Toll-like receptors

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Toll-like receptors Host-Pathogen Interaction
ONeill, Luke A.J. Immunitys Early-Warning
System. Scientific American, Jan (2005), 38-45.
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Microbe products recognized

• 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

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Pattern Recognition Receptors (PRRs)

• Toll-like receptors
• Natural history, function and regulation
• 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

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Nuesslein-Volhard Drosophila Toll

• Identified a protein she called Toll meaning
  weird
• Helps the Drosophila embryo to differentiate its
  top from its bottom
• (Neural tube development)

http//www.nature.com/genomics/papers/drosophila.h
tml
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Gay Toll and Inner Part of Human IL-1R is
Similar

• Searching for proteins similar to Toll
• Shows cytoplasmic domain of Toll related to that
  of
• hIL-1R
• Identity extends for 135 aa
• Didnt make sense

Why does a protein involved in human inflammation
look like one involved in fly neural tube
development?
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Toll Molecular Structure
IL-1R
Toll (will become TLRs)

• 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

Ig-like domain
LRRs
Box 1
TIR Domain
Box 2
Box 3
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Lemaitre Flies use Toll to Defend from Fungi

• Infected Tl-deficient adult flies with
  Aspergillus fumigatus
• All flies died after 2-3 days
• Flies use Toll to defend from fungi
• Thus, in Drosophila, Toll seems to be involved in
  embryonic development and adult immunity

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Lemaitre Flies use Toll to Defend from Fungi

• 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

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Survival rate of adult Drosophila infected with
Aspergillus fumigatus in Toll-
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Medzhitov Janeway Human Toll Discovery

• 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

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Medzhitov Janeway Human Toll Discovery

• Alignment of the sequences of human and
  Drosophila Toll proteins
• Homology over the entire length of the protein
  chains
• hToll gene most strongly expressed in Spleen and
  PBL (peripheral blood leukocytes)

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Rock Identification of hTLR1-5

• Identified 5 human Tolls, which they called Toll
  like receptors (TLRs)
• TLR4 same as Medzhitovs human Toll
• 4 complete - 1 partial hTLR
• 3 Drosophila TLRs

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Poltorak TLR4 Activated by LPS

• Normal mice die of sepsis after being injected
  with LPS
• C3H/HeJ mice have defective response to LPS and
  survive
• Missense mutation affecting the cytoplasmic
  domain of Tlr4
• Major breakthrough in the field of sepsis
  molecular mechanism that underlies inflammation
  revealed

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Takeuchi TLR6 discovery

• Murine TLR6 expression detected in spleen,
  thymus, ovary and lung

• Alignment of a.a. sequence of cytoplasmic
  domains TLR6 most similar to TLR1

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Chuang (2000) hTLR 7, 8 and 9

• Reported the cloning and characterization of 3
  hTLRs
• Ectodomain with multiple LRRs
• Cytoplasmic domain homologous to that of hIL-1R
• Longer ectodomain (higher MW) than hTLR1-6
• mRNA expression
  hTLR7 - lung, placenta
  and spleen hTLR8
  lung and PBL
  hTLR9 - spleen, lymph node, bone
  marrow and PBL

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Chuang (2001) hTLR10

• Expression of hTLR10 in human tissues and cell
  lines

• Isolation of cDNA encoding hTLR10
• Contains 811 aa, MW 94.6 kDA
• Architecture of hTLR10 same as in hTLR1-9

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Chuang hTLR10

• Phylogenetic tree of hTLR a.a. identity with
  hTLR1 (50) and hTLR6 (49)
• Only 30 with hTLR2 and 25 with the remaining
  ones

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TLR Roles
ONeill, Luke A.J. Immunitys Early-Warning
System. Scientific American, Jan (2005), 38-45.
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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
TLR4 Macrophages, PMLs, DCs, ECs, but not on lymphocytes
TLR5 Monocytes, immature DCs, epithelial, NK, and T cells
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
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Toll-Like Receptors and their Ligands
Receptor Ligand (PAMPs) Origin of Ligand
TLR1 Triacyl lipopetides Soluble factors Bacteria and Mycobacteria Neisseria meningitidis
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
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Converging Pathways
Beutler, Nature 2004

• Effects of signaling are cell specific
• NF-?B activation is the end result of
  TLR-signaling

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TLR Signaling Pathways
TLR2/TLR1 TLR2/TLR6
TLR4
Cell membrane
MAL MyD88
TRIF TRAM
MAL MyD88
TLR3 TLR7 TLR8 TLR9
IRF3
Endosome
TRIF MyD88
IRF7
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MyD88 Dependent and Independent Pathways Major
Role in Phagocyte Response
LPS
TLR4
Cell membrane
TLR4 MyD88-Dependent Signaling
TLR4 MyD88-Independent Signaling
MyD88
MAL
TNF COX2 IL-18 Chemokines
Chemokines Rantes, IP-10 IFN?
NF-?B
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LPS
TLR4 MyD88-Dependent Signaling
TLR4
Cell membrane
MyD88
MAL
IRAK4
IRAK1
IRAK2
TRAF6
IKK-?
IKK-?
IKK-?
Paz S., Nakhaei P,( 2005)
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LPS
TLR4
TLR4 MyD88-Independent Signaling
Cell membrane
TRAM
TRIF
TRAF6
IKK-?
Proteasome
TBK1
IKK-?
IKK-?
IKK?
Late induction
Paz S., Nakhaei P,( 2005)
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LPS
dsRNA
CpG DNA
TLR4
ssRNA
Cell membrane
TRIF
Tyk2
Jak1
TRAM
Endosome
STAT2
STAT1
ssRNA
CpG DNA
TBK1
TLR7/8
TLR9
STAT2
STAT1
IRF-9
IKK-?
MyD88
IKK-?
IKK-?
IRAK4
IRAK1
Proteasome
TRAF6
IFN-?
IFN-?
NF-?B
IFN Regulation
Inflammatory Cytokines
Paz S., Nakhaei P,( 2005)
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ST2
SIGIRR
Negative Regulation of TLR Signaling in Phagocytes
TLR4
Cell membrane
MAL
MyD88

• 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

IRAK4
IRAK1
UBC13
A20
TNF COX2 IL-18
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Phagocyte Sabotage Evading TLR Signaling
Yersinia LcrV

• 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.

Pseudomonas LPS
TLR2/TLR1 TLR2/TLR6 TLR4
Cell membrane
TRIF TRAM
MAL MyD88
Cytosolic Listeria
NF-?B
Nature Reviews Molecular Cell Biology 4 385-396
(2003)
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Leishmania-Induced Chemokine Expression
LPS
TLR4
MyD88 independent
MyD88
IRF-3
(-)
IRAK-1
SHP-1
TRAF6
?
IKKs
IkB-NFkB
NF-kB AP-1
Chemokines (MCP-1, MIP-1a/b, MIP-2) No NO No CD14
NO CD14 Chemokines (Rantes, IP-10, MCP-1,
MIP-1a/b, MIP-2, Eotaxin)
IFN-b
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Chemokines, linking innate and adaptive immunity