Plasma factors as endogenous agonists and modulators of TLR4 signaling in microglia [Elektronische Ressource] / submitted by Jörg Scheffel

Plasma Factors as
Endogenous Agonists and Modulators
of TLR4 Signaling in Microglia




Doctoral Thesis
In partial fulfillment of the requirements for the degree
“Doctor rerum naturalium (Dr. rer. nat.)”
in the Molecular Medicine Study Program
at the Georg-August University Göttingen


submitted by
Jörg Scheffel
born in
Berlin, Germany




Göttingen, April 2010


Members of the Thesis Committee:


Supervisor

Prof. Dr. U. K. Hanisch
Department of Neuropathology
University of Göttingen (Medical Faculty)


Second member of the Thesis Committee

Prof. Dr. J. Wienands
Department of Cellular and Molecular Immunology
University of Göttingen (Medical Faculty)




Third member of the Thesis Committee

Prof. Dr. KA. Nave
Department of Neurogenetics
Max Planck Institute for Experimental Medicine, Göttingen




Date of Disputation

21.06.2010 AFFIDAVIT

AFFIDAVIT

Here, I declare that my doctoral thesis entitled “Plasma Factors as Endogenous Agonists
and Modulators of TLR4 Signaling in Microglia” has been written independently with no
other sources and aids than quoted.



Göttingen, April 2010



(Signature)
- I - LIST OF PUBLICATIONS


Goos, M., Lange, P., Hanisch, U.K., Prinz, M., Scheffel, J., Bergmann, R., Ebert, S., Nau,
R., 2007. Fibronectin is elevated in the cerebrospinal fluid of patients suffering from
bacterial meningitis and enhances inflammation caused by bacterial products in primary
mouse microglial cell cultures. J Neurochem. 102, 2049-60.
Ribes, S., Ebert, S., Regen, T., Czesnik, D., Scheffel, J., Zeug, A., Bunkowski, S., Eiffert,
H., Hanisch, U.K., Hammerschmidt, S., Nau, R., 2010. Fibronectin stimulates Escherichia
coli phagocytosis by microglial cells. Glia. 58, 367-76.
- II - TABLE OF CONTENT
AFFIDAVIT I
LIST OF PUBLICATIONS II
ACKNOWLEDGEMENTS VI
ABSTRACT VII
LIST OF FIGURES IX
LIST OF TABLES XII
ABBREVIATIONS XIII
1. INTRODUCTION - 1 -
1.1 Innate and adaptive immunity – the two arms of (and for) defense - 1 -
1.2 Microglia as the innate immune cells of the CNS - 2 -
1.3 Microglial responses are highly regulated - 2 -
1.4 Toll-like receptors (TLRs) for pattern recognition - 5 -
1.5 TLRs are sensors of damage signals - 9 -
1.6 TLRs in the CNS – a double-edged sword - 10 -
1.7 Toll-like receptor 4 – a versatile sensor of PAMPs and DAMPs - 12 -
2. AIM OF THE STUDY - 14 -
3. MATERIAL AND METHODS - 15 -
3.1 Animals - 15 -
3.2 Primary microglial cultures - 15 -
3.3 Organotypic hippocampal slice cultures (OHSC) - 16 -
3.4 L929 mouse fibroblast cultures - 17 -
3.5 Generation and cultivation of mouse hybridoma cell cultures - 17 -
3.6 Microglial stimulation - 17 -
3.7 WST-1 cell viability assay - 20 -
3.8 Immunocytochemistry - 20 -
3.9 Cyto-/chemokine quantification (ELISA) - 21 -
3.10 Measurement of anti-DsRed IgG in hybridoma culture supernatants - 21 -
MAPK3.11 nt of phosphorylated NF κB p65 and p38 - 21 -
3.12 Citrullination of FN - 22 -
3.13 Protease digestion of TLR4 agonists - 22 -
3.14 Enzymatic deglycosylation - 23 -
- III - TABLE OF CONTENT
3.15 Conjugation of IgG to N-Hydroxysuccinimide-Fluorescein - 23 -
3.16 IgG refolding - 23 -
3.17 Matrix Assisted Laser Desorption/Ionization-Time of Flight
(MALDI-TOF) Mass Spectrometry (MS) - 24 -
3.18 SDS-PAGE and Imidazol–SDS–Zinc staining - 25 -
3.19 Immunoblot and Dot blot analyses - 26 -
3.20 Thrombin activity assay - 26 -
3.21 Gene expression analysis (4x44K Agilent microarray) - 27 -
3.22 HPLC and FPLC analyses - 27 -
3.23 Preparation of chemically competent cells - 29 -
3.24 Cloning of DsRed cDNA - 30 -
3.25 Expression and purification of recombinant DsRed protein - 31 -
4. RESULTS (PART A) - 32 -
4.1 Thrombin and fibronectin as DAMP signals for microglial cells - 32 -
4.2 Microglial release responses to thrombin preparations depend
HMWM on thrombin - 33 -
HMWM4.3 Thrombin critically depends on TLR4 and can interfere
with LPS signaling - 37 -
HMWM4.4 Thrombin activity for TLR4 does not derive from LPS contamination - 41 -
HMWM4.5 Thrombin signaling depends on a CD14/TLR4/MyD88/TRIF complex - 43 -
HMWM4.6 Thrombin is a complex of coagulation-associated proteins - 45 -
4.7 Microglia responses to pFN depend on CD14/TLR4/MyD88 signaling - 48 -
4.8 pFN can functionally displace LPS from its receptor - 53 -
HMWM4.9 Thrombin -, pFN- and LPS-induced microglial responses are
differently modulated by immunoregulatory cytokines - 55 -
4.10 Microglial responses to pFN involve integrin assistance - 57 -
4.11 The microglial activation by pFN requires conformational flexibility - 60 -
4.12 Microglia respond to LPS or FN with individual gene expression patterns - 61 -
RESULTS (PART B) - 64 -
4.13 Immunoglobulins as DAMP signals for microglial cells - 64 -
4.14 Microglia respond to IgG stimulation with release of cytokines
and chemokines - 64 -
4.15 Microglia respond to IgG isotypes across species borders - 66 -
4.16 G within a tissue context - 67 -
4.17 IgG-dependent activation of microglia reveals partial Fc R contributions - 68 -
- IV - TABLE OF CONTENT
4.18 IgGs utilize a CD14/TLR4/MyD88/TRIF complex to induce microglial
responses - 71 -
4.19 IgG-induced microglial responses are not caused by endotoxin
contamination - 75 -
4.20 IgG- and LPS-induced microglial responses are differently
modulated by immunoregulatory cytokines - 78 -
4.21 TLR4 agonistic activity of IgGs is influenced by minor conformational
changes - 80 -
4.22 Generation of monoclonal IgGs against a non-mammalian
fluorescent protein - 85 -
4.23 Transient pH shifts have lasting effects on the TLR4-agonistic
activity of IgGs - 89 -
4.24 Activation of microglial cells by IgGs depends on their
carbohydrate structures - 91 -
5. DISCUSSION - 98 -
5.1 Microglia as cellular and TLRs as molecular sensors of danger signals - 98 -
5.2 Thrombin as a potential signal for microglia - 100 -
5.3 pFN as a coagulation factor with DAMP character for microglia - 103 -
5.4 Ig(G) isotypes with DAMP signal function for microglia - 107 -
HMWM5.5 Thrombin , FN and IgGs as agonists for a CD14/TLR4
receptor complex - 111 -
5.6 TLR4 – decision maker in microglial responses to PAMPs and DAMPs - 113 -
5.7 TLR4 signaling to PAMPs and DAMPs - 116 -
5.8 Environmental factors controlling the character of DAMPs - 117 -
5.9 Microglial reactive phenotypes as controlled by DAMPs - 119 -
6. SUMMARY AND CONCLUSIONS - 121 -
REFERENCES XI
CURRICULUM VITAE XXIII
- V - ACKNOWLEDGEMENTS

I would like to thank my supervisor Prof. Dr. U. K. Hanisch for inviting me to his laboratory
and the interesting project(s). Special thanks for his excellent support and guidance, his
great patience and continuous good mood.

I also like to thank Prof. Dr. J. Wienands and Prof. Dr. KA. Nave for being members of my
Thesis Committee.

A special thanks goes to our collaboration partners in Göttingen (PD. Dr. Hassan Dihazi),
Berlin (Dr. Oliver Kann), Stockholm (Prof. Robert Harris and Roham Parsa – I really did
enjoy the time in your lab) and Atlanta (Dr. Nicole Poulsen) for sharing ideas, excellent
experimental support and discussion.

Moreover I would like to thank team members Denise, Tommy, Christiane, Jens, Martina,
Steffi and Arek for the great atmosphere and fun in the lab and the office. A special thanks
goes to Elke for her great patience and support in the cell culture and with the ELISAs.
Thanks for all the fruit gums and cakes. Moreover, thanks to Wiebke, Jutta, Tanja,
Claudia, Tobi and Mario, for having ‘after work beer’ and lots of fun (not only in the lab).

Very special thanks to Simone, Lilli, Matze and Axel for their great support.

I also like to thank my very good old friends back in Berlin (and now also Geneva) who
kept me grounded.

Finally, but most importantly, I like to thank my parents and my brother for their support
and motivation.
- VI - ABSTRACT
ABSTRACT

Toll-like receptors (TLRs) comprise a family of pattern recognition receptors (PRRs) which
recognize pathogen-associated molecular patterns (PAMPs) – an assortment of
evolutionary conserved structural motifs in microbes. Expressed by innate immune cells,
such as CNS microglia, TLRs trigger complex gene activations and functional
mechanisms of host defense. TLRs may additionally respond to certain endogenous
molecules which are produced, released or structurally modified upon cellular and tissue
injury and thereby serve as damage-associated molecular patterns (DAMPs). This study
focused on selected plasma proteins associating with the coagulation cascade
HMWM(thrombin , fibronectin) or belonging to the humoral limb of the adaptive immune
system (immunoglobulins). We found them to be TLR4-agonistic DAMP signal