Transcript TOLL-LIKE RECEPTORS Toll-like receptors & Host

TOLL-LIKE RECEPTORS
Toll-like receptors &
Host-Pathogen Interaction
O’Neill, Luke A.J. “Immunity’s Early-Warning System”. Scientific American, Jan (2005), 38-45.
Microbe products recognized
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Conserved amoung microbes
Known as pathogen-associated
molecular patterns (PAMPs)
PAMPs are recognized by plants as
well as animals, meaning this innate
response arose before the split
Only vertebrates have evolved an
adaptive immune response
Pattern Recognition Receptors (PRRs)
• Toll-like receptors
• Natural history, function and regulation
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Mannose binding lectin (MBL)
C-reactive protein
Serum amyloid –P
Functions of PRRs:
– Opsonization, activation of complement and
coagulation cascades, phagocytosis, activation of
pro-inflammatory signaling pathways, apoptosis
Nuesslein-Volhard: Drosophila Toll
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Identified a protein she
called “Toll” meaning “weird”
Helps the Drosophila embryo
to differentiate its top from
its bottom
(Neural tube development)
1985
1988 1989
1991
http://www.nature.com/genomics/papers/drosophila.html
1996 1997 1998 1999 2000 2001
Gay: Toll and Inner Part of Human IL-1R is
Similar
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Searching for proteins
similar to Toll
Shows cytoplasmic domain
of Toll related to that of
hIL-1R
Identity extends for 135 aa
Didn’t make sense
Why does a protein involved in human
inflammation look like one involved in fly neural
tube development?
1985
1988 1989
1991
1996 1997 1998 1999 2000 2001
Toll Molecular Structure
IL-1R
Toll (will become TLRs)
Ig-like
domain
LRRs
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Box 1
Box 2
Box 3
TIR
Domain
Toll receptor has an
extracellular region which
contains leucine rich repeats
motifs (LRRs)
Toll receptor has a
cytoplasmic tail which
contains a Toll interleukin-1
(IL-1) receptor (TIR) domain
Lemaitre: Flies use Toll to Defend from Fungi
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Infected Tl-deficient adult flies
with Aspergillus fumigatus
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All flies died after 2-3 days
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Flies use Toll to defend from
fungi
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Thus, in Drosophila, Toll seems
to be involved in embryonic
development and adult
immunity
1985
1988 1989
1991
1996 1997 1998 1999 2000 2001
Lemaitre: Flies use Toll to Defend from Fungi
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Drosophila has no adaptive
immune system
Therefore needs a rapid
antimicrobial peptide response
Two distinct pathways to activate
antimicrobial peptide genes in
adults
Mutations in Toll pathway reduce
survival after fungal infection
1985
1988 1989
1991
1996 1997 1998 1999 2000 2001
Survival rate of adult Drosophila infected
with Aspergillus fumigatus in Toll100
% survival
80
60
wild type
Tl-
40
20
0
0
1
2
3
4
Time (days)
5
6
Medzhitov & Janeway: Human Toll Discovery
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Ancient immune defence
system based on the Toll
signalling
In insect, IL-1 receptor and
the Toll protein are only
similar in the segments
within the cell
They searched for human
proteins that totally
resemble to Toll
1985
1988 1989
1991
1996 1997 1998 1999 2000 2001
Medzhitov & Janeway: Human Toll Discovery
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Alignment of the sequences of human
and Drosophila Toll proteins
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Homology over the entire length of the
protein chains
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hToll gene most strongly expressed in
Spleen and PBL (peripheral blood leukocytes)
1985
1988 1989
1991
1996 1997 1998 1999 2000 2001
Rock: Identification of hTLR1-5
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Identified 5 human Tolls,
which they called Toll like
receptors (TLRs)
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TLR4 same as Medzhitov’s
human Toll
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4 complete - 1 partial hTLR
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3 Drosophila TLRs
1985
1988 1989
1991
1996 1997 1998 1999 2000 2001
Poltorak: TLR4 Activated by LPS
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Normal mice die of sepsis after
being injected with LPS
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C3H/HeJ mice have defective
response to LPS and survive
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Missense mutation affecting the
cytoplasmic domain of Tlr4
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Major breakthrough in the field of
sepsis – molecular mechanism that
underlies inflammation revealed
1985
1988 1989
1991
1996 1997 1998 1999 2000 2001
Takeuchi: TLR6 discovery
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Alignment of a.a. sequence of
cytoplasmic domains: TLR6
most similar to TLR1
1985
1988 1989
1991
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Murine TLR6 expression
detected in spleen, thymus,
ovary and lung
1996 1997 1998 1999 2000 2001
Chuang (2000):
hTLR 7, 8 and 9
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Reported the cloning and characterization of 3 hTLRs
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Ectodomain with multiple LRRs
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Cytoplasmic domain homologous to that of hIL-1R
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Longer ectodomain (higher MW) than hTLR1-6
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mRNA expression:
hTLR7 - lung, placenta and spleen
hTLR8 – lung and PBL
hTLR9 - spleen, lymph node, bone marrow and PBL
1985
1988 1989
1991
1996 1997 1998 1999 2000 2001
Chuang (2001):
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Isolation of cDNA encoding
hTLR10
Contains 811 aa, MW 94.6
kDA
Architecture of hTLR10 same
as in hTLR1-9
1985
1988 1989
1991
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hTLR10
Expression of hTLR10 in
human tissues and cell lines
1996 1997 1998 1999 2000 2001
Chuang:
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hTLR10
Phylogenetic tree of hTLR:
a.a. identity with hTLR1
(50%) and hTLR6 (49%)
Only 30% with hTLR2 and
25% with the remaining
ones
1985
1988 1989
1991
1996 1997 1998 1999 2000 2001
TLR Roles
O’Neill, Luke A.J. “Immunity’s Early-Warning System”. Scientific American, Jan (2005), 38-45.
TLR Cell Type Distribution
Receptor
Cell Type
TLR1
Ubiquitous
TLR2
DCs, PMLs, and monocytes
TLR3*
DC and NK cells, upregulated on epithelial and endothelial
cells
Macrophages, PMLs, DCs, ECs, but not on
lymphocytes
Monocytes, immature DCs, epithelial, NK, and T cells
TLR4
TLR5
TLR6
High expression in B cells, lower on monocytes and NK
cells
TLR7
B cells, plasmacytoid percursor DCs
TLR8
Monocytes, low in NK cells and T cells
TLR9
Plasmacytoid percursor DCs, B cells, macrophages,
PMLs, NK cells, and microglial cells
TLR10
B cells, plasmacytoid precursor DCs
TLR11
Not Determined
Toll-Like Receptors and their Ligands
Receptor
TLR1
Ligand (PAMPs)
Origin of Ligand
Triacyl lipopetides
Soluble factors
Bacteria and Mycobacteria
TLR2
Heat Shock protein 70
Peptidoglycan
Lipoprotein/lipopeptides
HCV core and nonstructural 3 protein
Host
Gram-positive bacteria
Various pathogens
Hepatitis C Virus
TLR3
Double-stranded RNA
Viruses
TLR4
Lipopolysaccharides
Envelope protein
Taxol
Gram-negative bacteria
Mouse mammary-tumor virus
Plants
TLR5
Flagellin
Bacteria
TLR6
Zymosan
Lipoteichoic acid
Diacyl lipopetides
Fungi
Gram-positive bacteria
Mycoplasma
TLR7
Single-stranded RNA (ssRNA)
Imidazoquinoline
Viruses
Synthetic compounds
TLR8
Single-stranded RNA (ssRNA)
Imidazoquinoline
Viruses
Synthetic compounds
TLR9
CpG-containing DNA
Bacteria, Malaria and Viruses
TLR10
Not determined
Not Determined
TLR11
Profilin-like molecule
Toxoplasma gondii
Neisseria meningitidis
Converging Pathways
Beutler, Nature 2004
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Effects of signaling are cell specific
NF-B activation is the end result of TLR-signaling
TLR Signaling Pathways
TLR2/TLR1
TLR2/TLR6
TLR4
TLR3
Cell membrane
MAL MyD88
MAL MyD88
TRIF TRAM
TRIF
H+
H+
H+
TLR3
H+
TLR7
TLR8
TLR9
H+
H+
H+
H+
NF-B
H+
H+
IRF3
Interferon Pathway
Inflammatory Cytokines
H+
H+
Endosome
IRF7
TRIF
MyD88
NF-B
MyD88 Dependent and Independent Pathways:
Major Role in Phagocyte Response
LPS
LBP
MD-2
TLR4
MD-2
LPS
sCD14
Cell membrane
p50 p65
NF-kB
NF-B
p50
TLR4 MyD88-Independent
Signaling
MAL
MyD88
TLR4 MyD88-Dependent
Signaling
p50 p65
p50
IRF-3
PP
NF-kB
TNF
COX2
IL-18
Chemokines
P
P
NF-B
IFN-
Chemokines:
Rantes, IP-10
IFN
LPS
TLR4 MyD88-Dependent Signaling
MD-2
LBP
sCD14
Cell membrane
IRAK1
IRAK2
MKK3
MKK7
UBC13
TRAF6
TAB2 TAK1
p38
IKK-
TAB1
UBV1A
IKK-
IRAK4
MEKK3
IKK-
MAL
MyD88
TOLLIP
(-)
TLR4
MD-2
LPS
Proteasome
JNK
IB
IB
p50
NF-B
p65
TNF
COX2
IL-18
Paz S., Nakhaei P,( 2005)
LPS
LPS
TLR4 MyD88-Independent Signaling
MD-2
MD-2
LBP
sCD14
Cell membrane
IKK-
IB
IB
p50
IKK-
IKK-
TRAM
TRIF
TRAF6
Proteasome
TLR4
TBK1
IKK
P
IRF-3
P
p65
P
P
P
P
Late induction
NF-B
IFN-
Paz S., Nakhaei P,( 2005)
LPS
dsRNA
CpG DNA
TLR4
ssRNA
Cell membrane
TRIF
Tyk2
Jak1
TRAM
Endosome
STAT2
STAT1
ssRNA
CpG DNA
TLR7/8
TLR9
TBK1
STAT2
IRF-9 STAT1
MyD88
IKK-
IKK-
IKK-
IRF-3
IRAK4
IRAK1
Proteasome
TRAF6
IRF-7
IB
IB
p50
IFN-
IFN-
p65
Inflammatory Cytokines
IFN Regulation
NF-B
Paz S., Nakhaei P,( 2005)
ST2
SIGIRR
(-)
MD-2
MD-2
TLR4
(-)
IRAK4
IRAK1
TRAF6
(-)
UBC13
A20
UBV1A
Proteasome
IKK-
IKK-
IKK-
(-)
SOCS1
IRAK-M
MyD88
MAL
Cell membrane
IB
IB
p50 p65
NF-B
TNF
COX2
IL-18
Negative
Regulation of TLR
Signaling in
Phagocytes
Cytoplasmic molecules:
 IRAK-M (restricted to
monocytes and macrophages)
 SOCS1 (Supressor of cytokine
signaling 1)
 A20 (TNFAIP3)
Membrane bound molecules:
 SIGIRR (single
immunoglobulin IL-1R-related
molecule)
 ST2
Phagocyte Sabotage: Evading TLR Signaling
Yersinia
LcrV
TLR2/TLR1
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TLR2/TLR6
TLR4
Pseudomonas
LPS
Cell membrane
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TRIF TRAM
MAL MyD88
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NF-B
Cytosolic Listeria
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Changing the target:
Camouflaging or directly
modifying the molecules that
trigger TLR signaling (ex: P.
aeruginosa).
Crossing the wires:
Interfering with downstream
TLR-mediated signaling or to
express TLR agonists
(ex: Y. pestis).
Sneaking through the back
door:
Bacteria such as Shigella sp.
and Listeria sp. express
proteins that facilitate their
invasion of macrophages.
Inflammatory Cytokines
Nature Reviews Molecular Cell Biology 4; 385-396 (2003);
Leishmania-Induced Chemokine Expression
LPS
TLR4
MyD88 independent
MyD88
SHP-1
IRF-3
(-)
IRAK-1
TRAF6
?
IKKs
IB-NFB
NO
CD14
Chemokines
(Rantes, IP-10, MCP-1, MIP-1/,
MIP-2, Eotaxin)
NF-B
AP-1
Chemokines
(MCP-1, MIP-1/, MIP-2)
No NO
No CD14
IFN-
Chemokines, linking innate and adaptive immunity