Ruminant TLRs: What we know and what we dont know

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Ruminant TLRsWhat we know and what we dont know
Dirk Werling Department of Pathology and
Infectious Diseases Royal Veterinary College,
London
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One of the, now crumbling, hypotheses regarding
the innate immune system is/was that its
reactions are always the same, independent of
cell type, ligand binding to the same receptor,
or species.
This morning, I would not only like to tell you
where we stand regarding ruminant TLRs, but also
convince you that we really have to be precise
about these factors when we talk about activation
of the ruminant innate immune system via TLRs
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Starting at the cell surface pattern-recognition
receptors (PRR) and the recognition of
non-self
In order to protect against infection, one of the
things the body must initially do is detect the
presence of micro-organisms. The body does this
by recognizing molecules unique to
micro-organisms that are not associated with the
host cells (non-self). These unique molecules
are called pathogen-associated molecular patterns
(PAMPs).
There are two functionally different classes of
cell surface pattern-recognition receptors
endocytic PRRs and signaling PRRs.
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Pattern Recognition Receptors
3 major subclasses

• C-type lectins
• C-type lectin receptors

• Toll like receptors (TLRs)

Scavenger receptors
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The discovery of TOLL
Cell 1985 Oct42(3)791-8 Establishment of
dorsal-ventral polarity in the Drosophila embryo
the induction of polarity by the Toll gene
product. Anderson KV, Bokla L, Nusslein-Volhard
C.
Cell 1996 Sep 2086(6)973-83 The dorsoventral
regulatory gene cassette spatzle/Toll/cactus
controls the potent antifungal response in
Drosophila adults.Lemaitre B, Nicolas E,
Michaut L, Reichhart JM, Hoffmann JA
Nature 1997 Jul 24388(6640)394-7 A human
homologue of the Drosophila Toll protein signals
activation of adaptive immunity Medzhitov R,
Preston-Hurlburt P, Janeway CA Jr.
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TLRs are the principal source of host awareness
of infection
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Chromosome-mapping of bovine TLR1-10
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Bovine TLRs

• ten bovine TLRs known

Sus scrofa
Bos taurus
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SMART analysis of TLR2
bovine
human
SMART simple modular architecture research tool
http//smart.embl-heidelberg.de/
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On the cell surface.
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Spectrum of (functional) TLRs expressed on the
mRNA level in cattle
dsRNA
response to viral infection
Unmethylated CpG motifs
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Monocyte
Functional consequences?Cytokine Response to TLR
ligands
IL-4, GM-CSF
Macrophage
Myeloid dendritic cell
IL-12 IL-12 IL-12 -
TNF, IL-10 TLR2 TNF, IL-10 TLR4 IFN-a/b,
IL-10 TLR3 (?) TNF, IL-10
TLR9 (?)
IFN-g costimul.
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Cell-specific expression of TLR2 and TLR4 mRNA
transcripts
boMa
boDC
w/o
w
w/o
w
TLR2
TLR4
b-act.
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TLRs reside within surface lipid rafts in
macrophages
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Beyond the cell surface..
?
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Expression of bovine TLR2 in HEK cells
control
boTLR2-YFP
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Luciferase activity of HEK cells transfected with
TLR2 and a NFkB reporter gene
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The response to different TLR2 ligands seems to
be ligand- and species-specific
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CXCL8 response of HEK cells transfected with
BoTLR2
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CXCL8 response of bovine macrophages to TLR2
ligands
CXCL8 (ng ml-1)
Pam3CSK4
Time (hours)
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The LPS-TLR4-MD2 complex
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Bovine TLR4 recognises LPS in the presence of MD2
from different species
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The other ruminants..Detection of ovine TLR2 in
tissue by in-situ hybridization using a probe
generated using the bovine sequence
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So.what do we know?

• We have cloned and expressed several bovine TLRs
  and adaptor proteins
• Different TLRs are expressed by different bovine
  APC subsets
• Stimulation of bovine TLRs expressed in/on DC may
  stimulate a Th1-type of immune response
• Whereas the same ligand may activate a Th2-type
  of immune response in macrophages
• Questions timing effect? Localisation effect?

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Sowhat do we know (cont.)

• Studying bovine TLRs in HEK cells can provide a
  useful tool to establish ligand-TLR interaction
• There is evidence that bovine and human TLRs may
  react different to the same ligand
• However, this may not fully reflect the in vivo
  condition gt requirement of co-receptors?
• Additional molecules, like MD2, may influence the
  TLR-induced activation of cells

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What we dont know

• Which TLRs are really expressed?
• lack of convincingly cross-reacting antibodies
  (FACSWB?)
• Please check PS1-28 (Sauter and Jungi)
• TLR sequences/expression pattern in other
  ruminant species (some data available for the
  ovine system)
• Please check PS1-25 (NalubambaHopkins)
• No functional data for other ruminant species so
  far
• Please check PS1-26 (NalubambaHopkins)

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Convinced? If so..then the bad news is that
there is a lot to do and ruminant immunology is
getting more complex
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Thanks to