Response of the Innate Response of the Innate Immune System to Immune System to

Pathogens:Pathogens:Pattern Recognition Pattern Recognition

ReceptorsReceptors

What is the Innate Immune What is the Innate Immune Response?Response?

A universal and evolutionarily conserved mechanism A universal and evolutionarily conserved mechanism of host defense against infectionof host defense against infection

First line of DefenseFirst line of Defense Predates the adaptive immune responsePredates the adaptive immune response

Found in all multicellular organismsFound in all multicellular organisms Adaptive only in vertebratesAdaptive only in vertebrates

Uses receptors and effectors that are ancient in Uses receptors and effectors that are ancient in their lineagetheir lineage

Must provide protection against a wide variety of Must provide protection against a wide variety of pathogenspathogens

Distinguishes self from non-self perfectlyDistinguishes self from non-self perfectly Defects in innate immunity are very rare and almost Defects in innate immunity are very rare and almost

always lethalalways lethal

The Innate Immune Response:The Innate Immune Response:Common MisconceptionsCommon Misconceptions

The innate immune system is an evolutionary rudiment The innate immune system is an evolutionary rudiment whose only function is to contain the infection until the whose only function is to contain the infection until the “real” immune response can kick in.“real” immune response can kick in.

Adaptive immunity developed because of the inflexibility Adaptive immunity developed because of the inflexibility of the nonclonal receptors used by the innate immune of the nonclonal receptors used by the innate immune response. The innate system cannot cope with the high response. The innate system cannot cope with the high mutational rate and heterogeneity of pathogenic mutational rate and heterogeneity of pathogenic organisms.organisms.

The Innate immune system instructs the adaptive The Innate immune system instructs the adaptive immune response to respond to microbial infectionimmune response to respond to microbial infection

The major decision to respond or not respond to The major decision to respond or not respond to a particular ligand is decided by the genome-a particular ligand is decided by the genome-

encoded receptors of the innate immune systemencoded receptors of the innate immune system

Y

Endosome

YNaive T Cell

MHC B7

Inflammatory and effectorcytokines

PRR

PAMP

Activated T Cell

CD40L, FasL, CD30L, CD27L

B Cell

Adapted from Medzhitov and JanewayCur. Opin. Immunol. 1997 9:4-9

Phagocytosis

APC

Direct Bactericidal ActivityPhagocytosisOxygen burstAnti-microbial peptides

Pathogen-specific Antibody

Complement

Innate Immunity Adaptive Immunity

skin, gut villi, lung cilia,etc

many protein andnon-protein secretions

phagocytes, NK cell eosinophils

Physical Barriers

Soluble Factors

Cells

none

Immunoglobulins(antibody)

T and B lymphocytes

ComponentsComponents of Innate and Adaptive of Innate and Adaptive ImmunityImmunity

PAMPs: Pathogen Associated Molecular Patterns

PRRs: Pattern Recognition Receptors

Takeda and Akira Genes to Cells (2001) 6:733-742

The NF-kB Family of Transcription FactorsThe NF-kB Family of Transcription Factors

Eukaryotic transcription factor found in essentially all cell typesEukaryotic transcription factor found in essentially all cell types First described in 1986 as a nuclear factor required for the First described in 1986 as a nuclear factor required for the

transcription of the immunoglobulin kappa light chain in B transcription of the immunoglobulin kappa light chain in B cells.cells.

Binds to a 10-bp sequence GGGGYNNCCYBinds to a 10-bp sequence GGGGYNNCCY Important component in the inducible expression of many Important component in the inducible expression of many

proteins: cytokines, acute phase proteins, adhesion moleculesproteins: cytokines, acute phase proteins, adhesion molecules The NF-kB signaling system is evolutionarily conservedThe NF-kB signaling system is evolutionarily conserved

Three NF-kB molecules in Three NF-kB molecules in DrosophilaDrosophila dorsaldorsal

controls dorsal/ventral polarity during developmentcontrols dorsal/ventral polarity during development Regulates antifungal gene expressionRegulates antifungal gene expression

difdif andand relishrelish: : regulate expression of antifungal and antibacterial regulate expression of antifungal and antibacterial genesgenes

NF-NF-B exists in the cytoplasm as an inactive B exists in the cytoplasm as an inactive heterotrimer composed of 2 Rel family proteins and heterotrimer composed of 2 Rel family proteins and

an inhibitory IkB moleculean inhibitory IkB molecule

IkB

p65 p50

IKK

Stress, infection, or cytokine

P P (Ub)n

26S proteosome

NuclearTranslocation

Activation of NF-KB Responsive genes

NF-NF-B Family StructureB Family Structure

Toll

Pelle

tube??

cactuskinase

cactus

dorsaldifrelish

Conservation ofConservation ofsignaling signaling pathwayspathways

between flies between flies and humansand humans

Mutation of genes in the Toll signalingcascade in Drosophila block the expression of anti-fungal and anti-bacterial genes

Water

A. fumigatus

E. coli

Signaling to monocytic cells through the human Toll homolog induces accessory cell functions

NF-B Activation

Requirements for the recognition of targets by the innate immune

Molecular structures recognized by the immune Molecular structures recognized by the immune system must be shared by large groups of pathogenssystem must be shared by large groups of pathogens molecular patterns vs. particular structures (antigens) PAMPmolecular patterns vs. particular structures (antigens) PAMP

PAMPs must be conserved products of microbial PAMPs must be conserved products of microbial metabolism not subject to antigenic variability metabolism not subject to antigenic variability

The recognized structures must be absolutely distinct The recognized structures must be absolutely distinct from self antigens: discrimination of self vs. non-selffrom self antigens: discrimination of self vs. non-self

LPS Binding Protein (LBP)LPS Binding Protein (LBP) 60-kDa serum glycoprotein that binds with high affinity to LPS Sequence homology to bactericidal/permeability increasing

protein Acute phase protein secreted by hepatocytes

Serum levels between 1 and 10 ug/ml in normal human serum

Concentrations >300 ug/ml in acute phase serum Critical for rapid responses to small amounts of LPS or gram-

negative bacteria and for survival of Salmonella infection Expression of LBP in hepatocytes is regulated by LPS, IL-1, IL-6

and TNF

Molecules involved in the recognition Molecules involved in the recognition of LPS by Cellsof LPS by Cells

Molecules involved in the recognition Molecules involved in the recognition of LPS by Cellsof LPS by Cells

CD14, sCD14CD14, sCD14 55 kDa GPI-linked protein on the surface on monocytes and 55 kDa GPI-linked protein on the surface on monocytes and

PMNPMN Also found as a soluble protein in serum:sCD14Also found as a soluble protein in serum:sCD14 Each CD14 molecule binds 1 molecule of LPS complexed to Each CD14 molecule binds 1 molecule of LPS complexed to

LBPLBP Required for responses of genes to low concentrations of LPS Required for responses of genes to low concentrations of LPS No cellular signaling activityNo cellular signaling activity

CD11b/CD18 (Mac1)CD11b/CD18 (Mac1) Alternative cell surface receptor for LPSAlternative cell surface receptor for LPS Recognizes LPS at high concentrationsRecognizes LPS at high concentrations

For expression of a fullFor expression of a full repertoire of LPS-inducible genes, CD14 repertoire of LPS-inducible genes, CD14 and CD11b/CD18and CD11b/CD18 must be coordinately engaged to deliver must be coordinately engaged to deliver optimal signaling tooptimal signaling to the macrophagethe macrophage..

Search for the LPS Gene Search for the LPS Gene Endotoxin hyporesponsive mice

1. C3H/He 1947 C3H/HeN: Normal LPS response

C3H/HeJ: LPS hyporesponsiveSpontaneous mutationmid 1960’s

2. C57BL/10SnSometimeAfter 1953

C57BL10/ScCRLPS hyporesponsive

January 1998 DNAX January 1998 DNAX reports the cloning of reports the cloning of human TLR1-5human TLR1-5 No ligands No ligands

describeddescribed Janeway’s Janeway’s

hToll=TLR4hToll=TLR4

Rock, et al. 1998 PNAS 95:588

September 1998 Genentech identifies TLR2 as the LPS signaling molecule by transfection studies Didn’t look at TLR4

December 1998 Tularik also identifies TLR2 as LPS signaling molecule in transfection studies TLR4 didn’t work

September 1998: Bruce Buetler’s lab mapped LPS to a 1.2Mb region of chromosome 4. The only intact gene within this region is TLR4

December 1998: “Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: Mutations in Tlr4 gene” Poltarak et al. Science 282:2085

Poltarak et al 1998 Science 282:2085Poltarak et al 1998 Science 282:2085 A single nucleotide change in the gene for

TLR4 renders the C3H/HeJ mouse non-responsive to LPS

C3HHeN RFHLCLHYRDFI P GCAIAANIIQEGFHKC3HHeJ RFHLCLHYRDFI H GCAIAANIIQEGFHK

Hoshino et al 1999 J. Immunol. 162:3749Hoshino et al 1999 J. Immunol. 162:3749 TLR4 knockout mice are hyporesponsive to LPSTLR4 knockout mice are hyporesponsive to LPS

Dunne and O’Neil 2003 www.stke.org/cgi/content/full/sigtrans;2003/171/re3

ReceptorReceptor(Pattern Recognition (Pattern Recognition

Receptors)Receptors)

Agonist(s)Agonist(s)(Pathogen-Associated (Pathogen-Associated Molecular Patterns)Molecular Patterns)

TLR1TLR1 Heterodimerizes with TLR2Heterodimerizes with TLR2

TLR2TLR2 PGN, some LPS, some LTA, PGN, some LPS, some LTA, lipoproteins, AraLAMlipoproteins, AraLAM

TLR3TLR3 dsRNAdsRNA

TLR4TLR4 Gram(-) LPS, Taxol, some Gram(-) LPS, Taxol, some LTALTA

TLR5TLR5 FlagellinFlagellin

TLR6TLR6 Heterodimerizes with TLR2Heterodimerizes with TLR2

TLR7TLR7 ImidazoquinolineImidazoquinoline

TLR9TLR9 Bacterial DNA (CpG)Bacterial DNA (CpG)

TLR 8,10TLR 8,10 UnknownUnknown

Akira J.Biol.Chem. 2003 278:38105–38108

How do we explain the Genentech and Tularik How do we explain the Genentech and Tularik findings?findings?

How could TLR4 knockout mice be LPS-nonresponsive but How could TLR4 knockout mice be LPS-nonresponsive but TLR2 transfected cells LPS responsive? TLR2 transfected cells LPS responsive?

Why were TLR4-transfected cells LPS non-reponsive?Why were TLR4-transfected cells LPS non-reponsive?

1.1. In both cases, the LPS In both cases, the LPS preparations used by the preparations used by the investigators were investigators were contaminated with contaminated with bacterial lipopeptides: bacterial lipopeptides: TLR2 agonists!TLR2 agonists!

2.2. In Tularik’s experiments In Tularik’s experiments with transfected TLR4, with transfected TLR4, they lacked a critical they lacked a critical accessory protein required accessory protein required for efficient TLR4 for efficient TLR4 expression and function: expression and function: MD-2MD-2

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

TLR2Control

TLR2SigmaLPS

TLR2pureLPS

TLR2LTA

TLR2PGN

TLR4Control

TLR4SigmaLPS

TLR4pureLPS

TLR4LTA

TLR4PGN

Fo

ld In

du

ctio

n

0.0000

0.1000

0.2000

0.3000

0.4000

0.5000

0.6000

Control LPS PMA

Rel

ativ

e L

igh

t U

nit

s

Vector

MD2

MD-2

TLR4/4CD14IL-1RI IL-1RAcP

MyD88 MyD88

TIR domain

Death domain

IRAK IRAKTRAF6

TAK1/NIK

IKKComplex IkB

p65p50

Common Themes in IL-1, TLR, and IL-18 SignalingCommon Themes in IL-1, TLR, and IL-18 Signaling

IL-1RAcP

MyD88

IL-18R

IRAK

NF-B activation

Nat Immunol. 2002 Apr;3(4):392-8

Nature 2003 420:329

Nature 2003 420:324

TIRAP=MAL

www.stke.org/cgi/content/full/sigtrans;2003/171/re3Luke A.J. O’Neil

Science. 2003 Aug 1;301(5633):640-3

Barton and Medzhitov Science. 2003 Jun 6;300(5625):1524-5

MD-2

TLR4/4CD14TLR1/6TLR2

MyD88MyD88

IRAK IRAKTRAF6

TAK1/NIK

IKKComplex

IkBp65p50

Common and Distinct Themes in TLR SignalingCommon and Distinct Themes in TLR Signaling

TIRAP

TRIF

IFN-

TIRAP IRF3

MAP kinases

Rac

PI3K

AKT

TIR domain

Death domain

PI3K

CommonResponses

TLR4-Specific

TLR2-Specific

Single ligand-Single response vs. Multiple Ligands-complex response

Ozinsky and Underhill Current Opinion in Immunology 2002, 14:103–110

Other PRRsOther PRRs

Dectin-1Dectin-1 C-type lectin that binds b-glucan-containing particles including C-type lectin that binds b-glucan-containing particles including

zymosan and zymosan and C. albicansC. albicans Macrophage Mannose ReceptorMacrophage Mannose Receptor

Cell bound C type lectin that binds sugar molecules on the Cell bound C type lectin that binds sugar molecules on the surface on many bacteria and viruses.surface on many bacteria and viruses.

Macrophage Scavenger Receptor (SR-A)Macrophage Scavenger Receptor (SR-A) Recognize certain anionic polymers and low-density Recognize certain anionic polymers and low-density

lipoproteinslipoproteins Peptidoglycan Recognition Proteins (PGRP)Peptidoglycan Recognition Proteins (PGRP)

4 forms in humans:4 forms in humans: PGRP-L: liverPGRP-L: liver PGRP-Ia and PGRP-Ib: esophagus PGRP-Ia and PGRP-Ib: esophagus PGRP-S: bone marrowPGRP-S: bone marrow

AdjuvantsAdjuvants

Functionally defined as any substance that, when Functionally defined as any substance that, when mixed with antigen, increases the mixed with antigen, increases the immunogenicity of the antigenimmunogenicity of the antigen

Injection of unmodified, nonself proteins in the Injection of unmodified, nonself proteins in the absence of adjuvant results in tolerance.absence of adjuvant results in tolerance.