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