Insight into the activation mechanism of Toll-like receptor 4 by diC14-amidine

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Insight into the activation mechanism

of Toll-like receptor 4 by diC14-amidine

Boris Iker SCHMIDT

Promoteur:

Dr. Michel

VANDENBRANDEN

Co-promoteur: Prof. Jean-Marie RUYSSCHAERT

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1 Acknowledgements

Pursuing my Ph.D. would not have been possible without the help and support of

many people – in scientific questions as well as in matters concerning the day-to-day

issues of coming to live in a foreign country without actually ever having been there

before or knowing anybody. Unfortunately it is impossible to appreciate each of them

with the detail they would deserve – this would require countless pages.

I would like to start with my thesis supervisor Dr. Michel VANDENBRANDEN. Without

him and his constant support for my scientific work and his help concerning (not only)

administration and bureaucracy encounters my life would have been much more

difficult. My sincere thanks for always being willing to listen to my problems and for

always trying to help. Prof. Jean-Marie RUYSSCHAERT also deserves special

thanks, for being my co-supervisor, for always checking on the progress of my work

and for pushing me onwards, above all after setbacks, and for his honest and

down-to-earth feedback.

My special gratitude also goes to Dr. Caroline LONEZ, who, thanks to her

knowledge, contacts and gentleness and will to motivate me, had the misfortune to

end up as my personal emergency call service. Without my supervisors and her this

research would not have been possible.

I also wish to thank Dr. Erik GOORMAGHTIGH and Dr. Fabrice HOMBLÉ for

welcoming me to their laboratory and for making it possible to conduct my Ph.D.

here. Dr. Goormaghtigh together with Dr. Cédric GOVAERTS also formed part of my

supervisory committee and they deserve my thanks for supporting me throughout my

thesis. I also would like to thank Dr. Vincent RAUSSENS, Dr. Guy

VANDENBUSSCHE, Dr. Rabia SARROUKH and Dr. Eva-Maria KRAMMER for

interesting discussions and their helpful tips and suggestions.

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2 Libre de Bruxelles, for letting me use his laboratory's equipment and for providing me

with Dynasore.

Thank you also to Prof. Daniel DESMECHT for taking an interest in my work and for

agreeing to be part of my thesis committee.

Last but not least the "lab population" (as far as not already mentioned before) who I

would like to thank: Mouna, for (at least quite a while) sharing an office, moods, jokes

and a lab room; Matthieu, for always helping me out with bureaucratic and other

issues from tax declarations to knowing which university form to look for; my "office

roommates" Nancy, Rosie, Nicolas, Anastassia, Magdalena, Allison, Pamela,

Grégory; Vinciane, whose indirect "fault" (via a shared acquaintance) it basically was

that I ended up in Brussels; Benjamin and Malvina who worked in the same group as

me; and all the other staff and students I encountered during my time at the S.F.M.B.

And of course a thesis cannot be conducted without financial support and so I would

like to thank the F.R.S.-F.N.R.S. for allocating me a F.R.I.A. grant and Baron André

JAUMOTTE, president of the Fonds David et Alice Van Buuren, for supporting me

towards the end of my thesis.

The last years have been one hell of a ride

– not only inside but also outside the

laboratory. So I will take the chance to thank the Orient gang

– without them my life

would certainly have taken a completely different turn and they were – for a while – a

home away from home, for which I remain more than grateful.

And now to the most important people in my life: my wife and my family. Thank you

for always supporting me and for simply always being there for me, no matter what.

"Hättsch un wärsch" and "Renn em noach" are sensible pieces of advice to consider when retrospectively

pondering one's choices and decisions.

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I

II

I.4. diC14-amidine ... 51

I.4.1. Cationic lipids ...52

I.4.2. diC14-amidine ...55

II. Purpose of the work ...60

III. Results and discussion ...62

III.1. The role of the TLR4 co-receptor CD14 in the activation of the MyD88 pathway and the TRIF pathway – the two TLR4-dependent signaling cascades – by diC14-amidine ... 63

III.1.1. The HEK293 cell model ...63

III.1.2. Adaptation of the HEK293 cell culture parameters and of the transient transfection technique ...65

III.1.3. The role of CD14 in the activation of the MyD88 and TRIF pathways by diC14-amidine ...70

III.1.3.1. The influence of the membrane form of CD14 (mCD14) on the activation of the MyD88 pathway by diC14-amidine in HEK293 cells ...71

III.1.3.2. Blocking CD14 with neutralizing antibodies – effect on the activation of the MyD88 and the TRIF pathways of THP1-XBlueTM and THP1 cells by diC14-amidine ...74

III.1.3.3. The influence of the soluble form of CD14 (sCD14) on the activation of the MyD88 pathway in HEK293 cells by diC14-amidine ...79

III.1.3.4. Studying the interaction between soluble CD14 and diC14-amidine by infrared spectroscopy ...84

III.1.3.4.1. Comparison of the secondary structure of CD14 before and after incubation with diC14-amidine and LPS ...85

III.1.3.4.2. Comparison of the solvent accessibility of CD14 before and after incubation with diC14-amidine and LPS ...92

III.1.4. Discussion and preliminary conclusion part I: The role of CD14 with regard to the activation of the TLR4-dependent signaling cascades by diC14-amidine ...97

III.1.4.1. diC14-amidine does not require CD14 to activate the MyD88 and TRIF pathways ...97

III.1.4.2. Dual concentration-dependent effect of CD14 on the activation of the MyD88 pathway by diC14-amidine: enhancing at moderate concentrations, detrimental at high doses ...99

III.2. The role of the endocytosis in the activation of the TLR4-dependent signaling cascades by diC14-amidine ... 103

III.2.1. Dynasore ... 104

III.2.2. Bafilomycin A1 ... 107

III.2.3. Chloroquine ... 110

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III

IV. General conclusion and perspectives ... 116

V. Experimental procedures ... 122

V. 1. HEK293 ... 122

V.1.1. Cell maintenance ... 122

V.1.1.1. Creating HEK293 cell stocks ... 122

V.1.1.2. Initial Culture Procedure ... 123

V.1.2. Plasmids for transient transfection ... 123

V.1.2.1. Transformation into competent cells ... 123

V.1.2.2. Plasmid purification ... 124

V.1.2.3. Determining plasmid DNA concentrations ... 124

V.1.2.4. Preparing glycerol stocks ... 124

V.1.3. Transient transfection of the HEK293 cells ... 125

V.1.4. Recombinant protein treatment ... 126

V.1.4.1. Endotoxin removal ... 126

V.1.4.2. Determining protein concentrations ... 127

V.1.5. Stimulation of the HEK293 cells ... 127

V.1.5.1. diC14-amidine ... 128

V.1.5.2. LPS (bacterial lipopolysaccharide) ... 128

V.1.6. HEK293 cell lysis ... 129

V.1.7. Quantification of luciferase activity ... 129

V.2. THP1-XBlueTM and THP1 ... 130

V.2.1. Cell maintenance (THP1-XBlueTM) ... 130

V.2.2. Cell maintenance (THP1) ... 130

V.2.3. Priming the THP1-XBlueTM and THP1 cells ... 131

V.2.4. Experiments with neutralizing antibodies against CD14 on THP1-XBlueTM and THP1 ... 131

V.2.4.1. Quantification of SEAP ... 132

V.2.4.2. Quantification of hTNF-α and hIP10 ... 132

V.2.5. Experiments with endocytosis inhibitors on THP1 cells ... 133

V.2.5.1. Assessing the cell viability ... 133

V.3. Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) ... 134

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IV

Summary of the thesis

The bacterial lipopolysaccharide (LPS)-sensing machinery with Toll-like receptor 4

(TLR4) at its centre has been the subject of extensive research in the last decades

and considerable progress has been made in the elucidation of this intricate

process. However we are still far from a complete understanding of the roles and

effects of all the molecules involved in the activation of the innate immune system

via TLR4. While it has been established that TLR4 and myeloid differentiation

factor 2 (MD2) are both essential for the host cells' ability to respond to an LPS

challenge, the role of other, so-called "accessory", molecules is much less clear.

The co-receptor cluster of differentiation 14 (CD14) has been widely perceived as

being a mere facilitator for the capture and transfer of LPS to TLR4, until recent

studies suggested that its role in the LPS-caused immune response goes far

beyond that. It was proposed that CD14 might have a determining influence on

which TLR4-dependent signaling cascades are triggered (the so-called MyD88

and TRIF pathways) and to be crucial for the endocytosis of TLR4.

In our laboratory the TLR4-receptor complex was shown to be also triggered by

diC14-amidine, an amphiphilic cationic lipid originally synthesized for its carrier

properties. Unlike cationic lipids we tested, diC14-amidine is able to specifically

activate cells through a TLR4-dependent pathway, resulting in the secretion of

proinflammatory cytokines and in the production of type I IFNs. This suggests that

the stimulatory activity of diC14-amidine extends to both signaling cascades

downstream of TLR4, which depend on the TLR4 adaptor molecules MyD88 and

TRIF respectively.

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immunostimulatory activity. But despite diC14-amidine's ability to elicit a basal

activation level in the absence of CD14 it was also found that the presence of the

co-receptor modulates the TLR4 activation. Infrared spectroscopy experiments

conducted on CD14 and both diC14-amidine-CD14 and LPS-CD14 mixtures

suggest that both immunostimulants affect CD14 in a similar manner and lead to

similar structural modifications, indicating direct interaction and actual binding of

the cationic lipid to the TLR4 co-receptor. The fact that diC14-amidine is able to

activate both TLR4-dependent signaling cascades in the absence of CD14 raised

questions as to potential consequences of this finding.

Physiologically, i.e. in the case of sensing LPS, CD14 was demonstrated to be at

least required for the endocytosis of TLR4 and the subsequent activation of the

TRIF pathway. We therefore hypothesized that diC14-amidine possibly can be

internalized without requiring CD14 to trigger the TRIF pathway. When studying

the effect of several endocytosis blockers on the activation of the TLR4-dependent

cascades by the cationic lipid, we found that the blocking of the endocytosis

mechanism at different stages affected the immunostimulatory activity of

diC14-amidine and LPS in different ways. It turned out that blocking early clathrin-

or caveolae-dependent endocytosis inhibited not only the cationic lipid's activation

of the TRIF pathway, but also the one of the MyD88-dependent signaling cascade.

This constitutes another major difference to LPS that only requires endocytosis for

its triggering of the TRIF pathway, but not for the MyD88 one. Blocking the

maturation of the early endosomes into the late endosomes on the other hand only

inhibited diC14-amidine's activation of the TRIF pathway but did not compromise

its initiation of the MyD88 pathway. This suggests that the cationic lipid generally

enters the cell via endocytosis or an endocytosis-like mechanism and that it

activates

– unlike LPS – both TLR4-dependent signaling cascades from inside

endosomal vesicles, albeit at different stages of the endocytosis process.

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VI

List of abbreviations

AP: activator protein APC: antigen-presenting cell

ATR-FTIR: Attenuated total reﬂection - Fourier-transform infrared spectroscopy BAF: Bafilomycin A1

BCA: bicinchoninic acid

BMDC: bone marrow-derived dendritic cell BMDM: bone marrow-derived macrophage

BPI: bactericidal/ permeability-increasing protein C/EBP: CCAAT-enhancer-binding protein

CCP: clathrin-coated pit CCV: clathrin-coated vesicle CD: cluster of differentiation cDNA: complementary DNA chemokine: chemotactic cytokine CLR: C-type lectin receptors CLQ: Chloroquine

CpG: -C-phosphate-G-: cytosine and guanine separated by one phosphate CME: clathrin-mediated endocytosis

CREB: cAMP response element-binding protein Cxcl: chemokine (C-X-C motif) ligand

DAMP: damage-associated molecular pattern DDAB: dimethyldioctadecylammonium bromide

diC14-amidine: N-t-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine DLEPC: 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine

DMEM: Dulbecco's Modified Eagle's Medium

DMEPC: 1, 2-dimyristoyl-sn-glycero-3-ethylphosphocholine DMTAP: 1,2-dimyristoyl-3-trimethylammonium-propane DNA: deoxyribonucleic acid

DOEPC: 1, 2-dioleoyl-sn-glycero-3-ethylphosphocholine DOTAP: 1,2-dioleoyloxy-3-trimethylammoniumpropane DPEPC: 1, 2-dipalmitoyl-sn-glycero-3-ethylphosphocholine DPTAP: 1,2-dipalmitoyl-3-trimethylammonium-propane ds: double-stranded

DSEPC: 1, 2-distearoyl-sn-glycero-3 -ethylphosphocholine DSTAP: 1,2-stearoyl-3-trimethylammonium-propane DYN: Dynasore

E. coli: Escherichia coli

ELISA: enzyme-linked immunosorbent assay ER: endoplasmatic reticulum

ERK: extracellular-signal-regulated kinase FBS: fetal bovine serum

GlcNAc: N-acetyl-D-glucosamine GPI: glycosylphosphoinositol

H/D: hydrogen/deuterium (exchange) HDL: high density lipoproteins

HEK: human embryonic kidney (cells)

HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (buffer) HSP: heat shock protein

iE-DAP: γ-D-glutamyl-meso-diaminopimelic acid IFN: interferon

IKK: inhibitor-κB kinase IκB: NF-κB-inhibitor IL: Interleukin

IL1R1: Interleukin 1 receptor type 1 IRAK: IL1 receptor associated kinase IR: infrared

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VII

KDO: 2-keto-3-desoxyoctonate LBP: LPS-binding protein

LGP: laboratory of genetics and physiology LPS: lipopolysaccharide

LRR: leucine-rich repeat MAL: MyD88-adapter-like MAP: mitogen-activated protein

MAP3K8: mitogen-activated protein kinase kinase kinase 8 MAPK: mitogen-activated protein (MAP) kinase

mCD14: membrane-associated form of CD14 MD: myeloid differentiation factor

MDA: melanoma differentiation factor MDP: muramyldipeptide

MHC: major histocompatibility complex MINCLE: macrophage-inducible C-type lectin MKK: mitogen-activated protein kinase kinase mRNA: messenger RNA

MyD88: myeloid differentiation factor 88 NEMO: NF-κB essential modulator

NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells NLR: NOD-like receptor

NOD: nucleotide-binding oligomerization domain PAMP: pathogen-associated molecular pattern

PAPC: 2-arachidonoyl-1-palmitoyl-sn-glycero-3-phosphocholine PBS: phosphate buffered saline

PIP2: phosphatidylinositol 4,5-biphosphate PMA: phorbol 12-myristate 13-acetate PRR: pattern recognition receptor RIG: retinoic acid-inducible gene RIP: receptor-interacting protein rLPS: "rough" LPS (also R-LPS) RLR: RIG-I-like receptor RNA: ribonucleic acid

ROS: reactive oxygen species

RPMI: Roswell Park Memorial Institute developed media rRNA: ribosomal RNA

SAP130: Sin3A-associated protein, 130 kDa sCD14: soluble form of CD14

SEAP: secreted embryonic alkaline phosphatase sLPS: "smooth" LPS (also S-LPS)

sMD2: soluble form of MD2

SOC: Super Optimal broth (SOB) with Catabolite repression srLPS: "semirough" LPS (also SR-LPS)

ss: single-stranded TAB: TAK1 binding protein

TAK: transforming growth factor β activated kinase TBK: TANK-binding kinase

TIL: Toll/IL1R like protein

TIR: Toll/Interleukin 1 receptor (originally Toll-Interleukin 1- resistance) TLR(1-13): Toll-like receptor (1-13)

TMAG: didodecyl N-(α-(trimethylammonio)acetyl)-D-glutamate chloride TMB: 3,3′,5,5′-Tetramethylbenzidine

TPL2: see MAP3K8

TRADD: TNF receptor type 1-associated DEATH domain protein TRAF: TNF-α receptor-associated factor

TRAM: TRIF-related adaptor molecule

TRIF: TIR-domain-containing adaptor inducing interferon-beta

V. cholerae: Vibrio cholerae

Insight into the activation mechanism of Toll-like receptor 4 by diC14-amidine

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