innate immunity -pathogen recognition by innate … immunity acquired immunity cells involved...

© 2015 Osaka University. All rights reserved.

Laboratory of Host Defense,WPI Immunology Frontier Research Center (IFReC)

Osaka University

Shizuo AKIRA

Innate Immunity

- Pathogen Recognition by Innate Immunity-

Innate and adaptive immunity

Engulfment and digestionof pathogens,Inflammation(formerly called non-specific immunity)

Antibody(Humoral immunity)

Killer T cell(Cellular immunity)

Innate immunity

Immunity

Adaptive immunity


Cells involved in adaptive immunity

T cell

Cellular immunity

B cell

Antibody production

Adaptive immunity can respond to millions of different foreign antigens in a highly specific manner. Each cell makes one particular antigen-specific receptor protein.

Cells involved in innate immunity

Macrophage Dendritic cell (DC)

Antigen presentation to T cells

Engulfment and digestion of pathogensCytokine production

Neutrophil

Phagocytes


NK cell

Pathogen

Innate and adaptive immunity

Neutrophil

Dendritic cell（Antigen presenting cell）

Detection of pathogens and initial attack

Macrophage

Innate Immunity

Moving to lymph node

Specific attack against pathogens

Antigenic peptide

Adaptive Immunity

T cell

B cell

T-B interaction

Plasma cell

T cell

Antibody

Toll-like receptors (TLRs) are adjuvant receptors

Adjuvants are oily substances which, when mixed and injected with antigen, nonspecifically enhance the immune response.

Complete Freund’s Adjuvant (CFA): a water-in-oil emulsion containing killed mycobacteria

used for the potentiation of antibody production in experimental animals.


Innate immunity specifically recognizes pathogens

pathogen

Macrophages, Dendritic Cells

Non-specific engulfment

and digestion

Old idea

Macrophages, Dendritic Cells

Specific recognition by TLRs

pathogen

New idea

DC

PathogensT cell

Regionallymph node

Infectedtissue

Draining lymphatics

Efferentlymphatics

T cell activationAntigen presentation by DC

Phagocytosis of pathogens(Non-specific)

Old Model


Pathogen Recognition by Innate Immunity

DC

Pathogen T cell

Regional lymph node

Infected tissue

DC activation andantigen presentation

T cell activation

Efferent lymphatics

New Model

Draining lymphatics

Antigenpresentation

TLRs

Dendritic cell NaïveT cell

IFN-

Th2 cell

Th1 cell

IL-4

Co-stimulatorymolecules

(CD40, CD80, CD86)

Pro-inflammatory cytokines(IL-12, TNF-)

Pathogens


Cancer Immunotherapy

Dr. William B. Coley (1862-1936), New York City surgeon and Sloan-Kettering researcher, examined the records of all bone cancer patients at New York Hospital. Most cases ended in death. However, he discovered one patient who had conquered cancer. This patient had suffered two attacks of bacterial infection.

Dr. Coley started to treat advanced cancer patients with live or killed bacteria, or bacterial components (Coley’s toxin).

By 1953, however, all the production of the toxins in the United States stopped.

(the cell wall skeleton from Bacille

Calmette-Guerin) : TLR2, TLR4

(lyophilized prep of a low virulence strain

of Streptococcus pyogenes) : TLR4, TLR9

BCG-CWS

OK432


Naïve CD8+

T cellDC

CytolyticT cell

Phagocytosis

Tumor cell

No activation of cytolytic T cells in cancer patients

Tumor cell

IL-12

Inactive DC

TLRs

Activation and proliferation of cytolytic T cells

Active DC

Activation via TLRs

Induction of antitumor action

Tumor Cell

Phagocytosis

Tumor cell

Naïve CD8+

T cell

Bacterial components


Innate Immunity Acquired Immunity

Cellsinvolved

Receptors

Recognition

MacrophagesDendritic cells T and B cells

Not rearranged(Toll-like receptors)

Pathogen-associatedMolecular patterns

(PAMPs)(ex. LPS, PGN)

Rearranged

Fine structures

(ex. Peptides)

PAMP and PRR

Pathogens have unique and conserved molecular patterns (pathogen-associated molecular patterns; PAMP)Innate immune system detect pathogen invasion by recognizing PAMPs via pattern recognition receptors (PRRs)

Most microbes, not only pathogens, express PAMPs.


Lemaitre et al. Cell 86, 973-83 (1996)

Innate Immune Response in Drosophila

Spatzle

Toll

Tube

Cactus

Dorsal

Fungi

Pelle

Anti-fungal peptide genes

Mammalian Toll-like receptors (TLRs)

TIR domainLeucine Rich Repeats Ig-like domain

IL-1R family

dTo

ll

TL

R1

TL

R2

TL

R3

TL

R4

TL

R5

TL

R6

TL

R7

TL

R8

TL

R9

TL

R10

TL

R11

TL

R12

TL

R13

IL-1

RI

IL-1

R A

cp

T1/

ST

2

IL-1

8R

IL-1

8R A

cp

TIG

IRR

-1

SIG

IRR

IL-1

R A

pl

TLR


Structure of the Leucine-rich repeats

motif: xLxxLxLxxNxLxxLxxxxxxxLx-sheet -helix

Structures of the cell surfaces of bacteria

Teichoic acid

Gram-positive bacteria

Gram-negative bacteria

Mycoplasma

Lipoteichoic acid (LTA) Lipopolysaccharide (LPS)

LipoproteinsPorin

Peptidoglycan (PGN)


Structure of LPS

O-antigen

Core

Lipid A

LPS recognition via CD14

TNF-IL-1IL-6IFN-NO

.

.

.

Liver

LBP

LPS

CD14

solubleCD14

Endothelial cell

Macrophage

Gram negative


TLR4

MD-2

CD14

LPSLBP

Signal transduction

LPS recognition and signaling

Bacterial lipopeptide (Pam3CSK4)

OO

OOSCH2CH

N CO

SerLysLysLysLysH

O

Mycoplasma lipopeptide (MALP-2)O

O

OOSCH2CH

N CO

HH GlyAsnAsnAspGlu-

TLR6TLR1

Pam3CSK4 MALP-2

TLR2 recognizes lipoproteins together with TLR1 or TLR6

TLR2 TLR2


Flagellated bacteria

pilus

nucleoidflagellum

DNA

Cell wall

capsule

1. Unmethylated CpG motifs are much more common inbacterial DNA than in vertebrate DNA.

2. CpG motifs are often methylated in vertebrate DNA.

3. DNA containing unmethylated CpG motifs activatemacrophages, dendritic cells, NK cells and B cells.

4. CpG DNA stimulates a Th1 immune responsedominated by the release of IL-12 and IFN-g.

5. CpG DNA is now promising for immunotherapy ofcancer, allergy and infectious diseases.

CpG DNA


TLR9 recognizes bacterial and viral DNA

Unmethylated CpG-DNA

TLR9

Inflammation

CpG

Th1 differentiation

Activation of adaptive immunitycytokineinterferonschemokines

bacteria virus

Bacterial flagella and flagellin

Bacteria with Flagella Flagellium

Flagellin

monotrichous

lophotrichous

peritrichous

amphitrichous

Hook

Basal Body


Flagellin recognition by intestinal epithelial cells and dendritic cells

Intestinal lumenEpithelial cells Apical surface

Basolateral surface

M cell

Flagellin

TLR5 TLR5

Accumulation of leukocytes

macrophagesDendritic cells

Attack

Infected sites

Cytokines and chemokines

IL-8

The imidazoquinolines

The imidazoquinolines are synthetic compounds that have a capacity to induce IFN-a and other cytokine synthesis in a variety of cell types.

Imiquimod is now approved for treatment for external genital warts caused by human papillomavirus infection.

The imidazoquinolines have potent anti-viral and anti-tumor properties in animals.

R-848 is more potent derivative of imiquimod and anticipated to be used for clinical treatment.

Imiquimod’s activity was discovered while screening for anti-herpes virus activity.

Imiquimod

N

NN

NH2

O

OH

N

NN

NH2

R-848


TLR7 and TLR3 recognize single-stranded and double-stranded RNA respectively

Imidazoquinoline derivatives

TLR3

Viral single-stranded

RNA

interferons

TLR7

Viral double-stranded

RNA

Imiquimod

N

NN

NH2

O

OH

N

NN

NH2

R-848

LipidLipoprotein

Nucleic acids

TLR ligands

TLR2/6TLR1/2

TLR4

TLR3TLR7

TLR8TLR9

TLR13

Protein

DNARNA

LipoproteinLPS

Flagellin

TLR5 TLR11


TLR ligands

Bacteria Fungus Virus ParasiteTLR

Triacyl lipopeptide - - -TLR1/2

TLR2

TLR2/6

TLR3

TLR4

TLR5

TLR7

TLR8

TLR9

TLR11

TLR13

PGN, LTA

Diacyl lipopeptide

-LPS

Flagellin

RNA

DNA

Flagellin

RNA

-

--

Zymosan

β-glucan Glycoinositolphospholipids

DNA

--

-

-

--

Structural protein

RNA

Structural protein

RNA

RNA

DNA

-

-

DNA, Hemozoin

-

-

tGPI-mutin

-

--

-

Profilin-like molecule

Song DH, Lee JO.Immunol Rev. 250:216-29 (2012)

TLR shows horseshoe-like structureForms a hetero- or homo-dimer (M-shaped

structure) to bind to PAMPs

Structure of TLR and ligand complex

TLR4

Triacyl lipopeptide Diacyl lipopeptide dsRNA

TLR3TLR6TLR2TLR1TLR2

TLR5TLR4

FlagellinLPS


In addition to responding to PAMPs, TLRs respond to endogenoushost molecules and trigger inflammatory responses.

Most of these are produced as a result of cell death and injury or by tumor cells. These include degradation products of the extracellular matrix (ECM), heat-shock proteins and high-mobility group box 1 (HMGB1) proteins, which act as stimulators for cell surface TLRs.

In addition, chromatin-DNA and ribonucleoprotein complexes released by dying cells and immune complex–containing self antigens can stimulate TLR7 and TLR9 and lead to the development of systemic autoimmune disease.

In atherosclerosis and Alzheimer’s disease, oxidized low-densitylipoprotein and amyloid-β, respectively, trigger sterile inflammation and are recognized by TLR4 and TLR6.

Endogenous ligands for TLRs

Autoimmunity, Immune regulation

RAGE?FgRIIA

Immune complex

ECM

HSP

HMGB1β-amyloid

Ox-LDL

Inflammation, Repair

Dying cells

Ox-phospholipid

TLR2TLR4TLR6

β-defensin2

TLR9TLR7

Co-receptors

Mitochondria DNA

Peroxiredoxin

TLR3

U1 RNA

Endogenous ligands for TLRs

HMGB1& DNA

LL37& DNA or RNA


TLR4

MyD88

NF-B IRF

TLR3

TRIF

TIRAP

TRIF

TRAM

IKKiTBK1

TIRAP

IRAK

MyD88

Inflammatory cytokines IFN/

Selective usage of adaptors in TLR signalingTLR2TLR5TLR7TLR9

TLR7 TLR9

IFNα

RNA DNA

endosome

IRF7

MyD88IRAK1

IRF7

virusesType1 IFN production by TLR7 and TLR9

TLR7/9 induces type1

interferons through the

formation of MyD88-IRAK1-

IRF7. This pathway occurs in

plasmacytoid dendritic cells

which produce a huge amount

of IFNα during viral infection.


TLRs bridging between innate and adaptive immunity

Cytokines and chemokines Local inflammation（leukocytes migration etc) Antibacterial peptides Antibacterial responseInterferons Antiviral response

Innate immune response

PAMPS （Cell wall components, flagellin, nucleic acids etc）

Toll-like receptors

: receptors for pathogen specific peptide（T cell receptors and antibodies）

（Vaccine）

Innate immune cells

Antigen presentation by DC

Activation of adaptive Immunity

（Pathogen sensors）

Escape from recognition by TLRs

• A certain adenovirus decreases CpG motifs in the genome: Escape from TLR9 recognition.

• Poxvirus protein A52R blocks TLR signaling pathways.

• LPS of Prophyromonas gingivalis and of Leprospira are less toxic due to deviation from the typical TLR4-activating lipid A.

• Flagellin of Helicobacter pylori is poorly recognized by TLR5.


Single nucleotide polymorphism of TLRsTwo cosegregating polymorphisms of the human TLR4 gene-Asp299Gly

and Thr399Ile, present in approximately 10% of white individuals

hyporesponsive to inhaled LPS

susceptibility to Gram-negative bacterial infection

premature birth, which is frequently the result of infections

severe respiratory syncytial virus bronchiolitis in infants

less frequently in atherosclerosis

SNP within the C-terminal region of human TLR2 (Arg753Gln)

associated with Staphylococcal infection, tuberculosis and

acute rheumatic fever caused by Streptococcus pyogenes

A stop codon within the human TLR5 gene

association with pneumonia caused by Legionella pneumophila

TLR4

TLR2

TLR5

T cell

Th2

Th1 IFN-

IL-4

Tumor immunityResistance to bacterial

and viral infection Anti-allergy

Allergy

Resisitance to helminth

antigen

Th1 and Th2 responses in acquired immunityTLR ligands predominantly induce Th1 response.

Autoimmune disease


Clinical Trial using TLR ligands

Infection：Preventive vaccines, Therapeutic vaccines, General enhancement of resistance to infectious diseases Cancer：Tumor vaccine, Cellular immunotherapy

using dendritic cells, Recovery of immune function after anti-cancer drug treatment Allergy: Pollen allergy and asthma

Gram positivebacteria

Spatzle

Pro-Spatzle

Proteasecascade

dsRNALPS

flagellinCpG DNAlipoprotein

Drosophila Mammals

Pathogen Recognition in flies and mammals

Fungi


10

12

12

222

9

Deuterostomia

Protostomia

TLRs in various species

Human

Mouse

Fugu

Sea urchin

Mollusca

PlatyzoaNematoda

Echinoderm

UrochordataCephalochordata

HemichordataPriapulida

AnnelidaLophophorata

Sponge

Drosophila

Phylogenetic tree of insect and mammalian TLRs

Mammalian TLRs

Insect Tolls AgToll11AgToll10

AgToll7DmToll7DmToll2

AgToll8

AgToll6DmToll8

DmToll6

DmToll4DmToll3AgToll5B

AgTollBAgToll5A

AgTollDmToll5

DmToll1mTLR5

hTLR5 mTLR9

hTLR9

mTLR8hTLR8mTLR7

hTLR7

mTLR3hTLR3

mTLR4hTLR4

hTLR2mTLR2mTLR6mTLR1

hTLR1hTLR6

hTLR10AgToll9 DmToll9

mIL-1RAcPL

hIL-1RmIL-1R

hIL-18RmIL-18R

hIL-1RAcPmIL-1RAcP

hIL-1RAcPLDmMyD88

hMyD88mMyD88AgMyD88At1g52900(TX)At4g36140(TNL)

At4g36140(TNL)At1g72850(TN)


Innate and Adaptive Immunity

Innateimmunity

Adaptive immunity

Innate immunity is not a remnant system but activeas advanced system collaborating with adaptive immunity

Old idea New idea

Innateimmunity

Innateimmunity

Adaptive immunity

Cytoplasmic Pathogen Sensors


Signaling pathways mediated by RIG-I and Mda5

Mitochondria

IKK/

Nucleus

Cytoplasm

NF- B

Type I IFN

Inflammatory cytokines

TBK1IKKi

IBs

MDA5

CARDRNA helicaseRIG-I

IPS-1/MAVS

P

P

IRF3

P

P

CARD

dsRNA

RNA helicase CARD CARD

CARD

IKK

Viral Recognition in the cytoplasm

RIG-I Mda5

IBs

IKK

IKKIRFkinase（TBK1, IKKi)

IRF3

longdsRNA(>3kb)

Type I IFNInflammatory cytokines

IPS-1

dsDNA

DNA virus

DNA sensors

TBK1

IKKi

IRF3

Type I IFNInflammatory cytokines

a variety of RNA viruses Picornaviruses

Mitochondria

STINGCARD

NF-B

CARD

CARD

CARD

CARD

IKK

IKK

IBs

NF-B

Triphosphateshort ssRNA,dsRNA


Cytosolic DNA sensor: cGAS-cGAMP-STING axis

cGAS(cGAMP synthase)

dsDNA

cGAMP(cyclicGMP-AMP)

Type 1 IFNs

+ATP+GTP

cGAMP

STING

O

NH2

3’

5’

3’

5’ OH

O O-

P

GNH

N N

N

A

O

NH4+

OO

O O NH4+

OP

OOH

O

HN

O

N

NNH2N

TLR-dependent and -independent recognition of bacterial components

MyD88

PGN

NOD1

NOD2

CARD NOD LRR

CARD NOD LRRCARD

NF-B

TLR2

MDP

iE-DAP


Nod-like receptor and HIN-200 protein family

Gene name

NOD1NOD2

NLRC4NAIP5

NLRP1NLRP3NLRP6

AIM2

Core domain

NOD domain

HIN-200 domain

Functional domain

CARD, LRRs2CARD, LRRs

CARD, LRRs3BIR, LRRs

PYRIN, CARD, LRRsPYRIN, LRRsPYRIN, LRRs

PYRIN

NF-BMAPK

Inflammasome

IL-1 gene IL-18 gene

Pro-IL-1 Pro-IL-18

Caspase-1

Mature-IL-1 Mature-IL-18

Inflammatory Stimuli

IL-1, IL-18 activation pathways


Inflammasomes

Flagellin, etc.dsDNA ？

LRRs

NLRC4NLRP3

Caspase-1

ASC

NODPYRIN

PYRIN CARD

CaspaseCARD

LRRs

AIM2

NODCARDPYRIN HIN-200

Pro-IL-1b/IL-18

Processing

Mature IL-1b/IL-18

P. aeruginosaS. typhimurium

Influenza A virusC. albicans

Vaccinia virusF. tularensis L. monocytogenes

Lysosomal membrane disrupting agentsactivate NLRP3-inflammasome

Cholesterolcrystals

Uric acidcrystals

Human islet amyloidpolypeptides

Asbestos

Accumulation by hyper-nutrition Pulmonaryaspiration

Atherosclerosis Gout Type IIdiabetes

Lunginflammation

Inflammatory diseases caused by persistent activation of NLRP3-inflammasome


Syk

cytosol

Inflammatory cytokines

NF-kB

Syk

-Glucan

Pathogen recognition by C-type lectin receptors (CLRs)

P

P

P

P SykP

PFcR FcR

CCCARD CARD9CARDPYD BCL10

MALT1

MAPK

Dectin-1 Dectin-2 Mincle

-Mannan

MycobacteriaTrehalose dimycolate(TDM)

Candida albicansPneumocystis carinii Malassezia

Casp-L

Regulation of adaptive immunity by innate immunity

TLR RLR NLR

Pathogens

Adaptive immunity

antibody

Cytotoxic T cellHelper T cell

NK cell

Regulatory T cell

CLRInnate

immunity

innate immunity -pathogen recognition by innate … immunity acquired immunity cells involved...

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