Nobel Lecture slides [PDF]

The Antimicrobial Defense of Drosophila, A Paradigm for Innate Immunity

Jules Hoffmann, Strasbourg, France

Bacteria

Viruses

Protozoa Fungi

Jos Hoffmann 1911-2000

Pierre Joly 1913-1996

Antimicrobial Defenses in Insects : First Investigations

Metchnikoff

Paillot Phagocytosis « Cellular Immunity » Metchnikoff, 1880

Antimicrobial substances in the blood « Humoral Immunity» Paillot 1920-1935 Glaser

Induction of an antimicrobial activity in Drosophila by an immune challenge

1 Hyalophora cecropia

Injection of bacteria Antimicrobial activity in the cell-free hemolymph

2 Hans Boman 1924-2008

3 Control 0

3

6

9

12 24 Time (h)

48

Systemic (“humoral”) antimicrobial response in Drosophila – identification of antimicrobial peptides P

P P P GG P GG GGG

G

P

G

G

G G

G

G

G

Diptericin G

G G

G G GG G

G

G

Fat body cells

P

P P

G G G

P

G G G

G

Metchnikowin

G

Attacin

Cecropin Drosocin

Defensin

P

NF-κB response elements in the promoter of the diptericin gene -1 kb

enhancer

-150

-140 -62

-31

coding sequence of the diptericin gene

κB-Response element NLS

1

678

REL Stewart 1987

1

Ank

DORSAL CACTUS

Diptericin-LacZ reporter gene

Unchallenged

Challenged

482

Versailles, 18 years ago, Innate Immunity Conference Michael Zasloff Jean-Marc Reichhart Dan Hultmark

Klas Kärre

Danièle Hoffmann

Charlie Janeway Shunji Natori

Alan Ezekowitz Bob Lehrer Hans Boman Charles Hetru

Ingrid Faye

Gene cascade controlling the dorso-ventral axis in the Drosophila embryo CHORION

Easter

PERIVITELLIN SPACE Spätzle

Snake

EMBRYO

Gastrulation Defective Windbeutel and Pipe

Tube Toll

Cactus Pelle

Nudel

FOLLICLE CELLS

Christiane Nüsslein-Volhard

Cell membrane

Nuclear membrane

Dorsal

Do the genes of the Spätzle/Toll/Dorsal cassette control the challenge-induced expression of diptericin ? wild type

Infection

-

6h

Tolldeficient -

6h P

Diptericin

P P P GG P GG GGG

G

P

G

G

Diptericin 1990

rp49

G

G G

G

G

P

P P P GG P GG GGG

G

P

G

G

G G

G

G

G

Diptericin G

G G

G G GG G

G

G

Fat body cells

Drosomycin 1994 P

P P

G G G

P

G G G

G

Metchnikowin

G

Attacin

Cecropin Drosocin

Defensin

P

The challenge-induced expression of the Drosomycin gene is dependent on the Toll pathway. wild type

Infection

-

6h

Tolldeficient -

6h

Diptericin Drosomycin Drosomycin 1994 rp49

Two distinct pathways control the expression of antimicrobial peptides wild type

Infection

-

6h

Tolldeficient Cactdeficient -

-

6h

imd -

6h

Diptericin Drosomycin rp49

Toll

Imd

pathways

Imd pathway mutants are sensitive to bacterial infections 100

of survival

80

60

wild type Imd mutants

%

40

20

0

1

2

3

Time (days)

4

5

6

E. coli infection

Toll pathway mutants are sensitive to fungal infections 100

% survival

80

60

Wild type Toll mutants

40

20

0

1

2

3

4

Time (days)

5

6

Aspergillus infection

Overwhelming fungal infection in a Toll deficient background

Lemaitre B, Nicolas E, Michaut L, Reichhart JM, Hoffmann JA. Cell, 1996, 20:973-83

NF-κB activation by Toll and IL-1

Leucine rich repeat domains

CD14

Leucine rich repeat domains

Toll

Ig-like domains

IL-1R

GPI anchor TIR domain

NF-κB

TIR domain

NF-κB

Cell membrane

Activation of NF-κB by TLR family members Mycoplasmal Lipopeptide

LPS

Bacterial Lipopeptide

Flagellin

MD-2 TLR 2 TLR 4

TLR 6 TLR 2

TLR 1

TLR 5

Endosome

Adaptor proteins (MyD88, TRIF, TIRAP,TRAM)

NF-κΒ and IRFs Antimicrobial Peptides etc

Activation of Adaptive Immune Responses

TLR 3 dsRNA

TLR 7 ssRNA CpG DNA

TLR 9

Fungi Receptors

Gram positive bacteria

Proteolytic cascade

Spaetzle

Toll-4 Toll-5 Toll-6 Toll-3 Toll-7 Toll-2 Toll-8 Toll-9

Toll

NF-κB

A mutation in the PGRPSA gene (semmelweis) compromises the antiGram-positive defense

Survival rate

100

A mutation in the PGRP-LC gene compromises the defense against Gramnegative bacteria

Infection by

Infection by

Streptococcus faecalis

Enterobacter cloacae 100

wt

wt 50

50

seml PGRP-LC12

24

Time (h)

36

12

24 36 Time (h)

Royet and coll. 2001, Royet, Ferrandon and coll., Anderson and coll., Ezekowitz and coll. 2002

48

A mutation in the gene encoding GNBP3 compromises resistance to Candida infections

Survival (%)

100 80

wt Loss of function mutant of GNBP3

60 40 20 1

2

Time in days

3

4

Microbial Inducers of Immune Responses and Cognate Receptors in Flies: Peptidoglycan Recognition Proteins and Glucan Binding Proteins

Peptidoglycan MurNAc

GlcNAc

MurNAc

β-(1,3) -Glucan

GlcNAc

Glc

Lys

Lys

DAP

Glc

DAP

Glc

Glc

GNBP PGRP Roussel and coll. Werner and coll.; Kim and coll.; Reiser and coll.; Chang and coll.

Microbial Fungi proteases (β-Glucan) PGRP-SA

GNBP-3

Persephone (serine protease)

Gram positive bacteria (LYS-PGN) Gram negative bacteria (DAP-PGN)

Cascade of serine proteases Spaetzle

PGRP-LC Toll

NF-κB Dif

Effector genes

Relish

Effector genes

DD

Imd

NF-κB activation by Toll in Drosophila Microbial sensors Fungi

Spz TOLL

G+ bacteria

Proteasome

Spz

MyD88 TIR DD

DORSAL /DIF

Drosomycin and hundreds of genes

P

PELLE KD Cactus

TUBE Receptor/ adaptor complex

NF-κB activation by IMD in Drosophila G- Bacteria Dredd

Imd DD

Ub

FADD NF-κB / RELISH

P

IKK Signalosome P IKKβ P

Tak1

Ub

Tab2 IKKγ

Ub

Jnk pathway Diptericin and hundreds of genes Cytoskeletal proteins, proapoptotic signaling

PGRP-LC

TLR4

Toll Spaetzle

LPS

IMD TNF-α

PGN

Toll

TLR4

PGRP

MyD88

MyD88

Imd

Pelle kinase

IRAK

IKK complex

NF-κB (Dorsal, DIF)

TNF-R

RIP

TAK1

NF-κB Cactus

TNF

NF-κB IκB NF-κB (p65/p65)

TAK1 JNK

IKK complex

TAK1 JNK

IKK complex

NF-κB (Relish)

NF-κB IκB

NF-κB (cleaved Relish)

NF-κB

JNK

Phylogeny of Innate Immune Defenses AMP AMP NF-κB NF-κB TAK1 TAK1 TOLL TOLL Sponges Sea anemones (Porifera) (Cnidaria)

Radial diploblastic

AMP AMP AMP NF-κB NF-κB NF-κB TAK1 TAK1 TAK1 TOLL TOLL TOLL Insects Echinoderms Hemichordates Worms Molluscs

AMP NF-κB TAK1 TOLL Chordates

~ 450 million years

Protostomes Deuterostomes Bilateral triploblastic

Cambrian, ~ 550 million years

Precambrian, ~ 600 million years Precambrian, ~ 800 million years Multicellularity origin ~1 billion years

Acknowledgements D. Hoffmann

C. Hetru

JL. Dimarcq

J.M. Reichhart

B. Lemaitre

D. Ferrandon

J. Royet

J.L. Imler

E. Levashina

M. Lagueux

P. Bulet

USA,

Credits : Drosophila immunity

Kathryn Anderson Carl Hashimoto Steve Wasserman Tony Ip

Europe,

Ruth Stewart Shuba Govind

Hans Boman† Hakan Steiner Dan Hultmark Ingrid Faye Ylva Engström Ulli Theopold

Neal Silverman Tom Maniatis Alan Ezekowitz Nathalie Franc Linda Stuart Christine Kocks Norbert Perrimon Herve Agaisse Michael Boutros David Schneider

Bruno Lemaitre François Leulier Julien Royet Mika Ramet Nick Gay

Asia, Shoichiro Kurata Won-Jae Lee Young-Joon Kim

Acknowledgements D. Hoffmann

C. Hetru

B. Lemaitre JL. Dimarcq

J.M. Reichhart

D. Ferrandon

M. Meister

J.L. Imler

E. Levashina

M. Lagueux

J. Royet

P. Bulet

The sea anemone Nematostella

Nematostella Toll MyD88 TAK1

TLR TIR

TRAF6

TIR

MyD88

DD

IKK

Tak1

IKK

NLS RHD

G rich

Ank repeats

NF-κB IκB

NF-κB IκB NF-κB

Microbial ligands Cytokines

Hypothetical receptor

CD28

NF-κB

B.7 TCR

NF-κB

Denditric cell (Antigen Presenting Cells…)

MHC

Peptide

Naive T Cell

Activation of adaptive immunity by innate immunity