toll-like receptors. toll-like receptors & host-pathogen interaction o’neill, luke a.j....
TRANSCRIPT
TOLL-LIKE RECEPTORSTOLL-LIKE RECEPTORS
Toll-like receptors & Toll-like receptors & Host-Pathogen InteractionHost-Pathogen Interaction
O’Neill, Luke A.J. “Immunity’s Early-Warning System”. Scientific American, Jan (2005), 38-45.
Microbe products Microbe products recognizedrecognized
ConservedConserved amoung microbes amoung microbes Known as Known as ppathogen-athogen-aassociated ssociated mmolecular olecular ppatterns (atterns (PAMPsPAMPs))
PAMPs are recognized by PAMPs are recognized by plantsplants as well as as well as animalsanimals, meaning this , meaning this innate response innate response arose before the arose before the splitsplit
OnlyOnly vertebrates have evolved an vertebrates have evolved an adaptive immuneadaptive immune response response
Pattern Recognition Receptors (PRRs)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
Nuesslein-Volhard: Drosophila Nuesslein-Volhard: Drosophila TollToll
Identified a protein Identified a protein she called “Toll” she called “Toll” meaning “weird”meaning “weird”
Helps the Drosophila Helps the Drosophila embryo to embryo to differentiate its top differentiate its top from its bottom from its bottom
(Neural tube (Neural tube development)development)
1985 1991 20011996 1997 1998 1999 20001988 1989
http://www.nature.com/genomics/papers/drosophila.html
Gay: Toll and Inner Part of Human IL-1R Gay: Toll and Inner Part of Human IL-1R is Similaris Similar
1985 1991 20011996 1997 1998 1999 20001988 1989
Searching for Searching for proteins similar to proteins similar to TollToll
Shows Shows cytoplasmiccytoplasmic domain of Toll domain of Toll related to that of related to that of
hIL-1RhIL-1R Identity extends for Identity extends for
135 aa135 aa Didn’tDidn’t make sense make sense
Why does a protein involved in human inflammation look like one involved in fly neural
tube development?
Toll Molecular StructureToll Molecular Structure
Toll receptor has an Toll receptor has an extracellular region extracellular region which contains leucine which contains leucine rich repeats motifs rich repeats motifs ((LRRsLRRs))
Toll receptor has a Toll receptor has a cytoplasmic tail which cytoplasmic tail which contains a Toll contains a Toll interleukin-1 (interleukin-1 (IL-1IL-1) ) receptor (receptor (TIRTIR) domain) domain
IL-1R
TIR Domain
Toll (will become TLRs)
LRRsIg-like domain
Box 1
Box 2
Box 3
Lemaitre: Flies use Toll to Defend from Lemaitre: Flies use Toll to Defend from FungiFungi
1985 1991 20011996 1997 1998 1999 20001988 1989
Infected Infected TlTl-deficient -deficient adult flies with adult flies with Aspergillus fumigatusAspergillus fumigatus
All flies died after 2-3 All flies died after 2-3 daysdays
Flies use Toll to defend Flies use Toll to defend from fungifrom fungi
Thus, in Drosophila, Toll Thus, in Drosophila, Toll seems to be involved in seems to be involved in embryonic development and embryonic development and adult adult immunityimmunity
Lemaitre: Flies use Toll to Defend from Lemaitre: Flies use Toll to Defend from FungiFungi
1985 1991 20011996 1997 1998 1999 20001988 1989
Drosophila has Drosophila has no adaptiveno adaptive immune systemimmune system
Therefore needs a rapid Therefore needs a rapid antimicrobial peptideantimicrobial peptide responseresponse
TwoTwo distinct pathways to distinct pathways to activate antimicrobial activate antimicrobial peptide genes in adultspeptide genes in adults
MutationsMutations in Toll pathway in Toll pathway reduce survival after reduce survival after fungal infectionfungal infection
Survival rate of adult Survival rate of adult DrosophilaDrosophila infected infected
with with Aspergillus fumigatus in Aspergillus fumigatus in TollToll--
65432100
20
40
60
80
100
Tl-wild type
Time (days)
% s
urv
ival
Medzhitov & Janeway: Human Toll Medzhitov & Janeway: Human Toll DiscoveryDiscovery
1985 1991 20011996 1997 1998 1999 20001988 1989
AncientAncient immune defence immune defence system based on the system based on the Toll Toll signallingsignalling
In insect, IL-1 receptor In insect, IL-1 receptor and the Toll protein are and the Toll protein are only similaronly similar in the in the segments within the cellsegments within the cell
They searched for They searched for human human proteinsproteins that totally that totally resemble to Tollresemble to Toll
Medzhitov & Janeway: Human Toll Medzhitov & Janeway: Human Toll DiscoveryDiscovery
1985 1991 20011996 1997 1998 1999 20001988 1989
Alignment of the sequences of Alignment of the sequences of human and Drosophila Toll human and Drosophila Toll proteinsproteins
HomologyHomology over the entire length over the entire length of the protein chainsof the protein chains
hToll gene hToll gene most stronglymost strongly expressed in expressed in SpleenSpleen and and PBL PBL (peripheral blood leukocytes)(peripheral blood leukocytes)
Rock: Identification of hTLR1-5Rock: Identification of hTLR1-5
1985 1991 20011996 1997 1998 1999 20001988 1989
Identified 5 human Identified 5 human Tolls, which they Tolls, which they called Toll like called Toll like receptors (receptors (TLRsTLRs))
TLR4TLR4 same as same as Medzhitov’s human TollMedzhitov’s human Toll
4 complete - 1 partial 4 complete - 1 partial hTLRhTLR
3 Drosophila TLRs3 Drosophila TLRs
Poltorak: TLR4 Activated by Poltorak: TLR4 Activated by LPSLPS
1985 1991 20011996 1997 1998 1999 20001988 1989
NormalNormal mice mice diedie of sepsis of sepsis after being injected with after being injected with LPSLPS
C3H/HeJ C3H/HeJ mice have defective mice have defective response to LPS and response to LPS and survivesurvive
Missense mutationMissense mutation affecting affecting the cytoplasmic domain of the cytoplasmic domain of Tlr4Tlr4
Major breakthroughMajor breakthrough in the in the field of sepsis – molecular field of sepsis – molecular mechanism that underlies mechanism that underlies inflammation revealedinflammation revealed
Takeuchi: TLR6 discoveryTakeuchi: TLR6 discovery
1985 1991 20011996 1997 1998 1999 20001988 1989
Alignment of a.a. Alignment of a.a. sequence of sequence of cytoplasmic domains: cytoplasmic domains: TLR6 most similar to TLR6 most similar to TLR1TLR1
Murine TLR6 Murine TLR6 expression detected expression detected in spleen, thymus, in spleen, thymus, ovary and lungovary and lung
Chuang Chuang (2000)(2000): hTLR 7, 8 and 9: hTLR 7, 8 and 9
1985 1991 20011996 1997 1998 1999 20001988 1989
Reported the cloning and characterization of 3 hTLRsReported the cloning and characterization of 3 hTLRs
EctodomainEctodomain with multiple with multiple LRRsLRRs
Cytoplasmic domain homologous to that of hIL-1RCytoplasmic domain homologous to that of hIL-1R
LongerLonger ectodomain (higher MW) than hTLR1-6 ectodomain (higher MW) than hTLR1-6
mRNA expression: mRNA expression: hTLR7hTLR7 - lung, placenta and - lung, placenta and spleen spleen hTLR8hTLR8 – lung and – lung and PBL PBL hTLR9hTLR9 - spleen, lymph node, bone marrow and PBL - spleen, lymph node, bone marrow and PBL
Chuang Chuang (2001)(2001): hTLR10: hTLR10
1985 1991 20011996 1997 1998 1999 20001988 1989
Isolation of cDNA Isolation of cDNA encoding hTLR10encoding hTLR10
Contains 811 aa, MW Contains 811 aa, MW 94.6 kDA94.6 kDA
ArchitectureArchitecture of of hTLR10 same as in hTLR10 same as in hTLR1-9hTLR1-9
ExpressionExpression of hTLR10 of hTLR10 in human tissues and in human tissues and cell linescell lines
Chuang: hTLR10Chuang: hTLR10
1985 1991 20011996 1997 1998 1999 20001988 1989
Phylogenetic tree of Phylogenetic tree of hTLRhTLR: a.a. identity : a.a. identity with with hTLR1hTLR1 (50%) and (50%) and hTLR6hTLR6 (49%) (49%)
Only 30% with Only 30% with hTLR2hTLR2 and 25% with the and 25% with the remaining onesremaining ones
TLR RolesTLR Roles
O’Neill, Luke A.J. “Immunity’s Early-Warning System”. Scientific American, Jan (2005), 38-45.
TLR Cell Type DistributionTLR Cell Type Distribution
ReceptorReceptor Cell TypeCell Type
TLR1TLR1 UbiquitousUbiquitous
TLR2TLR2 DCs, PMLs, and monocytesDCs, PMLs, and monocytes
TLR3*TLR3* DC and NK cells, upregulated on epithelial DC and NK cells, upregulated on epithelial and endothelial cellsand endothelial cells
TLR4TLR4 Macrophages, PMLs, DCs, ECs, but Macrophages, PMLs, DCs, ECs, but not on not on lymphocyteslymphocytes
TLR5TLR5 Monocytes, immature DCs, epithelial, NK, Monocytes, immature DCs, epithelial, NK, and T cellsand T cells
TLR6TLR6 High expression in B cells, lower on High expression in B cells, lower on monocytes and NK cellsmonocytes and NK cells
TLR7TLR7 B cells, plasmacytoid percursor DCsB cells, plasmacytoid percursor DCs
TLR8TLR8 Monocytes, low in NK cells and T cellsMonocytes, low in NK cells and T cells
TLR9TLR9 Plasmacytoid percursor DCs, B cells, Plasmacytoid percursor DCs, B cells, macrophages, PMLs, NK cells, and microglial macrophages, PMLs, NK cells, and microglial
cellscellsTLR10TLR10 B cells, plasmacytoid precursor DCsB cells, plasmacytoid precursor DCs
TLR11TLR11 Not DeterminedNot Determined
ReceptorReceptor Ligand (PAMPs)Ligand (PAMPs) Origin of LigandOrigin of LigandTLR1TLR1 Triacyl lipopetides Triacyl lipopetides
Soluble factorsSoluble factorsBacteria and MycobacteriaBacteria and Mycobacteria
Neisseria meningitidisNeisseria meningitidis
TLR2TLR2 Heat Shock protein 70Heat Shock protein 70
PeptidoglycanPeptidoglycan
Lipoprotein/lipopeptidesLipoprotein/lipopeptides
HCV core and nonstructural 3 HCV core and nonstructural 3 proteinprotein
HostHost
Gram-positive bacteriaGram-positive bacteria
Various pathogensVarious pathogens
Hepatitis C VirusHepatitis C Virus
TLR3TLR3 Double-stranded RNADouble-stranded RNA VirusesViruses
TLR4TLR4 LipopolysaccharidesLipopolysaccharides
Envelope proteinEnvelope protein
TaxolTaxol
Gram-negative bacteriaGram-negative bacteria
Mouse mammary-tumor virusMouse mammary-tumor virus
PlantsPlants
TLR5TLR5 FlagellinFlagellin BacteriaBacteria
TLR6TLR6 ZymosanZymosan
Lipoteichoic acidLipoteichoic acid
Diacyl lipopetidesDiacyl lipopetides
FungiFungi
Gram-positive bacteriaGram-positive bacteria
MycoplasmaMycoplasma
TLR7TLR7 Single-stranded RNA (ssRNA)Single-stranded RNA (ssRNA)
ImidazoquinolineImidazoquinolineVirusesViruses
Synthetic compoundsSynthetic compounds
TLR8TLR8 Single-stranded RNA (ssRNA)Single-stranded RNA (ssRNA)
ImidazoquinolineImidazoquinolineVirusesViruses
Synthetic compoundsSynthetic compounds
TLR9TLR9 CpG-containing DNACpG-containing DNA Bacteria, Malaria and Bacteria, Malaria and VirusesViruses
TLR10TLR10 Not determinedNot determined Not DeterminedNot Determined
TLR11TLR11 Profilin-like moleculeProfilin-like molecule Toxoplasma gondiiToxoplasma gondii
Toll-Like Receptors and their LigandsToll-Like Receptors and their Ligands
Converging PathwaysConverging Pathways
Effects of signaling are cell specificEffects of signaling are cell specific NF-NF-B activation is the end result of TLR-B activation is the end result of TLR-signalingsignaling
Beutler, Nature 2004
TLR Signaling PathwaysTLR Signaling Pathways
Cell membrane
TLR3 TLR7 TLR8 TLR9
H+
H+
H+
H+
H+
H+
H+H+
H+
H+
H+
H+
TLR2/TLR1 TLR2/TLR6 TLR4
MAL MyD88 TRIF TRAM
NF-B
TRIF MyD88
NF-BInflammatory Cytokines
Interferon Pathway
Endosome
TLR3
TRIF
IRF3
IRF7
MAL MyD88
TLR4 MyD88-TLR4 MyD88-Dependent Dependent SignalingSignaling
MA
L
MyD
88
Cell membrane
LBP
sCD14
TLR4
LPS
MD
-2
MD
-2
LPS
p65p50
p50NF-kB
IRF-3
PP
PP
p65p50
p50NF-kB
TLR4 MyD88-TLR4 MyD88-IndependentIndependent SignalingSignaling
MyD88 Dependent and Independent Pathways: MyD88 Dependent and Independent Pathways: Major Role in Phagocyte ResponseMajor Role in Phagocyte Response
IFN-
NF-B
NF-B
TNFCOX2IL-18Chemokines
Chemokines:Rantes, IP-10IFN
TLR4LBP
sCD14
LPS
MD
-2
MD
-2
LPS
TLR4 MyD88-Dependent SignalingTLR4 MyD88-Dependent Signaling
MA
LM
yD88
IRAK4
IRAK2
IRAK1
TRAF6TAB1
TAB2 TAK1
IKK-
IKK
-
IKK
-
p65
IB
p50
NF-BTNFCOX2IL-18
UBC13
UBV1A
(-)
TOLLIP
MEKK3 MKK3
MKK7
JNK
p38
Cell membrane
IB
Proteasome
Paz S., Nakhaei P,( 2005)
TLR4LBP
sCD14
LPS
MD
-2
MD
-2
TR
IFT
RA
M
TRAF6
TBK1 IKK
LPS
IKK-
IKK
-
IKK
-
TLR4 MyD88-Independent Signaling
p65
IB
p50
NF-B
IFN-
P
P
IRF-3
P
P P
P
Cell membrane
IB
Proteasome
Paz S., Nakhaei P,( 2005)
Late induction
CpG DNA
dsRNA
ssRNA
ssRNA CpG DNA
MyD88
IRAK4
IRAK1
EndosomeEndosome
TLR7/8TLR7/8
IKK-
IKK
-
IKK
-
p65
IB
p50
LPS
NF-B
IFN-
TRIF
TLR4TLR4
TRAM
TBK1
IFN-
Tyk2 Jak1
STAT2STAT1
STAT2STAT1IRF-9
Cell membrane
TLR9TLR9
IRF-7
IRF-3
IB
Proteasome TRAF6
Inflammatory CytokinesInflammatory Cytokines IFN RegulationIFN Regulation
Paz S., Nakhaei P,( 2005)
TLR4
IKK-
IKK
-
IKK
-
p65
IB
p50
NF-BTNFCOX2IL-18
MD
-2
MD
-2
MA
LM
yD8
8
IRAK4
IRAK1UBC13
TRAF6
UBV1A
(-)
Cell membrane
ST2 SIGIRR
(-)
SOCS1
Cytoplasmic molecules:Cytoplasmic molecules: IRAK-MIRAK-M (restricted to (restricted to monocytes and monocytes and macrophages)macrophages)
SOCS1SOCS1 (Supressor of (Supressor of cytokine signaling 1) cytokine signaling 1)
A20A20 (TNFAIP3)(TNFAIP3)
Membrane bound molecules:Membrane bound molecules: SIGIRRSIGIRR (single (single immunoglobulin IL-1R-immunoglobulin IL-1R-related molecule)related molecule)
ST2ST2
Negative Negative Regulation of Regulation of
TLR Signaling in TLR Signaling in PhagocytesPhagocytes
IRA
K-M
(-)
IB
Proteasome
(-) A20
MAL MyD88
NF-B
TLR2/TLR1 TLR2/TLR6 TLR4
TRIF TRAM
Inflammatory CytokinesInflammatory Cytokines
(-)
PseudomonasLPS
Yersinia LcrV
Cell membrane
Cytosolic Listeria
Changing the target:Changing the target: Camouflaging or directly Camouflaging or directly modifying the molecules modifying the molecules that trigger TLR that trigger TLR signaling (ex: signaling (ex: P. P. aeruginosaaeruginosa).).
Crossing the wiresCrossing the wires:: Interfering with Interfering with downstream TLR-mediated downstream TLR-mediated signaling or to express signaling or to express TLR agonists TLR agonists
(ex: (ex: Y. pestisY. pestis).).
Sneaking through the back Sneaking through the back door:door:
Bacteria such as Bacteria such as ShigellaShigella sp. and sp. and ListeriaListeria sp. sp. express proteins that express proteins that facilitate their invasion facilitate their invasion of macrophages.of macrophages.
Nature Reviews Molecular Cell Biology 4; 385-396 (2003);
Phagocyte Sabotage: Evading TLR Phagocyte Sabotage: Evading TLR SignalingSignaling
TLR4TLR4
NF-NF-BBAP-1AP-1
ChemokinesChemokines(MCP-1, MIP-1(MCP-1, MIP-1//, MIP-2), MIP-2)
No NONo NONo CD14No CD14
LeishmaniaLeishmania-Induced Chemokine -Induced Chemokine ExpressionExpression
IKKs
MyD88
LPS
IRF-3IRAK-1
TRAF6
IB-NFB
NONOCD14CD14
ChemokinesChemokines(Rantes, IP-10, MCP-1, MIP-1(Rantes, IP-10, MCP-1, MIP-1//, ,
MIP-2, Eotaxin)MIP-2, Eotaxin)
?
IFN-
MyD88 independent
SHP-1(-)
Chemokines, linking innate and adaptive immunityChemokines, linking innate and adaptive immunity