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University of Veterinary Medicine Hannover
Immunology Unit &
Research Center for Emerging Infections and Zoonoses
Role of C-type lectin receptors in bacterial
recognition
THESIS
Submitted in partial fulfilment of the requirements for the degree
Doctor rerum naturalium
(Dr. rer. nat.)
awarded by the University of Veterinary Medicine Hannover
by
Sabine Mayer-Lambertz
from Prüm
Hannover, Germany 2019
Supervisor: Prof. Dr. Bernd Lepenies
Supervision Group: Prof. Dr. Bernd Lepenies
Prof. Dr. Maren von Köckritz-Blickwede
Prof. Dr. Bastian Opitz
1rst Evaluation: Prof. Dr. Bernd Lepenies
Immunology Unit & Research Center for Emerging Infections and
Zoonoses
University of Veterinary Medicine Hannover, Foundation
Hannover, Germany
Prof. Dr. Maren von Köckritz-Blickwede
Institute for Biochemistry, Department of Physiological Chemistry
University of Veterinary Medicine Hannover, Foundation
Hannover, Germany
Prof. Dr. Bastian Opitz
Medical Department, Division of Infectious Diseases and
Pulmonary Medicine
Charité - Universitätsmedizin Berlin
Berlin, Germany
2nd Evaluation: Prof. Dr. Thomas A. Kufer
Department of Immunology
University Hohenheim
Stuttgart, Germany
Date of final exam: 03.04.2019
„Imagination is more important than knowledge“
-Albert Einstein-
Parts of the thesis have been published previously:
Mayer S*, Raulf M-K*, Lepenies B (2017) C-type lectins: their network and roles in
pathogen recognition and immunity. Histochemistry and Cell Biology 147 (2):223-237.
doi:10.1007/s00418-016-1523-7
* S. Mayer und M-K. Raulf contributed equally as joined first authors to this work.
Mayer S, Moeller R, Monteiro JT, Ellrott K, Josenhans C, Lepenies B (2018) C-Type Lectin
Receptor (CLR)–Fc Fusion Proteins As Tools to Screen for Novel CLR/Bacteria Interactions:
An Exemplary Study on Preselected Campylobacter jejuni Isolates. Frontiers in Immunology
9:213. doi:10.3389/fimmu.2018.00213
Imai T, Matsumura T*, Mayer-Lambertz S*, Wells CA, Ishikawa E, Butcher SK, Barnett TC,
Walker MJ, Imamura A, Ishida H, Ikebe T, Miyamoto T, Ato M, Ohga S, Lepenies B, van
Sorge NM, Yamasaki S (2018) Lipoteichoic acid anchor triggers Mincle to drive protective
immunity against invasive group A Streptococcus infection. Proceedings of the National
Academy of Sciences 115(45):E10662-E10671. doi: 10.1073/pnas.1809100115
* T. Matsumura and S. Mayer-Lambertz contributed equally as joined second authors to this work.
Further publications:
Revised manuscript submitted to Cell Reports, 22.11.2018:
Raulf M-K, Johannssen T, Matthiesen S, Neumann K, Hachenberg S, Mayer-Lambertz S,
Steinbeis F, Seeberger P-H, Baumgärtner W, Strube C, Ruland J, Lepenies B The C-type
Lectin Receptor CLEC12A Recognizes Plasmodial Hemozoin and Contributes to Cerebral
Malaria Development.
Manuscript in preparation:
Achilli S, Monteiro J, Serna S, Mayer-Lambertz S, Thepaut M, Leroy A, Ebel C, Reichardt
N, Lepenies B, Fieschi F, Vivès C TETRALEC, Artificial Tetrameric Lectins: a tool to
screen ligand and pathogen interaction.
Table of content
List of abbreviations ................................................................................................................... I
Abstract .................................................................................................................................. - 1 -
Zusammenfassung.................................................................................................................. - 3 -
Chapter 1: General introduction............................................................................................. - 6 -
1. The immune system - essential for survival ................................................................... - 6 -
1.1 Innate Immunity .................................................................................................. - 7 -
1.2 Adaptive Immunity ............................................................................................. - 8 -
2. Of PAMPS, DAMPS and PRRs ................................................................................... - 11 -
2.1 TLRs: their discovery changed the understanding of pathogen sensing ........... - 13 -
2.2 NLRs and RLRs: soluble cytoplasmic sensors of PAMPs and DAMPs ........... - 14 -
2.3 CLRs: A diverse group that recognises sweet PAMPs ..................................... - 16 -
2.3.1 Role of CLRs in anti-bacterial immunity ....................................................... - 18 -
3. Aims of the study ......................................................................................................... - 22 -
Chapter 2: Paper I ................................................................................................................ - 24 -
C-type lectins: their network and roles in pathogen recognition and immunity .............. - 24 -
Chapter 3: Paper II ............................................................................................................... - 26 -
C-type lectin receptor (CLR)-Fc fusion proteins as tools to screen for novel CLR/bacteria
interactions: an exemplary study on preselected Campylobacter jejuni isolates ............. - 26 -
Chaper 4: Paper III ............................................................................................................... - 29 -
Lipoteichoic acid anchor triggers Mincle to drive protective immunity against invasive
group A Streptococcus infection ...................................................................................... - 29 -
Chapter 5: Discussion .......................................................................................................... - 32 -
Chapter 6: Future outlook .................................................................................................... - 41 -
Chapter 7: Concluding remarks ........................................................................................... - 43 -
References ............................................................................................................................ - 44 -
Affidavit ............................................................................................................................... - 61 -
Acknowledgement ............................................................................................................... - 62 -
List of abbreviations
I
List of abbreviations
ABTS 2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt
AIM 2 absent in melanoma 2
APC antigen-presenting cell
AP 1 activator protein 1
BMDC bone marrow-derived dendritic cell
BMM bone marrow-derived macrophage
CARD9 caspase recruitment domain family member 9
CD cluster of differentiation
CHO chinese hamster ovary
Clec C-type lectin
CLR C-type lectin receptor
CpG DNA CpG oligodeoxynucleotides
CRD carbohydrate-recognition domain
CTL cytotoxic T lymphocyte
CTLD C-type lectin-like domain
DC dendritic cell
DCAR dendritic cell immunoactivating receptor
DCIR dendritic cell immunoreceptor
DC-SIGN dendritic cell-specific ICAM-grabbing non-integrin
Dectin DC-associated C-type lectin
DGDG diglucosyldiacylglycerol
ds double-stranded
ECM extracellular matrix
ELISA enzyme-linked immunosorbent assay
ER endoplasmic reticulum
FC fragment crystallizable
FcR Fc receptor
FcRγ Fc receptor common gamma-chain
FITC fluorescein isothiocyanate
GAS group A Streptococcus
GalNAc N-acetylgalactosamine
List of abbreviations
II
GBS group B Streptococcus
G-CSF granulocyte-colony stimulating factor
GFP green fluorescence protein
GI gastrointestinal
Glu glutamate
HIF-1 hypoxia-inducible factor-1
HRP horseradish peroxidase
IFN interferon
Ig immunoglobulin
IL interleukin
IPS-1 interferon-beta promoter stimulator 1
ITAM immunoreceptor tyrosine-based activation motif
ITIM immunoreceptor tyrosine-based inhibitory motif
LBP lipopolysaccharide-binding protein
LOS lipooligosaccharide
LPS lipopolysaccharide
LRR leucine-rich repeat
LTA lipoteichoic acid anchor
ManLAM mannose-capped lipoarabinomannan
MAPK mitogen-activated protein kinases
MCL macrophage C-type lectin
MDA5 melanoma differentiation-associated gene 5
MDP muramyl dipeptide
MGDG monoglucosyldiacylglycerol
MGL macrophage galactose-type C-type lectin
MHC major histocompatibility complex
Mincle macrophage-inducible C-type lectin
MIP macrophage inflammatory protein
MR mannose receptor
MyD88 myeloid differentiation factor 88
NFAT nuclear factor of activated T cells
NF-κB nuclear factor kappa-light-chain-enhancer of activated B cells
List of abbreviations
III
NK cell natural killer cell
NLR NOD-like receptor
NO nitric oxide
NOD nucleotide-binding oligomerization domain
PAMP pathogen-associated molecular pattern
PE phycoerythrin
PHDs prolyl hydroxylase domain enzymes
Pro proline
Raf rapidly accelerated fibrosarcoma
RANTES regulated on activation normal T cell expressed and secreted
RIG-I retinoic acid inducible gene I
RLR RIG-I-like receptor
ROS reactive oxygen species
PRRs pattern recognition receptors
SAP130 Sin3A associated protein 130
SIGNR DC-SIGN-related protein
ss single-stranded
STING stimulator of interferon genes
Syk spleen tyrosine kinase
TAP transporter associated with antigen processing
TCR T cell receptor
TDB trehalose-6,6-dibehenate
TDM trehalose-6,6-dimycolate
Th T helper cell
TIR Toll-Interleukin 1 Receptor
TLR Toll-like receptor
TNF tumor necrosis factor
TRAF TNF receptor associated factor
Treg regulatory T cell
TRIF TIR-domain-containing adapter-inducing interferon-β
Abstract
- 1 -
Abstract
Sabine Mayer-Lambertz: Role of C-type lectin receptors in bacterial recognition
The most important function of the innate immune system is the recognition of pathogens that
may cause damage to the host. To sense foreign structures, host immune cells express so-
called pattern-recognition receptors (PRRs) on their surface to detect highly conserved
pathogen-associated molecular patterns (PAMPs) present in all classes of pathogens like
bacteria, viruses, parasites, and fungi. PRRs comprise soluble and transmembrane receptors,
including intracellular receptors as nucleotide-binding oligomerization domain-like receptors,
retinoic acid inducible gene I receptors and Toll-like receptors, but also the extracellularly
located C-type lectin receptors (CLRs). CLRs were originally described as calcium-
dependent carbohydrate-binding proteins. Nowadays, it is known that CLRs additionally
detect other ligands than carbohydrates such as lipids, proteins, ice crystals and inorganic
molecules. Furthermore, also calcium-independent ligand binding of CLRs has been
described. Macrophage-inducible C-type lectin (Mincle) is a type II transmembrane protein
expressed by monocytes, neutrophils, macrophages and dendritic cells. Activation of Mincle
by ligand recognition leads to phosphorylation of spleen-tyrosine kinase that in turn induces
the formation of the ternary CARD9/Bcl10/Malt1 complex and the activation of the
transcription factor NFκ-B. Finally, NFκ-B triggers effector functions such as the production
of cyto- and chemokines, cell degranulation and phagocytosis. Bacteria are known to express
various carbohydrate structures (glycans) on their surface that play important roles in host
cell adhesion, invasion and immune evasion. Since these structures are crucial for bacterial
colonization, survival, growth and initiation of pathogenesis, there is rising interest in
detecting novel CLR/bacteria interactions to prevent infections.
Chapter 3 of this thesis deals with the detailed description of three different methodologies to
detect yet unknown CLR/bacteria interactions using the very frequent human pathogen
Campylobacter jejuni as model organism. All methods presented can be easily applied to
other Gram-negative and Gram-positive bacteria and build on a comprehensive CLR-hFc
fusion protein library. CLR-hFc fusion proteins consist of the Fc part of a human IgG1 fused
to the carbohydrate-recognition domain (CRD) of the respective CLR. First, an ELISA-based
high-throughput assay is described that allows for an initial pre-screening for CLR/bacteria
interactions. This method relies on plate-coated bacteria that are incubated with the respective
Abstract
- 2 -
fusion proteins. Finally, binding is detected by reaction of the colorimetric substrate ABTS
with a secondary anti-human IgG1 Fc antibody coupled to horseradish peroxidase. Among
others, Dectin-1-hFc was found to bind to C. jejuni isolates MHH19 and MHH24. Since
false-positive CLR candidates may occur due to protein aggregation, a following flow
cytometry-based assay was established to confirm potential binding partners. Here, bacteria
were kept in solution, incubated with the CLR-hFc fusion proteins and binding was detected
by the usage of an anti-human IgG1-Fc antibody in a subsequent flow cytometric analysis.
Applying this protocol, binding to Dectin-1-hFc was confirmed for both C. jejuni isolates.
Finally, a confocal microscopy approach is presented that enables direct visualization of the
CLR binding to bacteria. Also, in this assay, Dectin-1-hFc was found to recognise both
C. jejuni isolates. In summary, the described methods based on CLR-hFc fusion proteins
offer the possibility to screen for various novel CLR/bacteria interactions. The detection of
these candidates presents the first step towards bacterial ligand identification and the analysis
of the biological function of the respective CLRs.
The identification of Mincle as CLR that recognises parts of the lipoteichoic acid anchor
(LTA) present in the cell wall of the human pathogen group A Streptococcus (GAS) and
thereby promoting antibacterial immunity is part of chapter 4. Gene expression analysis and
cell stimulation assays revealed that the Mincle adaptor protein CARD9 is involved in the
immune responses against GAS infection. Stimulation of bone marrow-derived dendritic cells
(BMDCs) with purified monoglucosyldiacylglycerol (MGDG) of LTA led to a Mincle-
dependent production of inflammatory cytokines, reactive oxygen species (ROS) and the
activation of inducible nitric oxide synthase (iNOS). In addition, the purified
diglucosyldiacylglycerol (DGDG) of the LTA is recognised by Mincle but does not initiate
signaling. Furthermore, it was found that DGDG serves as suppressor of Mincle whereas
MGDG mediates agonistic Mincle activation. In vivo, Mincle-deficient mice infected with
GAS strains others than the 5448 exhibited high mortality in line with severe bacteraemia and
an increased expression of proinflammatory cytokines. In summary, these results suggest a
protective role for Mincle in anti-GAS immunity.
This work presents comprehensive methods to detect novel CLR/bacteria interactions and
contributed to the identification of GAS-derived MGDG and DGDG as new Mincle ligands.
Zusammenfassung
- 3 -
Zusammenfassung
Sabine Mayer-Lambertz: Die Rolle von C-Typ Lektin Rezeptoren in der
Erkennung von Bakterien
Eine der wichtigsten Aufgabe des angeborenen Immunsystems ist die Erkennung von
Erregern, die in den Wirt eindringen und ihn schädigen. Um diese körperfremden Strukturen
zu detektieren, tragen die Immunzellen des Wirtes sogenannte Mustererkennungsrezeptoren
(engl.: „Pattern-recognition receptors“, PRRs) auf ihrer Oberfläche, die konservierte
Pathogen-assoziierte molekulare Muster (engl.: „Pathogen-associated molecular patterns“,
PAMPs) in allen Klassen von Erregern wie Bakterien, Viren, Parasiten und Pilzen erkennen.
PRRs umfassen lösliche und membrangebundene Rezeptoren sowie intrazelluläre Rezeptoren
wie nucelotide-binding oligomerization domain-like Rezeptoren, retinoic acid inducible gene
I Rezeptoren und Toll-like Rezeptoren. Zusätzlich zählen zu den PRRs auch die extrazellulär
lokalisierten C-Typ Lektin Rezeptoren (CLRs). Ursprünglich wurden CLRs als
kalziumabhängige Kohlenhydrat-bindende Proteine beschrieben. Dieser Kenntnisstand
änderte sich jedoch mit der Entdeckung, dass CLRs auch Lipide, Proteine, Kristalle und
anorganische Moleküle erkennen und dass diese Bindung nicht unbedingt durch Kalzium
vermittelt werden muss. Macrophage-inducible C-type lectin (Mincle) ist ein Typ II
Transmembranprotein, welches von Makrophagen, Dendritischen Zellen, Neutrophilen und
Monozyten exprimiert wird. Die Aktivierung von Mincle durch die Bindung von Liganden an
die Kohlenhydrat-erkennende Domäne (engl.: „Carbohydrate-recognition domain“, CRD)
führt zur Phosphorylierung der „Spleen-tyrosine kinase“ (Syk), die die Bildung des
CARD9/Bcl10/Malt1 Komplexes und die Aktivierung des Transkriptionsfaktors NFκ-B
initiiert. Letztlich werden NFκ-B-vermittelte Effektorfunktionen wie die Produktion von
Zytokinen und Chemokinen, Phagozytose und Degranulation ausgelöst. Bakterien besitzen
eine Vielzahl von Kohlenhydratstrukturen (Glykane) auf ihrer Oberfläche. Diese Strukturen
spielen eine wichtige Rolle in der Adhäsion und dem Eindringen in die Wirtszelle sowie bei
der Immunevasion. Da diese Strukturen essentiell für eine erfolgreiche bakterielle Besiedlung
und Pathogenese im Wirt sind, stellt die Entdeckung neuer CLR/Bakterien-Interaktionen
einen wichtigen ersten Ansatzpunkt dar, um eine bakterielle Infektion zu verhindern.
Das Kapitel 3 beschäftigt sich mit drei unterschiedlichen Methoden, um bislang unbekannte
CLR/Bakterien-Interaktionen aufzudecken. Dabei werden die Methoden anhand des
Zusammenfassung
- 4 -
humanpathogenen Bakteriums Campylobacter jejuni beschrieben. Allerdings können die
Methoden auf weitere Gram-negative sowie Gram-positive Bakterienstämme ausgedehnt
werden. Alle Techniken basieren auf einer CLR-hFc Fusionsprotein-Bibliothek. Die CLR-
hFc Fusionsproteine bestehen aus dem Fc-Teil eines humanen IgG1, das mit der CRD des
entsprechenden Rezeptors fusioniert wurde. Ein ELISA-basierter Hochdurchsatz-Assay wird
als erstes durchgeführt, der initiale Ergebnisse zu möglichen CLR/Bakterien-Interaktionen
liefert. Diese Methode basiert auf der Immobilisierung der Bakterien, die anschließend mit
den CLR-hFc Fusionsproteinen inkubiert werden. Die Umsetzung des Substrats ABTS durch
einen sekundären anti-human IgG1-Fc Antikörper, konjugiert an das Enzym
Meerrettichperoxidase, ermöglicht die Detektion von CLR/Bakterien-Interaktionen. Auf
diese Weise wurde Dectin-1 als Rezeptor für die C. jejuni Isolate MHH19 und MHH24
entdeckt. Da diese Methode durch die Bildung von Proteinaggregaten die Möglichkeit von
falsch-positiven Ergebnissen birgt, wurde ein nachfolgender durchflusszytometrischer Ansatz
zur Verifizierung der Kandidaten durchgeführt. Dabei wurden die Bakterien in Lösung mit
den Fusionsproteinen und einem sekundären anti-human IgG1 Fc Antikörper inkubiert, bevor
die Proben durchflusszytometrisch analysiert wurden. Hierbei konnte die Bindung von
Dectin-1-hFc an beide C. jejuni-Isolate bestätigt werden. Zum Schluss wurde die Dectin-1-
hFc/C. jejuni-Interaktion durch Konfokalmikroskopie visualisiert. Zusammenfassend
ermöglichen die drei auf CLR-Fusionsproteinen basierenden Methoden das Screening von
einer Vielzahl an Bakterienstämmen auf neue CLR-Interaktionen. Dies ist ein erster Schritt,
der zur Identifikation eines distinkten CLR-Liganden und zum besseren Verständnis von
dessen biologischer Funktion führt.
Das Kapitel 4 dieser These beschreibt die Identifikation von Strukturen des
Lipoteichonsäureankers (LTA) in Gruppe A Streptokokken (GAS) als Mincle-Liganden, die
in der Lage sind, eine Immunantwort gegen GAS auszulösen. Genexpressionsstudien und
Zellstimulationsexperimente zeigten, dass CARD9, ein Adaptermolekül von Mincle, an der
Immunantwort gegen GAS beteiligt ist. Die Stimulation von Makrophagen, die aus dem
Knochenmark differenziert wurden, mit aufgereinigtem Monoglucosyldiacylglycerol
(MGDG) aus dem LTA führte zu einer Mincle-abhängigen Produktion von
proinflammatorischen Zytokinen, reaktiven Sauerstoffspezies (ROS) und der Aktivierung der
Stickstoffmonoxid-Synthase (iNOS). Zusätzlich wurde gezeigt, dass von dem LTA
aufgereinigtes Diglucosyldiacylglycerol (DGDG) von Mincle erkannt wird, jedoch zu keiner
Zusammenfassung
- 5 -
Signalweiterleitung innerhalb der Zelle führt. Außerdem konnte gezeigt werden, dass DGDG
Mincle inhibiert, während MGDG als Mincle-Agonist fungiert. Die Infektion von Mäusen
mit einigen GAS-Stämmen zeigte, dass Mincle-Defizienz zu einer gesteigerten Anfälligkeit
auf Grund von erhöhter Bakterienlast und Expression von proinflammatorischen Zytokinen
führte. Alles in allem zeigen diese Ergebnisse, dass Mincle eine schützende Rolle in der
Immunabwehr gegen GAS besitzt.
Zusammenfassend präsentiert diese Arbeit umfangreiche Methoden, um neue Interaktionen
von CLRs und Bakterien zu detektieren. Außerdem trägt diese Arbeit zu der Entdeckung bei,
dass die beiden LTA Bestandteile MGDG und DGDG aus GAS als neue Mincle Liganden
fungieren.
Introduction
- 6 -
Chapter 1: General introduction
1. The immune system - essential for survival
“I find it astonishing that the immune system embodies a degree of complexity which suggests
some more or less superficial though striking analogies with human language, and that this
cognitive system has evolved and functions without assistance of the brain” -Niels K. Jerne-
The human body is challenged every day by multifaceted microorganisms, like fungi, viruses,
parasites and bacteria that attempt to invade and colonize the host. In addition to these
environmental pathogens, the body is naturally colonized by approximately 1014
microorganisms that harbor due to dysbiosis an increased risk for causing disease to the host
(1, 2). Nevertheless, it is the result of the immune system as a powerful defense system that
the host is usually able to limit the infection and eliminate pathogens fast. The immune
system (lat.: immunis; free from, devoid of) is one of the most complex systems in the human
body, forming a remarkable network of different tissues, cells and proteins that cooperate to
protect the body from foreign structures such as microbes, viruses, cancer cells and toxins
(3). The immune system is composed of two potent branches, the innate and the adaptive
immune system (figure 1). Whereas for fast and effective protection of the host both parts
have to collaborate using humoral and cellular components, each part clearly mediates
distinct functions. The innate immune system initiates the first responses after the host is
exposed to pathogens. To allow a fast detection of and response to invading pathogens, the
innate immune system provides various genetically predefined receptors, so-called pattern-
recognition receptors (PRRs), that recognize evolutionarily highly conserved microbial
structures on a broad spectrum of pathogens (Pathogen-associated molecular patterns,
PAMPs) or frequent signs of infections (Danger-associated molecular patterns, DAMPs).
However, this only induces a general immune response that includes the induction of
inflammatory responses, the process of tissue repair, the recruitment of immune cells and the
release of cytokines and chemokines. These processes are activated directly after the
infection occurred and they are not influenced in strength by repeated exposure to the same
antigen (3-5). In contrast, dependent on the activation by the innate immune system, the
adaptive immune system relies on a defined repertoire of specific receptors that enables
response to any antigen (6). Since the development of these highly specific components
requests time, the adaptive immune system is only activated after several days of infection.
The adaptive immune system displays memory, thereby providing an augmented immune
Introduction
- 7 -
reaction towards antigens that had already been encountered previously by the host (4, 7).
However, any disturbed immune function in parts of the immune system increases the
susceptibility of the host to infections, highlighting the importance of an efficient interaction
of the innate and adaptive immune system (3).
1.1 Innate Immunity
The innate immune system is the crucial first line of defence against infections and includes
the epithelium as the initial and main barrier against pathogens. The epithelium is located in
the respiratory tract, skin and gastrointestinal (GI) tract; hence, it is the part that is challenged
most frequently with invading pathogens. Thus, it offers multifaceted strategies to prevent
microbial entry and spread. Tight junctions connect cells with each other and provide an
important physical barrier (8) whereas mucus produced by the respiratory and GI tract
enables rapid entrapment of microbes and protects the epithelial surface. In addition, physical
and chemical barriers such as temperature, low pH or the secretion of antimicrobial
substances kill or neutralize pathogens (4, 9, 10). Importantly, tissue cells that encountered
pathogens produce cytokines that further activate innate immune cells and guide them to the
site of infection (11). The innate response also includes a soluble fraction that comprises
acute-phase proteins, interferons (IFNs), antimicrobial peptides and the complement system
(figure 1). Most notably, the complement system consists of more than 30 plasma proteins
that are cascade-like activated to identify, opsonize and kill pathogens or infected cells either
directly or by phagocytosis. Furthermore, it activates and mobilizes side by side with released
cytokines the cellular component of the innate immune system that comprises monocytes,
granulocytes, macrophages, mast cells, dendritic cells (DCs) and natural killer (NK) cells
(figure 1) (3, 12).
Of these cell subsets, DCs and macrophages are termed professional antigen-presenting cells
(APCs) since they are able to present antigens to T cells in the presence of co-stimulatory
molecules (figure 1), hence linking innate with adaptive immunity. Besides that,
macrophages are characterised by their pronounced phagocytic ability. If the epithelial barrier
is breached by physical damage and pathogens can invade the host, macrophages incorporate
intruders into large intracellular organelles, so-called phagosomes. This compartment fuses
with a lysosome containing enzymes, peptides and proteins to a phagolysosome and
subsequently leads to the degradation of the internal content (10, 13). In addition to the pure
Introduction
- 8 -
elimination of pathogens, peptide fragments can be presented by macrophages on major
histocompatibility complex (MHC) II molecules to T cells, leading to their activation. In
addition, macrophages initiate a potent response towards intracellular pathogens via the
IFNγ-mediated activation by Th1-cells (3, 4).
Whereas macrophages are defined by their phagocytic activity, the primary function of DCs
is the activation of the adaptive immune system by the presentation of antigens to T cells.
Immature DCs are resident throughout the tissues where they capture antigens with surface
located receptors, e.g. CLRs, but also via Fc receptors (FcRs) (14, 15). The processing of
respective antigens into proteolytic peptides is mediated in the next step via phagocytosis,
macropinocytosis or receptor-mediated endocytosis and leads to the maturation of DCs (16).
DCs take up larger organisms such as parasites or fungi and they are able to ingest various
Gram-positive and Gram-negative bacteria as well as mycobacteria (17). The uptake of
Salmonella by the gut epithelium is directly mediated by DCs that open tight junctions to
expose their dendrites to directly capture bacteria (18). In addition, DCs are often the target
of virus infections. Surface molecules on DCs enable viruses to enter the cells and start the
production of virus-associated particles in the cytoplasm. To ensure an effective immune
response towards these various extra- and intracellular stimuli, DCs express two different
classes of MHC molecules that trigger different immune response pathways. Whereas
endogenous (intracellular) peptides presented on MHC I molecules activate CD8+ cytotoxic T
lymphocytes (CTLs), MHC II molecules present exogenous peptides to CD4+ T cells (6, 19).
In addition to this classical antigen-presentation pathway, the mechanism of cross-
presentation enables presentation of extracellular peptides to naïve CD8+ T cells to become
activated CD8+ CTLs. This pathway is especially important for the initiation of immune
responses towards peripheral tissue cells others than antigen-presenting cells infected with
viruses or even against tumor cells (20).
1.2 Adaptive Immunity
Whereas more primitive organisms like invertebrates and plants exclusively rely on the innate
immune system, higher animals further developed the adaptive immune system: a clonal- and
antibody-based second branch that enables immunological memory, hence increasing the
reaction towards reoccurring intruders (12, 21). In parallel to the innate immune system, the
adaptive immune system is activated. The adaptive immune system is characterised by the
Introduction
- 9 -
formation of pathogen-specific pathways. Hallmark cells of this branch are antigen-specific T
cells and antibody-producing B cells that represent clonal lymphocyte populations (figure 1).
Whereas all lymphocyte progenitor cells develop from hematopoietic stem cells in the bone
marrow, where B cells persist to undergo maturation, immature T cells are subsequently
recruited to the thymus to reach their final maturation state (7).
T cells are characterised by specific surface-expressed T cell receptors (TCRs). These TCRs
recognize antigen fragments presented by APCs on MHC I or MHC II molecules. In addition
to the TCR/peptide/MHC interaction, a binding of the costimulatory molecules CD80 or
CD86 located on mature APCs to CD28 expressed on T cells is required to fully activate T
cells (22). Upon recognition of the antigen bound to MHC-I, naïve CD8+ CTLs differentiate
into mature CTLs. Once activated, mature CD8+ T cells release effector molecules, such as
perforin and granzyme B, that induce the apoptosis of infected and abnormal cells (6). After
successful elimination of foreign agents, only few cells survive as memory T cells that
protect the host against reinfection due to their rapid response (23). The presentation of
antigens bound to MHC-II molecules stimulates CD4+ T cells. These cells regulate in
particular the type of immune response by the release of cytokines that enable an enhanced
reaction to eliminate pathogens. Therefore, CD4+ T cells differentiate into various types of T
helper (Th) cells (figure 1) (24). Most notably, Type 1 helper (Th1) cells mediate a cellular
immune response by the release of IFNγ and interleukin (IL)-2, whereas a reaction initiated
by Th2 cells is characterized by IL-4, IL-5, IL-10 and IL-13 production, that in turn
efficiently activates naïve B cells to produce antibodies (humoral immune response) (6).
Th17 cells are characterized by their ability to produce IL-17. IL-17A and IL-17F are known
to be important proinflammatory cytokines that induce the production of IL6 and tumor
necrosis factor α (TNFα) as well as the recruitment of granulocytes (6). Th9 cells play an
important role in allergy and autoimmunity as well as in parasitic helminth infection (25) and
tumor eradication (26). Additionally, regulatory T cells (Tregs), another CD4+ T cell subset,
suppress or downregulate immune responses, thereby playing an important role in
maintaining immune homeostasis (27).
Introduction
- 10 -
Figure 1: Components of the innate and adaptive immune system. Both branches of the immune system
contain cellular and humoral components. The innate immune system resembles the first line of host defense
and comprises phagocytic immune cells like macrophages as well as classical antigen-presenting cells (APCs)
such as dendritic cells (DCs). The humoral part includes host defense peptides like antimicrobial peptides or the
LPS Binding Protein. If the innate immune response is unable to clear the pathogens, the adaptive immune
system is initiated after several hours. Crucial for the activation is the recognition of pathogen-associated
molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) by cellular receptors located on
APCs, e.g. C-type lectin receptors and the subsequent presentation of peptide fragments loaded on major
histocompatibility complexes (MHC) to T cells via T cell receptor (TCR)/MHC-interaction. Antigen
presentation stimulates immature T cells to differentiate into cytotoxic T cells (CTL) or T helper (Th) cells.
Activation of Th cells drives down one of several differentiation pathways. One subset represents the regulatory
T cells (Tregs) which dampen immune activation. In contrast, Th1 response activates killing of intruders, Th2
cells are involved in antibody production and clearance of helminths whereas Th17 cells induce inflammatory
responses. Th9 cells play a role in anti-tumor immunity, worm infections as well as in autoimmunity and
allergy. (4)
B cells mediate an important role in the humoral part of the adaptive immune system. Each B
cell expresses approximately 1.5x105 molecules of an unique antigen-binding receptor (BCR)
on its surface that allows a direct recognition and highly specific binding of an antigen (2).
Once activated by foreign antigens, naïve B cells divide rapidly by clonal expansion into
plasma cells or into memory B cells. Plasma cells produce a large amount of respective
Introduction
- 11 -
antibodies that in turn are able to bind to specific antigens on pathogens, hence flagging them
for uptake by phagocytes. Whereas these plasma cells die after one to two weeks, memory B
cells survive and continue the production of antibodies (28). Long-living memory T and B
cells present important survivors of an overcome infection and B cells produce antibodies
that allow immunological memory, enabling a faster and effective immune reaction towards
previously encountered antigens.
2. Of PAMPS, DAMPS and PRRs
For a long time the adaptive immune system was seen as the more powerful arm of
immunity. With the introduction of a model of microbial pattern recognition by Charles
Janeway Jr., the innate immune system got more into the focus of many research groups.
Janeway`s prediction relies on a limited number of germ line-encoded PRRs that have
genetically determined specificities for various microbes and other foreign molecules (5) and
are mainly present on APCs and other immune but also non-immune cells (29). PRRs
recognize highly conserved structures called PAMPs that are abundantly expressed by all
classes of pathogens including viruses, protozoa, fungi as well as Gram-positive and -
negative bacteria (30). Since PAMPs such as LPS or bacterial CpG DNA are exclusively
present in microbes and essential for pathogen survival and integrity, they represent suitable
molecules for microbial sensing by host cells (5, 29). However, besides the recognition of
external-derived PAMPs, PRRs also sense endogenous DAMPs. DAMPs represent host
factors that are either released by the extracellular matrix (ECM) or from cells upon infection,
inflammation or other types of cellular stress (31). Upon disruption of the cell, DAMPs that
for example are under healthy conditions attached to the cytoskeleton or the ECM, such as
actin (32) or fibrinogen (33), are proteolytically released into the extracellular space and are
involved in inflammation and cell regeneration processes in a PRR-dependent manner (34).
In addition to ECM-derived DAMPs, various soluble molecules are intracellularly generated
or accessible during cellular necrosis or apoptosis. According to their origin within the cell,
these molecules can be classified into different groups: mitochondria-derived DAMPs such as
mitochondrial DNA (35); DAMPs like ATP (36, 37) and IL-1β (38) that are produced during
the process of autophagy as well as nuclear (e.g. DNA (39), histones (40)) and cytosolic (e.g.
uric acid (41)) DAMPs (34).
Introduction
- 12 -
Up to now, the receptor system consists of several large families of PRRs (figure 2) including
Toll-like receptors (TLRs) and C-type lectin receptors (CLRs) that are membrane-bound,
hence they detect pathogens present in endocytic compartments or in the extracellular space.
Additionally, unbound intracellular receptors such as NOD-like receptors (NLRs), RIG-I-like
receptors (RLRs) and AIM2-like receptors (ALRs) are located in the cytoplasm, hence they
are responsible for the detection of intracellular agents e.g via the endoplasmic reticulum
membrane-resident adaptor stimulator of interferon genes (STING) (42). Remarkably, this
compartmentalization of the receptors provides important information on the class of detected
pathogens as well as their location, thus indirectly indicating their potential damage to the
host cell (43, 44). For example, it is known that the extracellular presence of Salmonella is
detected in a TLR-dependent manner leading to proinflammatory cytokine production and
phagocytosis whereas the intracellular detection of Salmonella leads to death of the
respective cell, initiated by NLRs (45). Along that line, the recognition of self-derived nucleic
acids is connected to multiple disorders (46, 47). Therefore, compartmentalization of TLRs
and their ligands provides a mechanism that enables the receptor to distinguish between
foreign and self-derived nucleic acids (48). For example, the expression of TLR9 in
intracellular compartments prevents the recognition of self-derived nucleic acids but enables
sensing of virus-derived DNA (49).
Stimulation of PRRs by ligand recognition leads to the activation of distinct adaptor
molecules that in turn trigger specific intracellular signaling pathways. Adaptor proteins play
a crucial role in signal integration from several receptors and enable the detection of various
ligands. Finally, the initiation of an enzymatic signal activates immune responses including
inflammatory pathways such as the stimulation of APCs by the production of
proinflammatory cytokines and IFNs (43, 50). This further elicits the adaptive immune
responses. In addition, the recruitment of PRRs also leads to direct events like autophagy
(51), cell death (52) and phagocytosis (53). These mechanisms finally orchestrate to allow for
a fast and efficient response towards the intruders (43).
Introduction
- 13 -
Figure 2: Pattern-recognition receptors (PRRs) with individual family members and their ligands. Pathogen-associated molecular patterns (PAMPs) are detected by several PRRs located in the cytoplasm of the
immune cells such as NOD-like receptors (NLRs) or RIG-I-like receptors (RLRs). Furthermore, members of the
Toll-like receptor (TLRs) and C-type lectin receptor (CLRs) families are expressed as transmembrane receptors.
This figure shows the main classes of PRRs including some members with their described ligands as examples.
(54, 55)
2.1 TLRs: their discovery changed the understanding of pathogen sensing
TLRs are probably the most intensively investigated class of PRRs. The first receptor
discovered was named Toll and mediates dorsoventral development during embryogenesis in
the fruit fly Drosophila melanogaster (56). Later, it was detected that Toll additionally plays
an important role in the protection of flies against fungal infections (57). Up to now, 11
human (TLR1-10) and 12 murine (TLR1-9 and TLR11-13) homologues of Toll are described
(58). TLRs are type I transmembrane glycoproteins and are mostly expressed by APCs like
macrophages, DCs, and B lymphocytes (48). TLRs are characterized by an extracellular
leucine-rich domain (LRR) that mediates ligand binding and a Toll/IL-1 receptor domain
(TIR) for signal transduction, hence located in the intracellular or cytoplasmic space (59). So
far, TLR ligands that are recognized by the LRR domain are described for most of the TLRs
as well as the role of TLRs in host response to various microbes such as viruses, bacteria,
Introduction
- 14 -
fungi and parasites (50, 60-64). PAMP binding to the respective TLR induces receptor
oligomerization followed by the induction of intracellular signaling that generates a potent
antimicrobial proinflammatory response (65). For most of the TLRs, except for TLR3, the
recruitment of the adaptor protein myeloid differentiation primary response gene 88 (MyD88)
is essential for induction of downstream signaling cascades (66). TLR3 in contrast interacts
with Toll/IL-1R-domain-containing adaptor-inducing IFN-β (TRIF) that together with a
bridging molecule is also used by TLR4. The engagement of various downstream adaptors
involved in signal transduction finally leads to the translocation of the nuclear factor kappa B
(NFκ-B) to the nucleus where it induces subsequently the expression of target genes involved
in inflammatory responses towards intruders (2, 54).
TLRs are classified according to the nature of their PAMP ligand into two subclasses.
Whereas TLR1, TLR2, TLR4, TLR5 and TLR6 recognise lipoproteins and lipoglycans,
TLR3, TLR7, TLR8 and TLR9 are involved in the detection of nucleic acids (50). Focusing
on bacterial TLR ligands, the interaction of TLR4 and bacterial lipopolyssacharide (LPS) that
is ubiquitously present in the cell wall of Gram-negative bacteria has been described most
intensively (67, 68). Since a TLR4/LPS interaction alone only leads to insufficient cell
signaling, the accessory proteins LPS-Binding Protein (LBP) and MD2 assist in LPS binding
and recognition by TLR4 (69, 70). In addition to many bacterial cell wall components that
serve as PAMPs for TLRs including lipoarabinomannan (TLR2 (71)), flagellin (TLR5 (72))
and lipoteichoic acid anchor (LTA, TLR2 (73-75), TLR2/TLR6 (76)), also bacteria-derived
DNA that contains hypomethylated CpG domains is recognized (TLR9 (77)). Furthermore,
TLRs are divided according to their localization within the cell. TLR1, TLR2, TLR4, TLR5
and TLR6 are expressed at the cell surface whereas the nucleic acid sensing TLRs (TLR3,
TLR7, TLR8, TLR9) are located in membranes of the endosomal compartment (48). To
enable sensing of intracellular cytosolic pathogen-derived PAMPs, like viral double-stranded
(ds)RNA or single-stranded (ss)RNA, cells express additionally a large repertoire of cytosolic
PRRs including RLRs and NLRs (65).
2.2 NLRs and RLRs: soluble cytoplasmic sensors of PAMPs and DAMPs
NLRs are a group of PRRs that have gained rising interest in the last years since all described
members are connected to certain human diseases. NLRs are important cytosolic sensors of
mainly bacteria-derived molecules present in the cytoplasm but also of environmental
Introduction
- 15 -
components and host cell molecules (50). Examples of ligands derived from bacteria
are: pore-forming toxins, flagellin proteins, muramyl dipeptide (MDP), anthrax lethal toxin or
microbial RNA and DNA (78). In general, NLRs are characterized by multiple LRRs that
mediate ligand recognition, a nucleotide-binding oligomerization domain (NOD) responsible
for oligomerization upon ligand binding and a protein-protein interaction domain, that is
specific for each NLR (79). Up to now, 22 NLRs are described that, based on their N-
terminal part, are divided into 4 subfamilies including CIITA, NLRB, NLRC and NLRP (80).
Additionally, NLRs can also be classified by their effector functions that they initiate upon
ligand binding (81). Accordingly, some members of NLRs including NLRP1, NLRP3 and
NLRC4 form a signaling complex, the inflammasome, that has a pivotal role in inflammatory
cell death as well as processing and maturation of the proinflammatory cytokines IL-1β and
IL-18 (82). Moreover, activated NLRs like NOD1 and NOD2 affect signal transduction via
downstream adaptor molecules that lead to the activation of NFκ-B or activator protein 1
(AP1), hence enhancing the transcription of various proinflammatory cytokines (83, 84). In
addition to the initiation of signal transduction, NLRs such as CIITA and NLRC5 are
described to induce with the transcription of MHC-II and I genes, respectively, as the lack of
MHC-II expression in patients with bare lymphocyte syndrome can be completely
compensated by CIITA (85-87). Furthermore, NLRs are also involved in the process of
autophagy, a fundamental cellular process of antigen elimination. NOD1 and NOD2 are
known to induce autophagy to remove bacterial components (88).
Whereas NLRs represent soluble receptors that recognise various cytoplasmic pathogens,
RLRs play a pivotal role in the detection and defense against viruses. Up to now, the family
consists of three members: RIG-I, MDA5 and LGP2 which commonly recognize RNA
viruses and subsequently lead to the induction of type I IFN, hence eliciting antiviral
response (54). MDA5 recognizes picornaviruses as encephalomyocarditis virus whereas RIG-
I is described to recognize several ssRNA viruses like Sendai virus, paramyxovirus and
influenza virus (89, 90). The detection of the 5’-triphosphate present in virus ssRNA enables
a selective discrimination of viral RNA from host RNA by RIG-I (91). Ligand recognition by
RIG-I or MDA5 leads to a complex downstream signaling that involves several signaling
adaptor proteins such as IFN-β promotor stimulator 1 (IPS-1) (92). Finally, the activated
signal cascades result in the expression of type I IFNs and proinflammatory factors that are
involved in efficient antiviral defense (54, 93).
Introduction
- 16 -
2.3 CLRs: A diverse group that recognises sweet PAMPs
CLRs represent a highly diverse group of promiscuous receptors that were originally
described to show a strong affinity for mannose, fucose and glucan carbohydrate structures
present in all classes of pathogens, including viruses, parasites, yeast, fungi as well as Gram-
positive and Gram-negative bacteria (94). In general, the group of CLRs comprises both,
soluble and cell membrane-bound proteins (95). CLRs are characterized by the expression of
one or more carbohydrate recognition domains (CRDs) or a C-type lectin-like domain
(CTLD). Classically, CRD/carbohydrate binding is mediated in a Ca2+-dependent manner as
Ca2+-ions are essential for ligand complexation and receptor integrity, hence determining its
activity. CTLDs are also present in CLRs that recognize other PAMPs than carbohydrates
such as proteins, crystals or lipid moieties. In addition, ligand recognition by these CLRs is
mainly mediated in a Ca2+-independent way (96, 97). Based on several criteria like
phylogeny and structure, vertebrate CLRs are divided into 17 groups (98). Commonly, CLRs
of the myeloid group are structured into an extracellular part with the CRD or CTL, a
transmembrane segment and a cytoplasmic domain that in most cases harbors the signaling
motifs of the respective CLRs. In the immune system, CLRs elicit various diverse functions
like cell-cell adhesion, signal transduction and turnover of plasma glycoproteins (99).
Nevertheless, one of the main function of CLRs is the recognition and subsequent uptake of
pathogens. After being internalized, pathogens are degraded in lysosomes and their fragments
are presented on MHC molecules by APCs to induce an adaptive immune response (100).
Due to the large number of members within the CLR superfamily, the following section
focuses on the subfamily of myeloid CLRs that consists of members with a pivotal role in
PAMP detection and subsequent modulation of immune responses.
These CLRs are expressed by myeloid cells, especially APCs like macrophages, DCs and B
cells and are involved in pathogen binding, uptake and degradation, thus playing a role in
activation and regulation of antimicrobial responses (101). Moreover, CLRs are subclassified
by their regulatory effect on the outcome of the initiated immune response. Accordingly,
CLRs such as Dectin-2, macrophage-inducible C-type lectin (Mincle) (figure 3) and DCAR
are associated with adaptor molecules (e.g. Fc Receptor common gamma-chain (FcRγ))
containing immunoreceptor tyrosine-based activation motif (ITAM, YxxL/I) domains that
activate downstream signaling pathways. Alternatively, CLRs like Dectin-1, SIGNR3 and
Introduction
- 17 -
Clec9a carry a single ITAM motif called hemITAM in their intracellular domain (102-104).
Upon ligand detection, ITAM- and also to a certain extent hemITAM bearing CLRs (105,
106) induce cell signaling by recruitment of spleen tyrosine kinase (Syk), that in turn leads to
the phosphorylation of tyrosines and activation of various intermediate molecules, including
the activation of transcriptional pathways such as mitogen-activated protein kinases (MAPK)
(107) and nuclear factor of activated T cells (NFAT) (102, 108). In addition, signaling via
Syk activates the NLRP3 inflammasome that induces the maturation and secretion of IL-1β,
hence playing a role in antifungal host defence (109-112). Essentially, Syk signaling leads to
the formation of an adaptor complex of Caspase recruitment domain-containing 9 (CARD9),
B cell lymphoma 10 (Bcl10) and mucosa-associated lymphoid tissue lymphoma translocation
protein 1 (Malt1) that triggers proinflammatory gene expression via activation of the NFκ-B
pathway (figure 3) (113, 114). CLRs with immunoreceptor tyrosine-based inhibitory motif
(ITIM, S/I/V/LxYxxI/V/L) domains mediate suppression of effector functions by activation
of tyrosine phosphatases (SHP-1, SHP-2, SHIP). This group comprises CLRs such as
Clec12a, DCIR and Clec12b. In addition, CLRs including Mannose-receptor (MR), SIGNR1
and DC-SIGN use complex signaling pathways independent of an ITAM or ITIM motif
(115). However, some CLRs are able to trigger a Syk-independent activation of the NFκ-B
pathway by recruitment of the serine/threonine kinase Raf1 (116). Finally, activation of CLRs
by ligand binding induces various cell responses like endocytosis, phagocytosis, production
of chemokines and cytokines, complement activation and induction of the adaptive immune
system (101).
Since pathogens express multiple PAMPs that can be recognized by several receptors, some
CLRs perform crosstalk with different PRRs, including TLRs to enable a response
concurrently against a broad range of pathogens (117). For example, it is well known that
only the cooperation of DC-SIGN with TLRs triggers a cellular response whereas DC-SIGN
signaling alone does not stimulate cytokine production in general, hence it rather modulates
TLR-mediated responses (118, 119). In addition to crosstalk with other PRRs than CLRs, the
formation of CLR-derived homo- or heterocomplexes, termed multimerization, affects
receptor signaling and effector functions. Especially CLRs with hemITAMs like Clec2 tend
to form homodimers since the successful activation of Syk requires two phosphorylated
tyrosines (120, 121). Moreover, DC-SIGN, a CLR that lacks signaling motifs, forms
tetramers that increases receptor avidity and specificity (122). However, the engagement of
Introduction
- 18 -
CLRs like Mincle (figure 3), Dectin-1 and Dectin-2 induces various immune responses upon
pathogen detection that are independent of other PRRs.
Figure 3: CLR engagement triggers various immune responses. This is displayed in this graphic for Mincle
as example. The Mincle carbohydrate-recognition domain (CRD) senses diverse glycan structures from several
pathogens like fungi and bacteria. Additionally, also danger-associated molecules released by damaged or dead
cells activate Mincle. Upon activation, the spleen-tyrosine kinase (Syk) is recruited via the ITAM-bearing FcRγ
stalk which leads to the formation of the CARD9/Bcl10/Malt1 complex and further to the activation of the
transcription factor NFκB. The subsequent production of cytokines and the activation of other effector functions
initiate important mechanisms involved in host defense, tissue repair mechanisms and autoimmune diseases.
(123)
The following section will provide a comprehensive introduction into the role of CLRs in
anti-bacterial immunity with a focus on the CARD9-dependent CLRs Mincle and Dectin-1.
These CLRs were identified to play an important role in bacterial recognition during the
projects that are included in this thesis.
2.3.1 Role of CLRs in anti-bacterial immunity
Bacteria express various virulence factors such as adhesins, toxins and enzymes that
contribute to pathogenesis in the host. Carbohydrate-based structures such as the M-protein,
lipoteichoic acid anchor (LTA), capsular polysaccharide (CPS) or O-antigen domains derived
from bacterial lipopolyssacharides (LPS) facilitate bacteria/host interactions (124-128).
Introduction
- 19 -
Hence, these structures are essential for adherence and subsequent colonization of host tissue
by bacteria, thus mediating the first and most important step in establishment of an infection.
Since bacterial glycosylation is usually the first “pattern” encountered by cells of the host
immune system, there is growing interest on the role of CLRs in bacterial recognition. The
impact of CLRs is best described for Mycobacterium tuberculosis. Up to now, several CLRs
including MR (129), DC-SIGN (130), Mincle (131, 132) and Dectin-1 (133) have been
described to interact with M. tuberculosis (134). However, whereas several reports on
CLR/M. tuberculosis interactions are described in vitro, the respective receptors showed only
limited to redundant role in clearance of the infection in vivo (130, 135, 136). MR was shown
to recognise mannose-capped lipoarabinomannan (ManLAM) present on the surface of
M. tuberculosis and affected the phagocytosis and assembly of the phagolysosome (129,
137). Additionally, it is reported that the tetrameric transmembrane protein DC-SIGN
recognises mannose moieties in ManLAM structures of mycobacteria and influences the
expression of costimulatory molecules and immune responses in human macrophages (138).
Although several murine homologs of DC-SIGN exist, SIGNR3 has been identified as the
closest functional orthologue of human DC-SIGN based on binding studies (139, 140).
Infection of SIGNR3-deficient mice with M. tuberculosis led to increased bacterial load in
the early phase of infection. However, in the later course of infection, these mice efficiently
exhibited an adaptive immune response, comparable to wild-type mice (130). Besides
M. tuberculosis, DC-SIGN is also described to interact with other bacteria like Helicobacter
pylori and a number of Lactobacillus strains (94). Here, the surface layer A protein of L.
acidophilus was detected as ligand for DC-SIGN (141).
Although contradictory reports on the role of Mincle in murine infection studies with
different bacterial species exist, its adaptor signaling protein CARD9 mediates an essential
role in defense against Mycobacterium species, since CARD9-deficient mice showed
exacerbated pathogenesis and rapid death in an infection study in vivo (142). The
mycobacterial cell wall glycolipid trehalose-6,6-dimycolate (cord factor, TDM) is a well-
known ligand for the CLR Mincle, capable of initiating an inflammatory response including
granuloma formation in mice (131, 132). In addition to TDM, Mincle also recognises the
synthetic analog trehalose-6,6-dibehenate (TDB) (131). The crystal structure of the
recognition sites of bovine and human Mincle binding to trehalose unraveled a canonical C-
type primary monosaccaride-binding site that complexes a Ca2+ ion (143, 144). Moreover, a
Introduction
- 20 -
primary and a proximal secondary binding site complex both glucose moieties in the head
group of the trehalose whereas a hydrophobic groove opposite of the primary binding side
was shown to sense fatty acids with a minimum of 10 carbon atoms (131, 143, 145).
Interestingly, the design of mono- and diacetylated derivatives of trehalose with different
numbers of attached acyl chains resulted in an increased affinity for Mincle binding with
rising number of carbon atoms (146) (for further structural information about Mincle, see
(147)). Mincle is a typ II transmembrane receptor that is mainly expressed by myeloid cells
like macrophages, DCs, monocytes and neutrophils (148-152). However, the receptor was
also found to be present on B cells (153). Activation of Mincle upon ligand binding to the
CRD initiates the phosphorylation of the ITAM motif present in the FcRγ receptor associated
to Mincle. Subsequently, the recruitment of Syk leads to the formation of the
CARD9/Bcl10/Malt1 complex that activates NFκB-signaling pathway resulting in the
expression of chemokines, proinflammatory cytokines such as IL-12 and TNF-α, induction of
phagocytosis and nitric oxide (NO) production (figure 3) (148, 154, 155). Whereas most of
the effector functions are proinflammatory, Mincle also induces the production of IL-10,
hence the activation of anti-inflammatory responses, thereby being involved in both, immune
homeostasis and immune modulation towards microbial pathogens (156). Mincle is described
to interact with a number of diverse bacterial strains, like Streptococcus pneumoniae (157,
158), Klebsiella pneumoniae (159), H. pylori (160) and several Corynebacterium (161) and
Lactobacillus strains (162, 163), but the actual Mincle ligand for most of the interactions in
still unknown. Recently, it was shown that glucosyldiacylglycerol from the important human
pathogens S. pneumoniae and group A Streptococcus (GAS, S. pyogenes) is recognised by
Mincle (157, 164). Glucosyldiacylglycerol is part of the LTA that is the major component of
the cell wall of Gram-positive bacteria. Besides binding of Mincle to the respective bacteria,
it was additionally demonstrated that the lack of Mincle in mice infected with the respective
bacteria resulted in increased mortality and dysregulated anti-bacterial immunity, hence
pinpointing to a protective role of Mincle (157, 164). Aside from sensing bacteria, Mincle
furthermore detects self-molecules such as SAP-130 (155) or β-glucosylceramide that derived
from damaged or necrotic cells (165) and pathogens like parasites (Leishmania major) and
fungi (101).
In fungi, the myeloid cell-derived CLR Dectin-1 is known to be the major receptor for β-1,3-
linked glucan, an ubiquitous cell wall component of fungi (166, 167). Hence, its role in
Introduction
- 21 -
sensing fungal pathogens such as Pneumocystis carinii (168), Candida albicans (168, 169),
Aspergillus fumigatus (170-172) and Cryptococcus neoformans (173) has been intensively
studied (174). Upon ligand detection by the single CTLD, Dectin-1 recruits Syk via
phosphorylation of the hemITAM motif present in the cytoplasmic tail, followed by the
engagement of the CARD9/Bcl10/Malt1 complex and subsequent activation of the NFκ-B
pathway with the production of inflammatory cytokines and induction of other effector
functions (175). Although several studies report a role for Dectin-1 in anti-mycobacterial
defense in vitro, its role in vivo as well as the definite ligand present in mycobacteria still
remains unclear since Dectin-1-deficient mice challenged with M. tuberculosis showed no
significant difference in pathology and induced immune responses compared to wild-type
mice (176). Moreover, Dectin-1 crosstalk with TLR2 is required for efficient response
including phagocytosis, inflammatory cytokine production and induction of the MAPK
pathways towards mycobacterial species in vitro (177).
Aims of the study
- 22 -
3. Aims of the study
CLRs play an important role in pathogen detection and the subsequent initiation and
modulation of various immune responses. Many interactions of CLRs and several pathogens
like viruses, bacteria, parasites and fungi are described. Bacteria present interesting study
targets since they express large numbers of glycoconjugates on their surface. In the field of
microbiology, the role of CLRs is best described for M. tuberculosis, whereas for other
bacteria the impact of CLRs still has to be elucidated more extensively. Importantly, some
bacteria such as S. pneumoniae or H. pylori are shown to interact with CLRs but for most of
the bacteria, distinct ligands of the respective receptors are still unknown. Moreover,
targeting specific CLRs presents an interesting strategy to deliver drugs or vaccine antigens
into specific cells to selectively modulate immune responses. Unraveling new CLR/bacteria
interactions presents the first step towards identification of new CLR ligands and to get better
insights into the biological relevance of the respective receptor.
Aim 1: Establishment of different in vitro methods to detect novel CLR/bacteria
interactions
Based on the usage of a comprehensive CLR-hFc fusion protein library (178) and
Campylobacte jejuni as representative model for various other bacteria, diverse methods have
been established to enable screening for novel CLR/bacteria interactions. First, an ELISA-
based assay allowed for high-throughput screening of plate-bound bacteria. Second, a flow
cytometric assay has been established to verify potential CLR binding candidates and finally,
the binding specificity of the respective CLR has been visualized using a confocal
microscopy-based method. The combination of these three distinct methods should allow for
identification of new CLR interactions with bacterial ligands, hence paving the way for
further functional studies.
Aim 2: Unraveling the role of the CLR Mincle in the recognition of group A
Streptococcus
GAS is an important human pathogen that can cause various diseases, ranging from self-
limiting infections like pharyngitis or impetigo to severe outcomes such as necrotizing
fasciitis or streptococcal toxic shock syndrome (179). Since several studies suggest that next
Aims of the study
- 23 -
to TLRs also other PRRs play an important role in the detection of GAS (180, 181), it was
investigated if CLRs may play a role in sensing GAS. In a collaborative approach,
transcriptome analysis and cell culture-based methods like stimulation of bone marrow-
derived DCs (BMDCs) and bone marrow-derived macrophages (BMMs) were applied to first
identify a novel CLR that interacts with GAS, followed by analysis of the antibacterial
response initiated by this CLR. Finally, the in vivo role of the respective CLR was analysed
by murine infection studies using the respective CLR-deficient mice.
Chapter 2 Paper I
- 24 -
Chapter 2: Paper I
C-type lectins: their network and roles in pathogen recognition and immunity
Histochem Cell Biol., (2017) | https://doi.org/10.1007/s00418-016-1523-7
Title: C-type lectins: their network and roles in pathogen recognition and immunity
Sabine Mayer1°, Marie-Kristin Raulf1,2°, Bernd Lepenies1*
1Immunology Unit & Research Center for Emerging Infections and Zoonoses, University of
Veterinary Medicine, Hannover, Germany
2Institute for Parasitology, Center for Infection Medicine, University of Veterinary Medicine,
Hannover, Germany
°These authors contributed equally to this manuscript.
*Corresponding author:
Prof. Dr. Bernd Lepenies, University of Veterinary Medicine Hannover, Immunology Unit &
Research Center for Emerging Infections and Zoonoses, Bünteweg 17, 30559 Hannover,
Germany
Phone: +49(0)511/953-6135; Email: [email protected]
The extent of Sabine Mayer-Lambertz´s contribution to the article is evaluated according to
the following scale:
A. has contributed to collaboration (0-33%).
B. has contributed significantly (34-66%).
C. has essentially performed this study independently (67-100%).
1. Design of the project including design of individual experiments: /
2. Performing of the experimental part of the study: /
3. Analysis of the experiments: /
4. Presentation and discussion of the study in article form: B
Chapter 2 Paper I
- 25 -
Abstract
C-type lectins (CTLs) represent the most complex family of animal/human lectins that
comprises 17 different groups. During evolution, CTLs have developed by diversification to
cover a broad range of glycan ligands. However, ligand binding by CTLs is not necessarily
restricted to glycans as some CTLs also bind to proteins, lipids, inorganic molecules, or ice
crystals. CTLs share a common fold that harbors a Ca2+ for contact to the sugar and about 18
invariant residues in a phylogenetically conserved pattern. In vertebrates, CTLs have
numerous functions, including serum glycoprotein homeostasis, pathogen sensing, and the
initiation of immune responses. Myeloid CTLs in innate immunity are mainly expressed by
antigen- presenting cells and play a prominent role in the recognition of a variety of
pathogens such as fungi, bacteria, viruses, and parasites. However, myeloid CTLs such as the
macrophage inducible CTL (Mincle) or Clec-9a may also bind to self-antigens and thus
contribute to immune homeostasis. While some CTLs induce pro-inflammatory responses
and thereby lead to activation of adaptive immune responses, other CTLs act as inhibitory
receptors and dampen cellular functions. Since CTLs are key players in pathogen recognition
and innate immunity, targeting CTLs may be a promising strategy for cell-specific delivery of
drugs or vaccine antigens and to modulate immune responses.
Chapter 3 Paper II
- 26 -
Chapter 3: Paper II
C-type lectin receptor (CLR)-Fc fusion proteins as tools to screen for novel
CLR/bacteria interactions: an exemplary study on preselected Campylobacter
jejuni isolates
Front Immunol., (2018) | https://doi.org/10.3389/fimmu.2018.00213
Title: C-type lectin receptor (CLR)-Fc fusion proteins as tools to screen for novel
CLR/bacteria interactions: an exemplary study on preselected Campylobacter jejuni
isolates
Sabine Mayer1, Rebecca Moeller1°, João T. Monteiro1, Kerstin Ellrott2,4, Christine
Josenhans2,3,4,5*, Bernd Lepenies1*
1 Immunology Unit & Research Center for Emerging Infections and Zoonoses, University of
Veterinary Medicine, Hannover, Germany
2 Institute for Medical Microbiology, Medical School Hannover, Hannover, Germany
3 Max von Pettenkofer Institute, Ludwig Maximilian University Munich, Munich, Germany
4 German Center for Infection Research (DZIF), partner site Hannover-Braunschweig
5 German Center for Infection Research (DZIF), partner site Munich
° Present address: Institute for Experimental Virology, TWINCORE, Centre for Experimental
and Clinical Infection Research, a joint venture between the Medical School Hannover and
the Helmholtz Centre for Infection Research, Hannover, Germany
*Corresponding authors:
Prof. Dr. Christine Josenhans, Medical School Hannover, Institute for Medical Microbiology,
Carl-Neuberg-Strasse 1, 30625 Hannover, Germany
Phone: +49511532-4354; Email: [email protected]
Prof. Dr. Bernd Lepenies, University of Veterinary Medicine Hannover, Immunology Unit &
Research Center for Emerging Infections and Zoonoses, Bünteweg 17, 30559 Hannover,
Germany
Phone: +49(0)511/953-6135; Email: [email protected]
Chapter 3 Paper II
- 27 -
The extent of Sabine Mayer-Lambertz´s contribution to the article is evaluated according to
the following scale:
A. has contributed to collaboration (0-33%).
B. has contributed significantly (34-66%).
C. has essentially performed this study independently (67-100%).
1. Design of the project including design of individual experiments: B
2. Performing of the experimental part of the study: C
3. Analysis of the experiments: C
4. Presentation and discussion of the study in article form: B
Chapter 3 Paper II
- 28 -
Abstract
C-type lectin receptors (CLRs) are carbohydrate-binding receptors that recognize their
ligands often in a Ca2+-dependent manner. Upon ligand binding, myeloid CLRs in innate
immunity trigger or inhibit a variety of signaling pathways, thus initiating or modulating
effector functions such as cytokine production, phagocytosis, and antigen presentation. CLRs
bind to various pathogens, including viruses, fungi, parasites, and bacteria. The bacterium
Campylobacter jejuni (C. jejuni) is a very frequent Gram-negative zoonotic pathogen of
humans, causing severe intestinal symptoms. Interestingly, C. jejuni expresses several
glycosylated surface structures, for example, the capsular polysaccharide (CPS),
lipooligosaccharide (LOS), and envelope proteins. This “Methods” paper describes
applications of CLR–Fc fusion proteins to screen for yet unknown CLR/bacteria interactions
using C. jejuni as an example. ELISA-based detection of CLR/bacteria interactions allows a
first prescreening that is further confirmed by flow cytometry-based binding analysis and
visualized using confocal microscopy. By applying these methods, we identified Dectin-1 as
a novel CLR recognizing two selected C. jejuni isolates with different LOS and CPS
genotypes. In conclusion, the here-described applications of CLR–Fc fusion proteins
represent useful methods to screen for and identify novel CLR/bacteria interactions.
Chapter 4 Paper III
- 29 -
Chaper 4: Paper III
Lipoteichoic acid anchor triggers Mincle to drive protective immunity against
invasive group A Streptococcus infection
PNAS, (2018) | https://doi.org/10.1073/pnas.1809100115
Lipoteichoic acid anchor triggers Mincle to drive protective immunity against invasive
group A Streptococcus infection
Takashi Imaia,b,c, Takayuki Matsumurad,1, Sabine Mayer-Lambertze,1, Christine A. Wellsf, Eri
Ishikawab,g, Suzanne K. Butcherf, Timothy C. Barnetth,i, Mark J. Walkerh,i, Akihiro Imamuraj,
Hideharu Ishidak,l, Tadayoshi Ikebem, Tomofumi Miyamoton, Manabu Atod, Shouichi Ohgac,
Bernd Lepeniese, Nina M. van Sorgeo, and Sho Yamasakia,b,g,p,2
aDivision of Molecular Immunology, Medical Institute of Bioregulation, Kyushu University,
Fukuoka 812-8582, Japan
bDepartment of Molecular Immunology, Research Institute for Microbial Diseases,
cDepartment of Pediatrics, Graduate School of Medical Sciences, Kyushu University,
Fukuoka 812-8582, Japan
dDepartment of Immunology, National Institute of Infectious Diseases, Tokyo 162-8640,
Japan
eImmunology Unit and Research Center for Emerging Infections and Zoonoses, University of
Veterinary Medicine, 30559 Hannover, Germany
fThe Centre for Stem Cell Systems, Anatomy and Neuroscience, Faculty of Medicine,
Dentistry and Health Sciences, University of Melbourne, Melbourne, VIC, Australia
gDepartment of Molecular Immunology, Immunology Frontier Research Center, Osaka
University, Osaka 565-0871, Japan
hSchool of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane,
QLD 4072, Australia
iAustralian Infectious Diseases Research Centre, The University of Queensland, Brisbane,
QLD 4072, Australia
jDepartment of Applied Bio-organic Chemistry, Gifu University, Gifu 501-1193, Japan.
kFaculty of Applied Biological Sciences and Center for Highly Advanced Integration of Nano
and Life Sciences (G-CHAIN), Gifu University, Gifu 501-1193, Japan
Chapter 4 Paper III
- 30 -
lCenter for Highly Advanced Integration of Nano and Life Sciences, Gifu University, 501-
1193 Gifu, Japan
mDepartment of Bacteriology I, National Institute of Infectious Diseases, Tokyo 162-8640,
Japan
nDepartment of Natural Products Chemistry, Graduate School of Pharmaceutical Sciences,
Kyushu University, Fukuoka 812-8582, Japan
oMedical Microbiology, University Medical Center Utrecht, Utrecht University, 3584 CX
Utrecht, The Netherlands
pDivision of Molecular Immunology, Medical Mycology Research Center, Chiba University,
Chiba 260-8673, Japan
1These authors contributed equally
2Correspondence to Sho Yamasaki: [email protected]
The extent of Sabine Mayer-Lambertz´s contribution to the article is evaluated according to
the following scale:
A. has contributed to collaboration (0-33%).
B. has contributed significantly (34-66%).
C. has essentially performed this study independently (67-100%).
1. Design of the project including design of individual experiments: A
2. Performing of the experimental part of the study: A
3. Analysis of the experiments: A
4. Presentation and discussion of the study in article form: A
Chapter 4 Paper III
- 31 -
Abstract
Group A Streptococcus (GAS) is a Gram-positive bacterial pathogen that causes a range of
diseases, including fatal invasive infections. However, the mechanisms by which the innate
immune system recognizes GAS are not well understood. We herein report that the C-type
lectin receptor macrophage inducible C-type lectin (Mincle) recognizes GAS and initiates
antibacterial immunity. Gene expression analysis of myeloid cells upon GAS stimulation
revealed the contribution of the caspase recruitment domain-containing protein 9 (CARD9)
pathway to the antibacterial responses. Among receptors signaling through CARD9, Mincle
induced the production of inflammatory cytokines, inducible nitric oxide synthase, and
reactive oxygen species upon recognition of the anchor of lipoteichoic acid,
monoglucosyldiacylglycerol (MGDG), produced by GAS. Upon GAS infection, Mincle-
deficient mice exhibited impaired production of proinflammatory cytokines, severe
bacteremia, and rapid lethality. GAS also possesses another Mincle ligand,
diglucosyldiacylglycerol; however, this glycolipid interfered with MGDG-induced activation.
These results indicate that Mincle plays a central role in protective immunity against acute
GAS infection.
Discussion
- 32 -
Chapter 5: Discussion
This work presents comprehensive methods to detect yet unknown CLR/bacteria interactions
that further enable to investigate the importance of CLRs in the recognition of and the
response to bacterial pathogens, as shown here for the interaction of LTA-derived glycolipids
from GAS and the CLR Mincle.
Sensing pathogens by CLRs expressed on APCs influences the immune responses in several
regards, including antigen presentation, phagocytosis, complement activation and immune
homeostasis (101). Therefore, identification of CLRs and their respective ligands that
enhance or modulate immune responses by inhibition or activation of signaling pathways is
of great interest. Bacteria express a huge repertoire of several carbohydrate-containing
structures on their surface that are essential for bacterial growth and survival (182). Since
CLRs are known to sense carbohydrate structures, bacteria present promising organisms to
study the impact of CLRs in pathogen recognition and induction of signaling pathways. As an
important initial step, the first project (chapter 3, paper II (183)) presents different in vitro
methods that allow for identification of novel CLR/bacteria interactions. It is worth to
mention that all methods can be easily applied to Gram-negative and Gram-positive bacteria,
but each method clearly has its own advantages and disadvantages. The ELISA-based method
relies on the immobilization of the bacteria on a plate which might lead to false-positive
CLR-candidates due to protein aggregation. Although the ELISA offers the possibility for
high-throughput screening, a following flow cytometry-based assay is recommended to
confirm the respective CLR candidates. Application of an adequate gating strategy enables
the identification of bacteria, hence ruling out debris or other contaminants in the sample.
Nevertheless, only bacteria that are detectable by size in the flow cytometer can be analysed.
The detection of small bacteria like mycoplasma may cause problems (184). Finally, a
confocal microscopy-based method allows for co-localisation studies that directly visualize
CLR/bacteria interactions (183).
All methods described are based on soluble fusion proteins, that consist of a Fc portion of a
human IgG1 and the CRD of a CLR (178, 185). In clinical applications, fusion proteins are
frequently used, since they offer several advantages. Favorably, the structure of the Fc part
enables bivalent presentation of the ligand, hence increasing receptor avidity. Additionally,
Discussion
- 33 -
the Fc part impacts important aspects of the fusion proteins like stability and solubility and
folds autonomously (186). Furthermore, purification of the proteins is performed fast, simply
and with low costs via the Fc region using well established protein-G/A affinity
chromatography methods (187). Since the first discovery of a CD4-Fc fusion protein that
prevented infection of T cells with human immune deficiency virus in 1989 (188), the interest
in Fc-fusion molecules has been constantly increasing. Up to now, several Fc-fusions are
already used in therapeutic applications (186). Here, fusion proteins are predominantly used
to target specific receptors and mediate agonistic or antagonistic functions to enhance or
inhibit immune responses, respectively. Among the approved Fc-fusion protein therapeutics,
etanercept is applied as TNF-α-inhibitor that prevents proinflammatory responses in patients
with severe, active rheumatoid arthritis (189). Furthermore, dulaglutide, a recombinant fusion
protein consisting of a glucagon-like peptide-1 analog linked to a modified IgG4 Fc-fragment
was recently approved as therapeutics to control the blood sugar level in patients with type 2
diabetes (190). A current approach is the fusion of peptides that specifically bind cancer cells
to the Fc-domain to induce antibody-dependent cellular cytotoxicity (191).
Apart from their relevance in clinical applications, the use of Fc-fusion proteins in biomedical
research has attracted increasing interest over the last decades. Since the interactions of CLRs
with their carbohydrate ligands are characteristically weak, the expression as Fc-fusion
proteins that form homodimers with bivalent CRD presentation increases the avidity of each
protein (192). This enables even the detection of low affinity receptor/ligand interactions and
renders Fc-fusion proteins interesting tools to analyse these interactions. For example, fusion
proteins are used in microarrays to detect novel receptor ligands. These protein, lectin and
glycan arrays rely on plate-coated biomolecules, like proteins as lectins, or carbohydrates.
The binding of other proteins, cells, carbohydrates, antibodies or Fc-fusion proteins to these
components is commonly evaluated by laser-based methods (193, 194). Recently,
carbohydrate arrays including synthetically produced glycan structures as potential ligands
and CLR-hFc fusion proteins were applied to evaluate the receptor binding preference in
regard to ligand structure and multivalency (195, 196).
Applying a set of recombinant fusion proteins, several CLRs were shown to interact with
bacteria like S. pneumoniae (158) or fungi such as Malassezia pachydermatis (197),
Gnadoderma lucidum (198) and Cordyceps sinensis (198). Although several interactions of
Discussion
- 34 -
CLRs with pathogens are described, the specific ligand involved is still unknown in most
cases. Since bacteria that express glycoconjugates in their surface structures harbor potential
ligands for CLRs, these bacteria are of special interest in the field of CLR research. For
instance, C. jejuni incorporates several different carbohydrates into important structures like
the lipooligosaccharides (LOS), N and O-linked glycoproteins as well as the peptidoglycan
and the polysaccharide capsule (199). One publication showed that N-glycosylated proteins
and LOS containing a terminal N-acetylgalactosamine (GalNAc) derived from C. jejuni were
recognized by the human (h) CLR macrophage galactose-type C-type lectin (MGL) (200).
However, in the study presented in chapter 3, only minor binding of the murine (m)MGL-1 to
the tested C. jejuni isolates was detected. One possible explanation for this might be the
different binding profiles of MGL isoforms that are not only described for mMGL-2, that is
more similar to hMGL, and mMGL-1 (201, 202) but also for other CLRs as the human DC-
SIGN and its eight murine orthologs DC-SIGN-related proteins (SIGNRs) (139, 140).
Despite SIGNR6, a pseudogene, and SIGNR4 all other SIGNR receptors are described to
generally recognize glycan ligands similar to DC-SIGN (139). A similar observation was
made for the human- and mouse-derived Dectin-1. Low-valency β-glucan parts of fucan and
laminarin were able to activate hDectin-1 expressing cells, but not cells expressing the
mDectin-1. Furthermore, stimulation studies with curdlan, a high-valency β-glucan, showed
binding to both human and murine Dectin-1. Further assays including reciprocal mutagenesis
analysis and substitution assays revealed that it is not the ligand-binding site of hDectin-1,
but the intracellular domain of this receptor that mediates its sensitivity to low-valency β-
glucan ligands. Interestingly, the substitution with the hDectin-1-specific amino acids in the
intracellular domain rendered mDectin-1 sensitive to low-valency β-glucans (203).
Dectin-1 is a type II transmembrane protein that is mainly expressed by myeloid cells from
the macrophage/monocyte and neutrophil lineages, but also to a lower extent by DCs and T
cell subpopulations (167, 204). Activation of Dectin-1 includes several cellular functions like
pathogen sensing, uptake and clearance. Since the cytoplasmic tail of Dectin-1 contains a
hemITAM-motif (205), engagement of Dectin-1 leads to the activation of effector functions
including the production of chemokines and cytokines, thus shaping immune responses (105,
174, 206). Applying the methods described in chapter 3 paper II and the CLR-hFc fusion
protein library, Dectin-1 was identified as a receptor for C. jejuni (183). Interestingly,
Dectin -1 is classically described as a major receptor that recognizes specifically β-1,3-
Discussion
- 35 -
glucans present in fungi like C. albicans or S. cerevisae (53). Zymosan, a cell wall polymer
of yeast like S. cerevisae, is recognized by TLR2 and Dectin-1. Whereas Dectin-1 induces
phagocytosis and ROS production that is enhanced by TLR2 signaling, TLR2 triggers
inflammatory cytokine production via NFκ-B that is further augmented by Dectin-1 in return
(207). Moreover, also a cooperation of TLR2 and Dectin-1 was described in the context of
mycobacterial species (133, 208). Interestingly, a study with several virulent, attenuated and
avirulent mycobacterial species showed that, in contrast to an anti-fungal immune response,
Dectin-1 was not able to induce phagocytosis in macrophages upon mycobacterial sensing.
However, the release of immune mediators by macrophages such as the cytokines IL-6,
TNFα, RANTES and G-CSF was impaired in a Dectin-1-dependent manner (208). Strikingly,
up to know there is no data suggesting that mycobacterial cell wall incorporates β-glucan.
Furthermore, Dectin-1 is also known to interact with other bacteria like Haemophilus
influenzae (209) or parasites, such as L. infantum and L. amazonensis (210, 211). These
reports clearly demonstrate that Dectin-1 plays a role in recognition of other pathogens than
fungi, but future work has to be performed to gain more insight into the relevance of Dectin-1
regarding recognition of bacteria, signal transduction and immune modulation. A huge
progress will be the identification of distinct Dectin-1 ligand(s) in these pathogens, which
facilitates research on how Dectin-1 affects immune responses towards pathogens beyond the
focus on fungi (212).
Besides the use of Fc-fusion proteins, also reporter cell line assays are commonly applied to
screen CLR/PAMP interactions, for their ligands and to validate CLR agonists or antagonists
(132, 155, 157, 164, 213-216). These reporter cells express distinct CLRs on their surface
coupled to an intracellular promotor-reporter expression cassette, e.g. NFAT-GFP. Here,
ligand binding to the CRD of the CLR induces the activation of the transcription factor
NFAT. Since subsequent activation of the GFP cassette leads to a fluorescence signal, CLR
activation can be determined by measuring the GFP intensity (155, 164, 216). The application
of reporter cell lines was for example used to extend the finding that S. pneumoniae is
recognized by Mincle (158) by the discovery of S. pneumoniae-derived
glucosyldiacylglycerol present in the LTA as Mincle ligand (157). Along that line, the second
project (chapter 4, paper III (164)) contributed to the finding, that two LTA-derived
components of GAS, namely DGDG and MGDG that differ in one glucose residue are
recognized by Mincle. Moreover, the data suggest that the increased susceptibility of GAS
Discussion
- 36 -
infected Mincle-deficient mice is probably due to an impaired ability to release inflammatory
mediators (164).
GAS is an important human pathogen that expresses several adhesion molecules, like LTA,
to mediate binding to host cells. In contrast, multiple host cells are known to play an
important role in anti-GAS immunity by the release of various inflammatory cytokines in
response to GAS infection to control bacterial growth and dissemination (217-219).
Additionally, the production of NO and ROS increases the clearance of GAS in early stages
of infection (220). Nonetheless, bacteria evolved during evolution efficient and complex
evasion mechanisms to escape the host immune response, such as the expression of nucleases
to prevent entrapment by neutrophil extracellular traps or the blockage of antimicrobial
peptides (221, 222). Modification or masking of critical virulence factors is a common
feature of pathogenic organisms to evade the immune system. For instance, it is assumed that
GAS covers TLR2 agonists like LTA and peptidoglycan by the expression of a thick capsule
to ensure immune evasion (223) until phagocytically active immune cells destroy the capsule
(224). Also the fact that Dectin-1 was not able to detect some pathogenic yeasts like
A. fumigatus or C. neoformans although they express β-glucans (225), argues for the
hypothesis that these strains are able to mask their β-glucan to avoid recognition by PRRs
(226). Moreover, phase variation is a common strategy of several C. jejuni strains to rapidly
adapt to environmental changes and avoid recognition by host cells by controlling the
expression of glycosylated LOS (227), capsule (228, 229) and several other bacterial
determinants (230). Strain-specific genetic differences as well as mechanisms like phase-
variation and antigenic variation that even frequently occur in each individual C. jejuni strain
may additionally explain different study outcomes (183, 200, 231). Recognition of LTA-
derived MGDG by Mincle initiated a protective immune response against GAS whereas
sensing of DGDG suppressed this response without further initiation of signal transduction.
Since it is hypothesize that DGDG serves as suppressor for agonistic PRR-mediated signaling
via Mincle, it is tempting to speculate that bacterial strains might vary their ratio of MGDG
and DGDG in the LTA to escape Mincle-mediated anti-bacterial immunity (164). Whereas it
is shown for group B Streptococcus (GBS) that blockage of the glucosyltransferase, the GBS-
DGDG synthesizing enzyme, led to attenuated virulence of the bacteria in vivo (232), there is
no data existing concerning the precise function of GAS-DGDG.
Discussion
- 37 -
The paper III presented in chapter 4 reported that invasive and noninvasive GAS strains
incorporate different DGDG/MGDG ratios in the LTA (164). Interestingly, amongst the
tested GAS strains, those with increased amounts of DGDG belong to the group that caused
invasive infections (5448, NIH34, NIH44), whereas strains with lower pathogenicity (SF370,
K33, Se235) harbored comparably less DGDG. In correlation with this finding, it was shown
that infection of mice with the different GAS strains influenced the clinical disease course,
which might be partially explained by differences in the DGDG/MGDG ratio (164).
Interestingly, infection of mice with the highly invasive strain 5448 (233, 234) that displayed
the most striking difference in DGDG/MGDG ratio neither significantly initiated Mincle-
dependent immune responses (164) nor impaired the disease progression of the infected mice
(unpublished data), although this GAS strain was shown to be recognized by Mincle. Indeed,
differences between in vitro and in vivo data are also well described in the context of other
CLRs. For instance, infection of mice with S. pneumoniae in a model of invasive
pneumococcal disease did not indicate a role for Mincle in anti-pneumococcal immune
response in vivo although binding of Mincle in a Ca2+-dependent manner to S. pneumoniae
was detected (158). The reasons for the discrepancy between in vitro and in vivo data are
manifold. One explanation might involve the accessibility of the ligand since some ligands
like MGDG and DGDG are incorporated into the LTA present in the bacterial membrane that
is covered by the thick peptidoglycan (235) but can also be shed or exposed to the outer space
of several streptococci (232, 236). Since Mincle did not recognise the whole LTA structure, it
is likely that only portions of shed and degraded moieties are recognized. Most convincingly,
the involvement of other PRRs than CLRs in signaling as well as the observation that other
CLRs can compensate for missing signaling pathways is well known in literature. Thus, we
cannot exclude the contribution of other PRRs in GAS recognition and subsequent initiation
of signal transduction. For example, the CLR MCL is described to be co-expressed and -
regulated with Mincle (237), both being upregulated upon MyD88 activation (238).
Furthermore, whole LTA and the peptidoglycan are well known ligands for TLR2 in Gram-
positive bacteria (239) and TLR2 has been shown to upregulate Mincle expression to induce
downstream signaling upon stimulation with Corynebacterial-derived glycans (161). A clear
role for the TLR adaptor protein MyD88 in initiating immune response towards GAS is
described, since the absence of MyD88 caused a significantly decreased production of TNFα,
type I IFNs, IL6 and IL12 by phagocytic immune cells. This impacted neutrophil recruitment
and led to a higher susceptibility of MyD88-deficient mice challenged with GAS (240-243).
Discussion
- 38 -
Additionally, impaired MyD88 expression in human patients affected TLR-mediated ligand
recognition and may result in severe life-threatening predisposition to several pyogenic
bacterial infections caused by e.g. Staphylococcus aureus and S. pneumoniae (244, 245). In
contrast, whereas TLR9 was shown to play a protective role against GAS in vivo (181) the
impact of TLR2 and TLR13 in anti-GAS immunity was not confirmed in a murine infection
study (180, 217). Moreover, the different pathogenicity profiles of bacterial strains may
explain the partially contradictory results. However, also technical features and the
experimental design like the infection model, infection route and dose might influence the
outcome of in vivo studies in general (246). Indeed, applying a model of focal pneumonia in
infection of mice with a non-invasive S. pneumoniae strain revealed a protective role for
Mincle as indicated by increased bacterial recovery in lungs and pro- and antiinflammatory
cytokine production and larger areas of inflamed tissues in Mincle-deficient mice (157).
Since CLR activation impacts both, the phagocytic activity of APCs and the initiated immune
responses, the knowledge about CLR/pathogen interactions and involved ligands was used in
the last decades to design CLR agonists that specifically target CLR-expressing cells (247).
Indeed, activation of CLRs has been shown to efficiently deliver drugs and genes into
specific cells to initiate immune responses towards vaccines or tumors and also to induce
immune tolerance (248). For example, antibody-mediated targeting of the plasmacytoid DC
(pDC)-specific CLR blood DC antigen 2 (BDCA-2) on BDCA-2-humanized mice impaired
CD4+ and Treg responses (249). Major research on glycan-based CLR targeting has been
performed for the CLR DC-SIGN. Due to its tetrameric CRD presentation, DC-SIGN allows
multivalent targeting (101). Modification of antigens, nanoparticles, liposomes or dendrimers
with DC-SIGN specific glycan ligands has already been successfully introduced to target DC-
SIGN and influence T cell responses (250-256). Furthermore, recognition of glycan-
harboring components as LPS, zymosan or TDB by PRRs is described to initiate expression
of cytokines, enhance internalization of antigens and their presentation on MHC molecules to
T cells, hence influencing the T cell fate (257). This modulatory function renders
carbohydrate ligands interesting candidates for vaccine adjuvants and paves the way for the
field of glycan-based vaccines. Vaccination of mice with liposomes harboring TDB as
Mincle ligand mediated strong anti-GAS immunity after intranasal infection which was
caused by a prominent Th17 immune response (258). GAS infection in vivo characteristically
leads to a Th17 response that plays an important role in the protection against GAS (259,
Discussion
- 39 -
260). Since MGDG was shown to induce a Th17 response (164), this novel Mincle ligand
harbors potential as a future adjuvant candidate. It is known that Mincle recognizes
mycobacterial-derived TDM as well as its synthetic analog TDB and plays an essential role in
enhancing protective immune response via FcRγ/Syk/CARD9 axis in immune cells,
emphasizing its potent adjuvant activity (131, 261). Studies using vaccines with TDB/TDM
as adjuvants showed that both glycolipids mediate immunostimulatory effects towards M.
tuberculosis that involves the FcRγ/Syk/CARD9 pathway and the activation of CD4+ T cells
(262, 263). Up to now, TDB and TDM are applied in liposome-based adjuvants in
experimental vaccines against pathogens like M. tuberculosis, Chlamydia and blood stage
malaria (263-266).
To further investigate the nature of different Mincle ligands, studies were performed to
analyse the crystal structures of human and bovine Mincle. Results showed that Mincle
harbors a CRD that contains a central Ca2+ ion to complex the 3- and 4-OH groups of the
glucose in the trehalose head-group. On the other site of this primary binding site, a
secondary binding site complexes the second glucose residue in the trehalose head-group.
Opposite to this binding pocket, a major hydrophobic groove binds to linear or branched
trehalose glycolipids (143-146). Further studies analysed the influence of the ligand structure
in regard to Mincle binding and signal transduction and showed that branched lipid
backbones (267) with long acyl chains (144) are better complexed by Mincle and induced
cytokine release by macrophages more efficiently (268, 269). Recently, different long-chain,
α-branched trehalose mono- and diesters were synthesized to unravel their capability to be
recognized by Mincle and to induce cell signaling. Interestingly, the prepared diesters of the
trehalose glycolipid mediated an increased agonistic activity of hMincle compared to the
adjuvant TDB (270). The knowledge from these structural data can be used in the future to
develop novel Mincle agonists that mediate efficient adjuvant properties.
In general, CLRs are described to be promiscuous receptors that are often able to detect more
than one ligand (147). For example, Mincle recognizes various nonself-derived glycolipids
and glycoproteins but also self-ligands such as sterols or β-glucosylceramides (213, 271,
272). Obviously, Mincle is also able to bind to the same ligand that derived from different
bacterial species, as shown for the glucosyldiacylglycerol of GAS (164) and S. pneumoniae
(157). Since glucosyldiacylglycerol is a part of the LTA which is a common feature of Gram-
Discussion
- 40 -
positive bacteria, further studies will elucidate if this glycolipid is generally recognized by
Mincle in all Gram-positive bacteria. However, there is insufficient knowledge about
differences in ligand preferences within pathogen species as shown for the recognition of β-
glucan harboring fungi by Dectin-1 (225). The mode of the initiated signal transduction by
CLR engagement is dependent on several factors like receptor density and the nature of the
respective ligands. For example, the affinity and avidity of ligand binding impairs signal
quantity and duration mediated by CLRs. But also the ligand properties like soluble or
particulate as well as the ligand size influence the resulting immune response (273). It was
shown for Mincle that the expression of this receptor in unstimulated cells is only weak,
whereas Mincle is upregulated upon stimulation and translocated to the cell surface via
MyD88 (238). Previous studies have shown that the constitutively expressed CLR MCL
enhances the expression of Mincle upon stimulation with TDM (274). In particular, upon
activation, MCL and Mincle form a complex heterodimer through the binding of the
hydrophobic repeat of MCL to the stalk region of Mincle, hence increasing the cell surface
expression of Mincle (237, 238, 275). These data and the finding that MCL and Mincle both
sense TDM (132, 274, 276) pinpoint to a synergistic relationship between these two
receptors. Furthermore, it is hypothesized that Mincle upon a certain threshold fine-tunes the
signaling that is initiated by MCL ligands to regulate host immune response (277).
In conclusion, the presented work highlights the role of CLRs in anti-bacterial immunity.
Methods were described to detect CLRs that recognize bacteria. This further allows for
functional characterization of CLR/bacteria interactions to unravel host/pathogen
interactions, bacterial pathogenesis and host immune responses. The identification of a
specific CLR ligand, as shown for GAS-derived MGDG and DGDG-binding to Mincle, as
part of a collaborative effort enables additional approaches like glycan-based targeting and
the production or the modification of potent vaccines against GAS.
Future outlook
- 41 -
Chapter 6: Future outlook
Although the previous chapters present examples that CLRs like Dectin-1 and Mincle
mediate an important role in anti-bacterial immunity, still intense work needs to be done to
identify the Dectin-1 ligand in C. jejuni, as well as the underlaying immune mechanisms and
signaling consequences of the MGDG and DGDG/Mincle interaction in GAS.
Bacteria like C. jejuni constantly change their surface structures to adapt to new
environments, including evasion of host immune responses. In addition to inter-strain specific
modifications, also changes of surface structures within one specific strain are described.
Studies are needed that reproducibly screen a broader set of C. jejuni strains that differ in
various characteristics like genetics or lifestyle as generalists or specialists (183). Since this
work only focused on the binding of Dectin-1 to C. jejuni, further studies are needed to show
if this recognition also induces cell signaling or if it is dispensable for the initiation of an
immune response as seen for C. neoformans (173) and other examples of CLR/pathogen
interactions (136, 158, 176). Moreover, the distinct ligand for Dectin-1 present in C. jejuni
has to be determined.
Regarding the detection of LTA-derived MGDG and DGDG as Mincle ligands as part of a
scientific collaboration, it would be helpful if also a larger number of GAS strains isolated
from clinical patients in various stages of infection are analysed concerning their
MGDG/DGDG ratio to enable association between virulence and Mincle-ligand expression.
Since LTA is a ubiquitous component of the Gram-positive cell wall (278), it is interesting to
investigate if Mincle also mediates recognition of other Gram-positive bacteria via LTA-
derived MGDG. The strain 5448 was the only GAS strain investigated with a minor role for
Mincle in vivo despite it was recognized by this receptor (164). The GAS strain 5448 was
isolated from a patient with severe, invasive necrotizing fasciitis (234). Since the infection
and inflammation of the skin is known to cause lower oxygen content as under normal
condition, so-called hypoxia, one should consider the oxygen concentration to obtain
physiologically relevant results. Indeed, publications report that hypoxia-inducible factor-1
(HIF-1), the main transcription factor that regulates the adaptive immune response to hypoxia
has an impact on both, the susceptibility to GAS and the PRR signaling pathways. On the one
hand, GAS 5448 induces HIF-1α (279). A murine infection study showed that HIF-1-
Future outlook
- 42 -
deficient mice are more susceptible to an invasive GAS 5448 infection, probably due to a
decreased phagocytic killing of the bacteria (279). On the other hand, crosstalk of HIF with
PRRs like TLR2 and TLR6 (280) is described in the literature as well as the HIF-induced
upregulation of TLR4 expression in macrophages (281). Interestingly, in response to
activation via TDB, Mincle signaling increased the transcription of the hif1α gene but was not
required for the expression of HIF-1α (282). Furthermore, key regulator molecules like the
adaptor molecule CARD9 and the transcription factor NFκ-B that play an important role in
signaling via Mincle and other CLRs were found to be involved in hypoxia-sensitive
pathways (283-285). Further studies should unravel the role of Mincle in GAS infection
under hypoxic conditions.
Concluding remarks
- 43 -
Chapter 7: Concluding remarks
The presented projects highlight the relevance of CLRs in sensing bacterial pathogens like
C. jejuni or GAS and contributed to unravelling that GAS-derived MGDG and DGDG serve
as novel ligands for Mincle.
CLR-Fc fusion proteins present powerful tools to identify yet unknown CLR/bacteria
interactions. Parts of this thesis describe ELISA-, flow cytometry- and confocal microscopy-
based methods that enable high-throughput screening and subsequent confirmation of CLR
candidates as innate bacterial sensors based on a set of human and murine CLR-Fc fusion
proteins. With the help of these methods, Dectin-1 was detected as a CLR that recognises
different C. jejuni isolates.
Additionally, this thesis contributed to the finding that the LTA-derived glycolipids MGDG
and DGDG from GAS act as novel ligands for Mincle. Whereas MGDG induced activation
of innate immune responses against GAS, DGDG interfered negatively with this response.
Moreover, in vivo data showed that Mincle mediated a protective role in anti-GAS immunity.
The identification of distinct bacterial CLR ligands is important for future vaccination
strategies to induce potent immune responses. Moreover, the analysis of initiated immune
responses by receptor/ligand interactions offers the possibility to therapeutically interfere
with the activated signaling pathway and thus shape immune responses.
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Affidavit
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Affidavit
I herewith declare that I autonomously carried out the PhD-thesis entitled “Role of C-type
lectin receptors in bacterial recognition”.
No third party assistance has been used.
I did not receive any assistance in return for payment by consulting agencies or any other
person. No one received any kind of payment for direct or indirect assistance in correlation to
the content of the submitted thesis.
I conducted the project at the following institution: Immunology Unit & Research Center for
Emerging Infections and Zoonoses, University of Veterinary Medicine Hannover,
Foundation.
The thesis has not been submitted elsewhere for an exam, as thesis or for evaluation in a
similar context.
I hereby affirm the above statements to be complete and true to the best of my knowledge.
Acknowledgement
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Acknowledgement
As we express our gratitude, we must never forget that highest
appreciation is not to utter words, but to live by them. -John F. Kennedy-
First of all, I would like to thank my supervisor Bernd Lepenies for giving me the opportunity
to perform a PhD in his research group. I am grateful for the support, novel ideas and
inspiring discussions throughout the time of the PhD. Thank you for having an open door and
open ears for various problems, small or large. Thank you for your guidance but also for
believing in me, giving me the freedom to do work my own way. Your passion for science
motivated and inspired me throughout the PhD times and I will never forget this.
I also want to thank Maren von Köckritz-Blickwede and Bastian Opitz for participating as
supervisors in my supervision group and their constant support with fruitful discussions and
comments. I am particularly grateful to Maren von Köckritz-Blickwede since you support me
from my time as a Master`s student onwards, hence you hugely contributed to my successful
scientific carrier in a hundred ways and I am immensely honored that you guided me
throughout the times!
Special thanks belongs to the whole immunology group, especially to:
Marie-Kristin Raulf for your practical and mental support during the whole PhD time and
even before that. Tim Ebbecke for the perfect times including a lot of laughing in the lab, the
office and off the work. Mariano Prado Acosta for your support in and off the lab and funny
spanish/german lessons. Dimitri Lindenwald for amazing stories and crucial fun facts of the
day. Guillaume Goyette-Desjardins for help with several computer programs and Silke
Schöneberg for technical support and always a smile on the face.
I am especially grateful to João Monteiro: Thanks so much for your constant encouragement
and your optimism. Your ideas have meant a lot to me and helped in so many ways! I will
never forget our time, especially the starting time. You have been a great colleague to me,
you will be still a great friend to me and for that I will be forever thankful. “In the end,
everything will be fine”
Acknowledgement
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A big thanks to present and former PhD and practical students, involved in my projects.
Especially Timo Johannssen, Rebecca Möller, Veronika Breitkopf, Katrin Opitz and Ina Ott.
I also want to thank the Hannover Graduate School for the financial support to attend
conferences.
Additional thanks goes to all collaborators, particularly to Christine Josenhans for providing
the C. jejuni strains and the support in the CLR/C. jejuni project. Furthermore, I would like to
thank Sho Yamasaki and Nina van Sorge for the successful GAS collaboration project. Also a
big thanks to Graham Brogden for the great introduction into the world of lipids.
Furthermore, I would like to thank my friends apart the lab, particularly Melissa Langer for
her support throughout the time and proofreading of this thesis.
Finally, I truely thank my family for their love, support and trust in me. I will be grateful
forever from the deepest of my heart.
