed less bacterial burden in the spleen one to 7 days

post infection in CD11c.DOG mice compared to wild type mice and a better survival of the

CD11c.DOG mice compared to the wild type mice. We also observed less viable intracellular

Y. enterocolitica in CD11b+ and Gr-1+ cells one day post infection in CD11c.DOG mice

compared to wild type mice. No difference in the cell populations in the spleen could be

observed one day post infection between the two mouse strains. However, the number of

CD11b+ and Gr-1+ cells was increased in the spleen of CD11c.DOG mice compared to wild

type mice at later time points. One day post infection CCL2, CCL4, CCL7, and IFN-gamma

mRNA expression was increased 12 to 35 fold in wild type mice compared to CD11c.DOG

mice. Adoptive transfer of CFSE-labeled OVA-specific CD4+ T cells into CD11c.DOG mice

treated with DT and infected with Y. enterocolitica revealed reduced proliferation rates

compared to control mice. Taken together, our data suggest that DC may play an ambiguous

role in Y. enterocolitica infection: DC contribute to increased cytokine and chemokine

production and T cell priming, but upon high dose Y. enterocolitica infection may contribute

to increased bacterial burden and mortality.


65

Injury-induced Th cell suppression after injury is restored through targeting dendritic

cells in the regenerating tissue

Florian Wirsdörfer, Jörg Martin Bangen, Michaela Bak, F. U. Schade, Stefanie Flohé

Surgical Research, Department of Trauma Surgery, University Hospital Essen,

Virchowstr.171, D-45147 Essen

Severe injury causes a broad deterioration of the immune system that leads to an enhanced

susceptibility to infectious complications. In parallel, repair/regeneration processes are

initiated in order to restore tissue integrity. We investigated whether dendritic cells (DC) that

appear in the regenerating tissue after skeletal muscle injury might modulate the immune

response to a microbial antigen. A blunt soft tissue trauma of the gastrocnemius muscles of

mice was induced through a single drop-mass impact. One, 4, or 7 d later, leukocyte

infiltration in the muscles and specific T cell responses to ovalbumin/LPS as model antigen in

the popliteal lymph nodes (LN) were characterized. DC in the damaged tissue increased in

number from day 4 after trauma and continuously increased the expression of MHC class II,

CD40, and CD86. After intramuscular application of FITC-labelled ovalbumin, the percentage

of ovalbumin-FITC positive DC was higher in draining LN from trauma than from sham mice

indicating migration of DC from the muscle into the LN. Ovalbumin-specific Th-cells in the LN

of trauma mice secreted higher levels of IFN-gamma than cells from sham mice when

ovalbumin/LPS was applied into the gastrocnemius muscle on day 7 but not earlier after

injury. The proliferation of antigen-specific T cells remained unaffected. In contrast, upon

application of ovalbumin/LPS distant to the site of injury, namely into the footpad, the

ovalbumin-specific IFN-gamma secretion in the popliteal LN was suppressed. Thus, Th cell

responses are impaired after skeletal muscle injury but can be restored through targeting DC

in the regenerating tissue.


66

TLR agonist R848 inhibits differentiation of mDCs and leads to differentiation of

tolerogenic APC from CD14+ monocytes

Sabine Woelfle1, Aline Gierschke1, Klaus Heeg1

1Dept. of Hygiene and Medical Microbiology, University of Heidelberg, Germany

We have shown previously that TLR agonists interfere with GMCSF and IL4 (G4) driven

differentiation of immature mDC from CD14+ monocytes in vitro. Among the TLR agonists

the TLR7/8 agonist R848 is the strongest inhibitor of mDC differentiation. Stimulation of

CD14+ monocytes with G4 and R848 resulted within 5 days in a cell type that is

characterized by an increased PDL-1 and a decreased CD1a expression. The induced

deviant APCs preferentially induce CD25+ FoxP3+ T regulatory cells. We have started to

analyse the mechanisms of action. Therefore we determined cytokine secretion and MAP-

kinases activation: In comparison to G4 stimulated monocytes, R848 plus G4 stimulated

monocytes released high IL-6 amounts and TNF, IL-10 and IL-12p40. Because of the ability

of IL-6 to prevent upregulation of CD1a we blocked the IL-6 signalling pathway and

investigated STAT-3 phosphorylation. IL-6 alone was not responsible for the decreased

CD1a expression. However STAT-3 phosphorylation was enhanced by R848 plus G4. There

were also differences in the MAPK activation pattern. Upon stimulation with R848 and G4 a

strong JNK phosphorylation was observed which was not seen with G4 alone. P38 was also

stronger phosphorylated in R848 plus G4 stimulated cells. P44/42 phosphorylation was

higher with R848 and G4 within the first 15 min. However, at later time points p44/42

phosphorylation was lower. Inhibition of p44/42 lead to downregulation of PD-L1 as STAT-3

inhibition did. We suggest a link between p44/42 and STAT-3 phosphorylation. Here we

present first hints, that this could be indeed the case.


67

LIST OF PARTICIPANTS

Name Institute Email

1. Alber, Gottfried Universität Leipzig, Veterinärmedizinische Fakultät, Immunologie [email protected]

2. Bang, Claudia Universität Lübeck, Institut für Anatomie [email protected]

3. Bank, Elena Universitätsklinikum Ulm; Medizinische Mikrobiologie und Hygiene

[email protected]

4. Bart, Thomas Universität Regensburg, Institut für Immunologie [email protected]

5. Barthelmann, Julia Universität Lübeck; Institut für Anatomie [email protected]

6. Bauer, Judith

Universität Heidelberg, Institut für Hygiene und Medizinische Mikrobiologie

[email protected]

7. Behrends, Jochen Forschungszentrum Borstel; Infektionsimmunologie [email protected]

8. Bekeredjian-Ding, Isabelle Universität Heidelberg; Medizinische Mikrobiologie [email protected]

9. Bogdan, Christian Universität Erlangen; Medizinische Mikrobiologie [email protected]

10. Bohn, Erwin

Universität Tübingen, Institut für Medizinische Mikrobiologie und Hygiene

[email protected]

11. Borkens, Stephanie Universität Düsseldorf; Medizinische Mikrobiologie [email protected]

12. Bruder, Dunja Helmholtz-Zentrum für Infektionsforschung, Braunschweig [email protected]

13. Chittappen Kandiyil, Prajeeth

Universität Erlangen, Institut für Klinische Mikrobiologie, Immunologie und Hygiene

[email protected]

14. Dalpke, Alexander


[email protected]

15. Degrandi, Daniel Universität Düsseldorf; Medizinische Mikrobiologie [email protected]

16. Deininger, Susanne

Universitätsklinikum Bonn, Institut für Medizinische Mikrobiologie, Immunologie und Parasitologie

[email protected]

17. Didovic, Sonja

Universität Düsseldorf, Düsseldorf Institut für umweltmedizinische Foschung

Sonja. [email protected]

18. Dietze, Kirsten Universitätsklinikum Essen, Institut für Virologie [email protected]

19. Dresing, Phillip Universität Düsseldorf; Medizinische Mikrobiologie [email protected]

20. Durlanik, Sibel


[email protected]

21. Eberle, Florian Universität Heidelberg; Medizinische Mikrobiologie [email protected]

22. Echtennacher, Bernd Universität Regensburg; Immunologie [email protected]

23. Ehlers, Stefan Forschungszentrum Borstel, Molekulare Entzündungsmedizin [email protected]

24. Enzenmüller, Stefanie

Universitätsklinikum Ulm, Medizinische Mikrobiologie und Hygiene

[email protected]

25. Erdmann, Hanna Forschungszentrum Borstel, Infektionsimmunologie [email protected]

26. Fischer, Anna Universität Düsseldorf, Institut für medizinische Mikrobiologie [email protected]

27. Fischer, Michael Universität Mainz, Institut für Dermatologie [email protected]

28. Flohe, Stefanie Universitätsklinikum Essen; Klinik und Poliklinik für Unfallchirurgie [email protected]

29. Florian, Christian Universität Regensburg, Institut für Immunologie [email protected]

30. Friedrich, Diana


[email protected]

31. Geginat, Gernot

Universität Mannheim, Institut für Medizinische Mikrobiologie und Hygiene

[email protected]

32. Gentil, Katrin

Universitätsklinikum Bonn, Institut für Medizinische Mikrobiologie, Immunologie und Parasitologie

[email protected]

33. Gibbert, Kathrin Universitätsklinikum Essen, Institut für Virologie [email protected]

34. Grauling-Halama, Silke Institut für Medizinische Mikrobiologie und Hygiene, Mannheim [email protected]

35. Griewank, Klaus Universität Mainz, Institut für Dermatologie [email protected]

36. Häberlein, Susanne


[email protected]

37. Händel, Ulrike Universität Magdeburg, Institut für Medizinische Mikrobiologie [email protected]

38. Haroon, Fahad Universität Magdeburg, Institut für Medizinische Mikrobiologie [email protected]

39. Heeg, Klaus


[email protected]

40. Heitmann, Lisa Forschungszentrum Borstel, Infektionsimmunologie [email protected]

41. Hölscher, Christoph Forschungszentrum Borstel, Infektionsimmunologie [email protected]

42. Holz, Kristian Forschungszentrum Borstel, Infektionsimmunologie [email protected]

43. Jantsch, Jonathan


[email protected]

44. Jugert, Christiane Universität zu Lübeck; Medizinische Mikrobiologie und Hygiene [email protected]

45. Kalies, Kathrin Universität Lübeck; Institut für Anatomie [email protected]

46. Kaschel, Mariel Esther

Universitä Heidelberg, Institut für Hygiene und Medizinische Mikrobiologie

[email protected]


68


47. Kautz-Neu, Kordula Universität Mainz; Institut für Dermatologie [email protected]

48. Kocur, Magdalena Universität Düsseldorf; Medizinische Mikrobiologie [email protected]

49. Kravets, Elisabeth Universität Düsseldorf, Institut für medizinische Mikrobiologie [email protected]

50. Kubatzky, Katharina


[email protected]

51. Lang, Roland Universität Erlangen; Medizinische Mikrobiologie [email protected]

52. Limmer, Andreas Universitätsklinik Bonn; Molekulare Medizin und Experimentelle Immunologie

[email protected]

53. Lopez, Susanne Universität Mainz; Institut für Dermatologie [email protected]

54. Lorenz, Beate Universität Mainz; Institut für Dermatologie [email protected]

55. Luhn, Kerstin

University of Oxford, John Radcliffe Hospital, MRC Human Immunology Unit

[email protected]

56. Masic, Anita Universität Würzburg, Institut für Molekulare Infektionsbiologie [email protected]

57. Metzdorf, Daniela


[email protected]

58. Minsinger, Andreas


[email protected]

59. Mostböck, Sven Universität Regensburg, Institut für Immunologie [email protected]

60. Munoz, Melba Charite Berlin; Institut für Mikrobiologie [email protected]

61. Neumann, Jan Forschungszentrum Borstel; Mikrobielle Grenzflächenbiologie [email protected]

62. Nickel, Daniel


[email protected]

63. Nippe, Nadine Universität Münster, Institut für Immunologie [email protected]

64. Opitz, Bastian Charite Berlin, Med. Klinik m. S. Infektiologie und Pneumologie, [email protected]

65. Parcina, Marijo


[email protected]

66. Pastille, Eva Universität Duisburg-Essen; Klinik und Poliklinik für Unfallchirurgie

[email protected]

67. Pfleiderer, Lisa


[email protected]

68. Plickert, Rita Charite Berlin; Institut für Mikrobiologie [email protected]

69. Raczkowski, Friederike Universitätsklinikum Hamburg Eppendorf, Institut für Immunologie [email protected]

70. Reiling, Norbert Forschungszentrum Borstel; Mikrobielle Grenzflächenbiologie [email protected]

71. Remer, Katharina Universität Würzburg; Molekulare Infektionsbiologie [email protected]

72. Ritter, Uwe Universität Regensburg, Institut für Immunologie [email protected]

73. Schale, Kolja Forschungszentrum Borstel, Mikrobielle Grenzflächenbiologie [email protected]

74. Scheu, Stefanie Universität Düsseldorf; Medizinische Mikrobiologie [email protected]

75. Schleicher, Ulrike Universität Erlangen; Medizinische Mikrobiologie [email protected]

76. Schlüter, Dirk Universität Magdeburg; Medizinische Mikrobiologie [email protected]

77. Schmidt, Kim Universitätsklinik Bonn; Molekulare Medizin und Experimentelle Immunologie

[email protected]

78. Schmidt, Lotte Universität Heidelberg; Medizinische Mikrobiologie [email protected]

79. Schnare, Markus


[email protected]

80. Schneider, Bianca LSHTM London; Infectious and Tropical Diseases [email protected]

81. Schneider, Michael Universitätsklinikum Hamburg Eppendorf, Institut für Immunologie [email protected]

82. Schneider, Wulf

Universität Regensburg, Institut für Medizinische Mikrobiologie und Hygiene

[email protected]

83. Schoenen, Hanna


[email protected]

84. Schumak, Beatrix Universitätsklinik Bonn; Molekulare Medizin und Experimentelle Immunologie

[email protected]

85. Schwandt, Timo

Universitätsklinikum Bonn, Institut für Molekulare Medizin und Experimentelle Immunologie

[email protected]

86. Schwonberg, Kirsten Universität Mainz, Institut für Dermatologie [email protected]

87. Siegfried, Alexandra


[email protected]

88. Sodenkamp, Jan Christian Forschungszentrum Borstel; Infektionsimmunologie [email protected]

89. Stamm, Claudia Universität Lübeck; Institut für Anatomie [email protected]

90. Steinhoff, Ulrich MPI Infektionsbiologie [email protected]

91. Stenzel, Werner

Klinikum Virchow, Campus Charite, Berlin, Institut für Neuropathologie

[email protected]

92. Strebovsky, Julia


[email protected]

93. Stutz, Andrea


[email protected]


69


94. Tschernig, Thomas Medizinische Hochschule Hannover, Institu für Anatomie [email protected]

95. Van Laak, Vincent Charite Berlin, Med. Klinik m. S. Infektiologie und Pneumologie [email protected]

96. Van Zandbergen, Ger Universitätsklinikum Ulm; Medizinische Mikrobiologie und Hygiene

[email protected]

97. Von Stebut, Esther Universität Mainz, Institut für Dermatologie [email protected]

98. Warnke, Philip


[email protected]

99. Weighardt, Heike Universität Düsseldorf, Institut für umweltmedizinische Foschung [email protected]

100. Wirsdörfer, Florian Universitätsklinikum Essen, Klinik für Unfallchirurgie [email protected]

101.Wölfle, Sabine


[email protected]

Documents

13. Minisymposium „Infektion und Immunabwehr“ Burg … 2009 - Programm... · Werner Stenzel et al., Berlin 8 Neuron and astrocyte –specific function of IKK-2 and NEMO in Toxoplasma