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Insect Immunity & Drosophila Gut Immune Response

Insect Immunity & Drosophila Gut Immune Response

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Bruno Lemaitre<br />

Global Health Institute EPFL


<strong>Insect</strong> Characteristics & Peculiarities<br />

One of the most diverse animal group (> 70% of animal species)<br />

Include *many vectors of human diseases (mosquito, tse-tse flies…)<br />

*many damaging crop species<br />

* many beneficial microbes (bee, silkworm….)<br />

Many adaptive traits, thriving on varied ecological habitats and diets<br />

Animal feeding Parasitoid Plant feeding<br />

=>Strong capacity to deal with microbes


The enemies of insects<br />

• Viruses: Baculovirus<br />

• Bacteria: Bacillus thuringiensis<br />

• Fungi : Beauveria bassiana<br />

• Parasites : Plasmodium falciparum<br />

• Other insects: parasitoid wasps


Baculovirus infection of an insect host<br />

-circular, double stranded DNA<br />

- 80 -180 kbp<br />

- mostly restricted to invertebrates<br />

- used in biotechnology<br />

- biopesticide


Bacillus thuringiensis<br />

- Gram-positive, soil-bacterium,<br />

- express a pore forming toxin : the<br />

Cry toxin<br />

- used as Biopesticide<br />

- used in transgenic crop (Bt Maize)<br />

Crystal<br />

Spore


Beauveria Bassiana: an entomopathogenic fungus<br />

- produces lipase, chitinase<br />

and proteases<br />

- crosses through the cuticle<br />

of insects<br />

- used in field to protect<br />

crop from insects


Plasmodium development in Anopheles<br />

Derived from Riehle et al, 2003<br />

48 hours : Initiation of<br />

Oocysts development<br />

14 hours : ookinetes<br />

formation<br />

24 hours :<br />

Ookinetes penetration


Strategies to block malaria transmission<br />

- Mosquito Nets<br />

- Reduce standing water<br />

- <strong>Insect</strong>icide<br />

- Transgenic mosquitoes<br />

Shin et al, J. Exp. Biol., 2003


A wasp parasitizing of <strong>Drosophila</strong><br />

larvae<br />

Parasitoids wasps<br />

A wasp parasitizing a moth caterpillar


II - How insects (<strong>Drosophila</strong>)<br />

defend themselves<br />

• Antiviral defense<br />

• Physical and Chemical barriers<br />

– Reactive oxygen species<br />

• Cellular <strong>Immunity</strong><br />

– Phagocytosis<br />

– Capsule formation<br />

– Hemolymph coagulation and nodule formation<br />

• Humoral <strong>Immunity</strong><br />

– Melanization<br />

– Antimicrobial peptides


<strong>Drosophila</strong> antiviral defense<br />

1) RNA interference<br />

2) Endosymbiont-mediated immunity<br />

- Wolbachia are common intracellular bacteria of arthropods<br />

Purified particles of DCV<br />

- Wolbachia are transmitted vertically through host eggs, and manipulate host<br />

reproduction to increase transmission to the next generation.<br />

=> New studies suggest that Wolbachia infections in <strong>Drosophila</strong> can<br />

confer resistance to viruses and therefore act as mutualists


I - How insects defend themselves<br />

• Antiviral defense<br />

• Physical and Chemical barriers<br />

– Reactive oxygen species<br />

• Cellular <strong>Immunity</strong><br />

– Phagocytosis<br />

– Capsule formation<br />

– Hemolymph coagulation and nodule formation<br />

• Humoral <strong>Immunity</strong><br />

– Melanization<br />

– Antimicrobial peptides


Physical and chemical<br />

barriers<br />

Chitin-containing membranes<br />

- Cuticle<br />

- Peritrophic matrix<br />

In the gut:<br />

- Acidity<br />

- Digestive enzymes and lysozymes<br />

- Production of Reactive Oxygen<br />

Species (ROS) by DUOX<br />

PM:peritrophic matrix<br />

Mv: microvilli B: bacteria


I - How insects defend themselves<br />

• Antiviral defense<br />

• Physical and Chemical barriers<br />

– Reactive oxygen species<br />

• Cellular <strong>Immunity</strong><br />

– Phagocytosis<br />

– Capsule formation<br />

– Hemolymph coagulation and nodule formation<br />

• Humoral <strong>Immunity</strong><br />

– Melanization<br />

– Antimicrobial peptides


Cellular immunity<br />

Cell type<br />

plasmatocyte<br />

lamellocyte<br />

Bacterial infection<br />

Parasitoid Egg<br />

Function<br />

Phagocytosis<br />

Encapsulation


I - How insects defend themselves<br />

• Antiviral defense<br />

• Physical and Chemical barriers<br />

– Reactive oxygen species<br />

• Cellular <strong>Immunity</strong><br />

– Phagocytosis<br />

– Capsule formation<br />

– Hemolymph coagulation and nodule formation<br />

• Humoral <strong>Immunity</strong><br />

– Melanization<br />

– Antimicrobial peptides


Activation of melanization<br />

Melanization:<br />

activation of a<br />

proteolytic<br />

cascade


The <strong>Drosophila</strong> immune system: conclusions<br />

• <strong>Drosophila</strong> is now one of the best-characterized host defense systems<br />

among the metazoans.<br />

• <strong>Drosophila</strong> has multiple defense “modules” that can be deployed in a<br />

coordinated response against distinct pathogens => activation of the<br />

appropriate response<br />

• There are similarities between <strong>Drosophila</strong> host defense and essential<br />

facets of vertebrate innate immunity (signalling pathways)<br />

Future studies should investigate the links between <strong>Drosophila</strong><br />

immune defense and the ecology of this insect


The <strong>Gut</strong><br />

- Site of digestion<br />

- Major entry site of pathogens<br />

- Exposed to commensals<br />

(yeast, bacterial flora)<br />

Epithelial<br />

immunity<br />

Innate <strong>Immunity</strong><br />

Yersinia (Plague)<br />

Rickettsia (Typhus)<br />

Plasmodium (Malaria)<br />

Bacterial<br />

tolerance


- A Gram-negative, phytopathogenic bacteria<br />

- A natural <strong>Drosophila</strong> pathogen<br />

- Induces an Imd-dependent gut immune response upon oral<br />

infection<br />

Natural infection<br />

Filter paper<br />

+ sucrose
<br />

+ bacteria<br />

Wild-type<br />

Wild-type<br />

Imd mutant<br />

Control<br />

Ecc15<br />

Ecc15<br />

Diptericin-lacZ


Wild-type<br />

E. coli<br />

DAP-PGN<br />

LYS- PGN<br />

PGRP<br />

-LC E12<br />

E. coli<br />

Diptericin-lacZ<br />

No role for the Toll pathway in the gut<br />

Imd<br />

Relish<br />

κB κB κB<br />

DAP PGN<br />

AMPs<br />

PGRP-LB<br />

PGRP-LC


PGRP-LB is en enzymatic PGRP regulated<br />

by the Imd pathway<br />

PGRP-LB<br />

<strong>Immune</strong><br />

non-reactive<br />

fragments<br />

Imd<br />

Relish<br />

κB κB κB<br />

DAP PGN<br />

AMPs<br />

PGRP-LB<br />

PGRP-LB


PGRP-LB<br />

Western Blot<br />

(gut extracts)<br />

wild-type<br />

C NI NI<br />

Relish E20<br />

Larval guts<br />

(fed with Ecc15)<br />

Wild-type PGRP-LB RNAi<br />

Dpt-LacZ Dpt-LacZ Dpt-GFP


Absence of infection Severe infection<br />

bacteria<br />

Imd<br />

pathway<br />

RELISH<br />

Imd<br />

Imd<br />

Imd<br />

pathway<br />

RELISH<br />

pathway pathway<br />

RELISH RELISH


The Imd pathway is activated throughout the gut,<br />

BUT<br />

Diptericin shows patterned expression<br />

Diptericin-lacZ<br />

?<br />

PGRP LC<br />

Imd pathway<br />

κB κB κB<br />

Control<br />

Caudal<br />

Diptericin<br />

Caudal RNAi<br />

Ryu et al (2008) Science 319<br />

⇒ additional regulatory elements<br />

restrict Diptericin expression<br />

in some gut cells.


PGRP-LC<br />

Wild-Type<br />

Imd<br />

Relish<br />

κB κB κB<br />

Caudal<br />

AMP Genes<br />

Ryu et al Science 2008<br />

PGRP-LC<br />

Caudal knock down<br />

Imd<br />

Relish<br />

κB κB κB<br />

AMP Genes


Tolerance to gut indigenous flora<br />

• Compartmentation: restriction of antimicrobial<br />

peptide production in the hindgut to protect the<br />

bacterial flora<br />

• Negative feed-back: Tight regulation of the Imd<br />

pathway to prevent its activation by indigenous<br />

bacteria


Both invasive and indigenous bacteria<br />

stimulate epithelium renewal<br />

Nicolas Buchon<br />

Nichole Broderick<br />

Buchon et al., Cell Host & Microbe 2009<br />

Buchon et al., Genes and Dev 2009


Major Remodeling of the <strong>Gut</strong> upon gut infection is<br />

required for barrier integrity


lumen<br />

Ohlstein et al. 2006, Nature<br />

Micchelli et al. 2006, Nature<br />

Stem cell<br />

Epithelial cells


Mitosis marker staining : phospho histone H3<br />

An#‐PH3
<br />

staining
<br />

An/
PH3
<br />

DAPI
<br />

Unchallenged
 Oral
infec/on
with
Ecc15
<br />

UC
<br />

An/
PH3
<br />

An#
PH3
<br />

E1
 E2
<br />

Number of PH3<br />

Positive cells / midgut<br />

UC
 Ecc15

inf
<br />

lumen



Duox
<br />

1
<br />

Imd
<br />

H2O2 /O ‐<br />

2 
<br />

AMPs
<br />

1:

Recogni/on
of
bacteria
&
immune
response
<br />

2:
Bacteria
destruc/on
&
damage
to
gut
cells
<br />

3:
Cell
repair
&
epithelium
renewal
<br />


What
causes
damage
and
epithelium
renewal
?
<br />


What
are
the
pathways
?
<br />


What
is
the
relevance
upon
infec/on
?
<br />


What
happens
with
different
types
of
bacteria
?
<br />

?<br />

2
<br />

?<br />

?<br />

3
<br />

Buchon et al., 2009, Cell Host & Microbe


Epithelium stress & damage<br />

Bacterial direct stress “Collateral damage”<br />

Oxidative<br />

Stress ???


WT
<br />

Unchallenged
<br />

WT+Glutathione
<br />

WT
+
Paraquat
<br />

Proliferation : clones of lacZ positive cells Number of mitotic cells<br />

Ecc15
<br />

PH3
posi#ve
cells
<br />

WT
 WT
 dUOX‐
IR
 UAS‐IRC
 WT
 W<br />

UC
 Paraqua<br />

Ecc15 



WT<br />

JAK-STAT<br />

deficient<br />

Unchallenged Ecc15 infection<br />

esg‐gal4;
UAS‐GFP
<br />

esg‐gal4,
UAS‐GFP;
uas‐Socs36E



upd3-Gal4 UAS-GFP


Intestinal<br />

muscles<br />

Intestinal<br />

epithelium<br />

<strong>Gut</strong> lumen<br />

Regula/on
of
ep/helium
renewal
upon
Ecc15
infec/on
<br />

Upd3<br />

DUOX







<br />

Oxida/ve
burst
<br />

JAK/STAT
?
<br />

Vein<br />

JAK/STAT
 EGFR/MAPK



Percentage of survival<br />

100<br />

80<br />

60<br />

40<br />

20<br />

0<br />

<strong>Immune</strong> response<br />

0 2 4 6 8 10<br />

Days post treatment<br />

Ecc15<br />

esg-gal4 uas Socs36E / DAPI<br />

WT<br />

UAS-dFADD Relish IR<br />

UAS-upd3 IR<br />

UAS UAS-STAT-IR Dome(dom<br />

neg)<br />

In enterocytes<br />

In ISCs<br />

Epithelium renewal


Pe-GFP<br />

Control P. entomophila


Control<br />

Ecc15<br />

P. entomophila<br />

Control<br />

P.entomophila<br />

Lack of gut integrity<br />

death<br />

DAPI


Two important host defense mechanisms:<br />

- ROS production by DUOX<br />

- Antimicrobial peptide production by the Imd pathway<br />

Negative feedback on the Imd pathway and compartimentation of Imd<br />

pathway activation are important for tolerance and the establishment of<br />

a beneficial flora<br />

A successful response to infection requires coordination of BOTH immune<br />

and homeostatic (repair) pathways<br />

Commensal bacteria stimulate epithelium renewal at a basal level<br />

Epithelium renewal as a target for gut pathogens


A Bacterial Effector Targets Mad2L2, an APC<br />

Inhibitor, to Modulate Host Cell Cycling<br />

Hiroki Iwai et al. 2007 Cell, Vol 130, 611-623, 2007<br />

Summary<br />

…Here, we show that the Shigella effector IpaB,<br />

when delivered into epithelial cells, causes cellcycle<br />

arrest by targeting Mad2L2, an anaphasepromoting<br />

complex/cyclosome (APC) inhibitor. ..<br />

These results strongly indicate that Shigella<br />

employ special tactics to influence epithelial<br />

renewal in order to promote bacterial<br />

colonization of intestinal epithelium<br />

microvilli<br />

Tight Jonction<br />

crypt<br />

α-defensin<br />

Paneth Cells<br />

Lymphocyte<br />

intra-épithélial<br />

Stem Cell<br />

Richard Locksley et al, <strong>Immunity</strong>.1er édition, p57.)


Collaborators<br />

GHI,
EPFL,
Lausanne
(CH)
<br />

Nicolas
Buchon
<br />

Nichole
Broderick
<br />

Takayuki
Kuraishi
<br />

Onya
Opota
<br />

David
Welchman
<br />

Claudine
Neyen
<br />

Sveta
Chakrabar/
<br />

Mathilde
Gendrin
<br />

Jeremy
Herren
<br />

Olivier
Binggeli
<br />

Jean
Philippe
Boquete
<br />

Allison
Bardin
(Ins/tut
Pasteur,
France)
<br />

Dominique
Mengin‐Lecreulx
(Orsay,
France)
<br />

Sylvain
Praverdan
(CIG,
Lausanne)
<br />

CGM,
Gif‐sur‐Yveae
(F)
<br />

Anna
Zaidman‐Rémy
<br />

Mickael
Poidevin



- A successful response to infection requires coordination of BOTH<br />

immune and homeostatic pathways<br />

- Commensal bacteria stimulate epithelium renewal at a basal level<br />

- Epithelium renewal as a target for gut pathogens<br />

Buchon et al.; Genes and Dev 2009


Imd deficient flies have a higher<br />

bacterial load AND an altered flora<br />

composition<br />

PH3
posi/ve
cells
<br />

**<br />

***<br />

This higher bacterial load leads to<br />

increased epithelium renewal


<strong>Gut</strong>s of 30-day-old Relish E20 flies displayed altered gut<br />

morphology that are not observed in axenic conditions.<br />

Relish E20 ,30 days<br />

Conventionally raised Axenic


The EGFR pathway coordinates <strong>Drosophila</strong> stem cell<br />

proliferation and gut remodeling in response to infection


EC<br />

PM<br />

B<br />

Crop<br />

midgut<br />

cardia<br />

DAPI<br />

Pe rfp<br />

Hml-gal4, UAS-GFP<br />

Malpighian<br />

tubules<br />

rectum<br />

Humans <strong>Drosophila</strong><br />

Physical barriers Mucus Peritrophic matrix<br />

Chemicals, enzymatic barriers Low pH, proteases, lysozymes<br />

Flora<br />

Complex (>500 sp)<br />

Dense (10 12 /ml)<br />

<strong>Immune</strong> Defense AMPs, GALT, M cells<br />

Simple (30sp)<br />

Rare<br />

ROS, AMPs,<br />

<strong>Gut</strong>-associated Hemocytes


Esg‐GFP
:
<br />

Stem
cells,
<br />

enteroblasts
<br />

newly
<br />

generated
<br />

enterocytes
<br />

Micchelli
et
al.2006
<br />

Mosaic
<br />

analysis
<br />

lacZ+
clones
<br />

Harrison
and
Perrimon
1993
<br />

Unchallenged
 Oral
infec/on
with
Ecc15


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