The Antimicrobial Defense of Drosophila, A Paradigm for Innate Immunity

Jules Hoffmann, Strasbourg, France Viruses Bacteria

Protozoa

Fungi

Jos Pierre Hoffmann Joly 1911-2000 1913-1996 Antimicrobial Defenses in I nsects : First Investigations

Metchnikoff Paillot

Phagocytosis Antimicrobial substances in the blood « Cellular Immunity » Metchnikoff, 1880 « Humoral Immunity» Paillot 1920-1935 Glaser

Induction of an antimicrobial activity in Drosophila by an immune challenge

1 Hyalophora Injection cecropia of bacteria Antimicrobial activity in the 2 cell-free Hans Boman hemolymph 1924-2008

3 Control

0 3 6 9 12 24 48 Time (h) Systemi c (“ humoral”) antimicrobial response in Drosophila – identification of antimicrobial peptides

P G G P P G P P G G G P GG G GG G G G G Diptericin

G G G Fat body G G G G G G G cells P P G P P P G G G G G

G G Metchnikowin Attacin

Cecropin Defensin Drosocin NF-κB response elements in the promoter of the diptericin gene

-1 kb -150 -140 -62 -31 coding sequence of enhancer the diptericin gene κB-Response element

1 NLS 678 REL 1 Ank 482 Stewart 1987 DORSAL

CACTUS

Diptericin-LacZ reporter gene

Unchallenged Challenged Versailles, 18 years ago, Innate Immunity Conference

Michael Zasloff Klas Kärre Alan Ezekowitz Jean-Marc Reichhart Danièle Hoffmann Bob Lehrer Hans Boman Dan Hultmark Shunji Natori Charlie Janeway Charles Hetru Ingrid Faye Gene cascade controlling the dorso-ventral axis in the Drosophila embryo

CHORION Christiane Easter PERIVITELLIN Nüsslein-Vol hard SPACE Spätzle Snake EMBRYO

Gastrulation Defective Tube Cactus Dorsal Windbeutel and Toll Pelle Pipe Nudel

Cell FOLLICLE CELLS Nuclear membrane membrane Do the genes of the Spätzle/Toll/Dorsal cassette control the challenge-induced expression of diptericin ?

wild type Toll deficient

Infection - 6h - 6h P G G P P G P P G G G P GG G Diptericin GG G G G G Diptericin 1990

rp49 P G G P P G P P G G G P GG G GG G G G G Diptericin

G G G Fat body G Drosomycin 1994 G G G G G G cells P P G P P P G G G G G

G G Metchnikowin Attacin

Cecropin Defensin Drosocin The challenge-induced expression of the Drosomycin gene is dependent on the Toll pathway.

wild type Toll deficient

Infection - 6h - 6h Diptericin

Drosomycin Drosomycin 1994 rp49 Two distinct pathways control the expression of antimicrobial peptides

deficient wild type Toll Cactdeficient imd

Infection - 6h - 6h - - 6h Diptericin

Drosomycin

rp49 Tol l Imd pathways Imd pathway mutants are sensitive to bacterial infections

100

80

60 wild type Imd mutants of survival of

40 %

20

0 1 2 3 4 5 6 Time (days) E. coli infection Toll pathway mutants are sensitive to fungal infections

100

80

60 survival

40 Wild type % Toll mutants

20

0 1 2 3 4 5 6 Time (days) 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 Leucine Ig-like rich CD14 rich Toll domains IL-1R repeat repeat domains domains

GPI anchor Cell membrane TIR TIR domain domain

NF-κB NF-κB Activation of NF-κB by TLR family members

Mycoplasmal Bacterial LPS Lipopeptide Lipopeptide Flagellin

MD-2 TLR 2 TLR 6 TLR 4 TLR 2 TLR 1 TLR 5

Endosome Adaptor proteins TLR 3 ( M yD 88, TR I F, TI R AP, TR AM ) dsRNA

TLR 7 NF-κΒ and IRFs ssRNA Antimicrobial Activation of Adaptive CpG DNA Peptides TLR 9 etc I mmune Responses Fungi Gram positive Receptors bacteria Proteolytic cascade

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

Toll

NF-κB A mutation in the PGRP- A mutation in the PGRP-LC SA gene (semmelweis) gene compromises the compromises the anti- defense against Gram- Gram-positive defense negative bacteria

Infection by Infection by Streptococcus faecalis Enterobacter cloacae 100 100

wt wt

50 50 Survival rate seml PGRP-LC-

12 24 36 12 24 36 48 Ti me ( h) Ti me (h) Royet and coll. 2001, R oyet, Ferrandon and coll., Anderson and coll., Ezekowitz and coll. 2002 A mutation in the gene encoding GNBP3 compromises resistance to Candida infections

100

80

60 wt Loss of function Survival (%) 40 mutant 20 of GNBP3

1 2 3 4 Time in days Microbial I nducers of Immune Responses and Cognate Receptors in Flies: Peptidoglycan Recognition Proteins and Glucan Binding Proteins Peptidoglycan MurNAc GlcNAc MurNAc GlcNAc β- (1,3) -Glucan

Glc Glc Glc Glc Lys Lys DAP DAP

GNBP PGRP

Roussel and coll. Werner and coll.; Kim and coll.; Reiser and coll.; Chang and coll. Microbial Fungi Gram positive proteases (β-Glucan) bacteria PGRP-SA GNBP-3 (LYS-PGN) Gram negative bacteria Persephone (DAP-PGN) (serine protease) Cascade of serine proteases

Spaetzle

PGRP-LC Toll NF-κB

Dif Relish DD Imd

Effector genes Effector genes NF-κB activation by Toll in Drosophila

Microbial sensors Spz Spz TO L L Fungi G+ bacteria Proteasome MyD88 TIR

DD TUBE P PELLE DORSAL Cactus KD Receptor/ /DIF adaptor complex

Drosomycin and hundreds of genes NF-κB activation by IMD in Drosophila

G- Bacteria

Dredd Imd PGRP-LC

DD Ub FADD NF-κB / RELISH IKK Tak1 P Ub Signalosome P IKKβ P Tab2 Ub IKKγ

Jnk pathway Diptericin and hundreds of genes Cytoskeletal proteins, proapoptotic signaling Toll TLR 4 IMD TNF Spaetzle LPS PGN TNF-α To l l TLR 4 PGRP TNF-R

MyD88 MyD88 Imd RIP Pelle kinase IRAK TA K 1 TA K 1 TA K 1 JNK JNK JNK IKK IKK IKK complex complex complex

NF-κB Cactus NF-κB IκB NF-κB (Relish) NF-κB IκB

NF-κB NF-κB NF-κB (Dorsal, DIF) (p65/p65) NF-κB (cleaved Relish) Phylogeny of I nnate I mmune Defenses AMP AMP AMP AMP AMP AMP NF-κB NF-κB NF-κB NF-κB NF-κB NF-κB TA K 1 TA K 1 TA K 1 TA K 1 TA K 1 TA K 1 TO LL TO LL TO LL TO LL TO LL TO LL Sponges Sea anemones I nsects Echinoderms Hemichordates Chordates (Porifera) (Cnidaria) Worms Molluscs ~ 450 million years Protostomes Radial diploblastic Deuterostomes Cambrian, ~ 550 million years Bilateral triploblastic Precambrian, ~ 600 million years

Precambrian, ~ 800 million years

Multicellularity origin ~1 billion years Acknowledgements

D. Hoffmann C. Hetru JL. Dimarcq

B. Lemaitre J.M. Reichhart 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 Hans Boman† Shuba Govind Hakan Steiner

Neal Silverman Dan Hultmark Asia, Tom Maniatis Ingrid Faye Ylva Engström Shoichiro Kurata Alan Ezekowitz Ulli Theopold Won-Jae Lee Nathalie Franc Linda Stuart Bruno Lemaitre Young-Joon Kim Christine Kocks François Leulier Julien Royet Norbert Perrimon Herve Agaisse Mika Ramet Michael Boutros Nick Gay

David Schneider

Acknow- ledge- ments

D. Hoffmann C. Hetru B. Lemaitre JL. Dimarcq

M. Meister J.M. Reichhart D. Ferrandon J. Royet

J.L. Imler E. Levashina M. Lagueux P. Bulet Nematostella The sea anemone Nematostella

To l l

MyD88

TA K 1 TLR

TIR TR AF6 IKK TIR Tak1

MyD88 DD IKK NLS RHD G rich NF-κB NF- B I B IκB κ κ Ank repeats

NF-κB

Microbial ligands

Cytokines

Hypothetical receptor

CD28 NF-κB B.7

TCR

MHC Peptide NF-κB Nai ve T Cell Denditric cell (Antigen Activation of adaptive Presenting immunity by innate Cells…) immunity