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Innate immunity
Toll signaling and related topics
Antimicrobial peptides
• Insects produce antimicrobial peptides in
response to infections
• The peptides can be: 1 Secreted into the
circulation, 2. Produced by barrier epithelia
and 3. Produced by blood cells.
• These processes are regulated by rel related
signaling events.
Gastrulation in Drosophila
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windbeutel
pipe
Dorsal group
nudel
Gastrulation defective
snake
easter
spaetzle
Toll
pelle
tube
cactus
dorsal
Injection into the perivitelline
space to monitor
“polarizing activity”
Polarizing activity is processed
spaetzle
• Polarizing activity is found in pip, ea, and
Toll mutants but not spz.
• Anti-spz antibodies recognize a protein that
co-purifies with polarizing activity
• Acid boiling reduces the size of spz
mimicking a presumed natural proteolytic
process
Ordering genes in the Toll
pathway
spaetzle -
-
W.T.
easter -
TlD
V
V
V
D
D
eaD
V
V
D
D
D
cac
V
V
V
V
D
pelle
dorsal
-
windbeutel
pipe
Dorsal group
nudel
Gastrulation defective
snake
easter
spaetzle
Toll
pelle
tube
cactus
dorsal
Is spaeztle the Toll ligand?
• There is no physical evidence for such an
association.
• Many have tried to demonstrate such an
association.
• Beware of those who call spaetzle “the Toll
ligand”.
Toll protein structure
Extracellular
domain
Intracellular
domain
Some Interesting Toll Mutants
C
Y
Dominant
activated
Dominant
Activated - but
Requires wt allele
and ligand
Dominant
Negative
Main points
• These genes were identified in studies
involving the maternal contribution to
dorsal-ventral pattern formation.
• The pathway was ordered almost entirely by
genetic techniques.
• EMS mutagenesis can give you very
important tools.
We should have known Toll was
involved in immunity
• Toll mutants form melanotic tumors.
• Tissue is encapsulated by Drosophila blood
cells - just like parasitic wasp eggs.
• People didn’t make the connection.
Antimicrobial peptides have kB
binding sites and dorsal has a
homolog
• Peptide chemists were studying insect
antimicrobial peptides and their expression.
• Hans Boman. Purified from Cecropia moth
pupae.
• Ylva Engstrom noticed the enhancers.
• Tony Ip found a dorsal homolog that wasn’t
involved in d/v patterning.
• No functional data from these experiments
Identification of imd
• While testing a mutation called black cell,
Lemaitre found a closely associated mutant
which made flies sensitive to bacterial
infections.
Testing other known mutants
• Look at dorsal group genes.
• Induction of peptide genes by mixed gram
+ and - bacterial infections.
• Toll affects ability to fight a fungal
infection.
• Toll does not affect gram negative bacterial
infections
Forward genetic screens
• Louisa Wu and Kathryn Anderson
• Use diptericin-LacZ promoter
• Look for larvae that did not turn on the gene
when infected
• Found, Dif, ikk beta, modulo.
• Some genes affect signaling
• Some genes affect development of immune
organs
Second generation screen
• Survival of a bacterial infection
• Found mutations in Dredd - a caspase and dTAK1
an Map kkk
• Enhancing an immune phenotype
Particles are taken up by hemocytes
Phagocytosis Assay
Cells Phagocytose
Particles
Trypan Blue Quenches
Extracellular Fluorescence
3 minutes post E.coli injection
No trypan blue
3 minutes post E. coli injection
Plus trypan blue
30 minutes post E.coli injection
Plus trypan blue
3 minutes post E.coli injection
No trypan blue
3 minutes post E. coli injection
Plus trypan blue
30 minutes post E.coli injection
Plus trypan blue
30 minutes post E.coli injection
Plus trypan blue
Pre-injected with plastic beads
Wild type survival curve
Beads
Bacteria
Bacteria and beads
Day post infection
Beads
Bacteria
Bacteria and beads
Day post infection
Humoral immunity
Bacteria
Bead
Treatment
Death
Cellular immunity
Humoral immunity
imd
Bacteria
Death
Cellular immunity
Main points
• Demonstrate a number of forward genetic
approaches to identifying genes involved in
immunity.
Recent findings upstream of Toll
• Seml: Semmelweiss
• Sensitivity to bacterial infection
• Blocks drosomycin expression from gram
positive bacteria but not fungi.
Pathway upstream of Toll in flies
Fungi
Gram positive bacteria
Semmelweiss
Necrotic
(serpin)
Protease “X”
Spaetzle
Toll
Redundancy: A genetic point
• Easter, snake and gastrulation mutants
respond to infections.
• Does this mean the genes are not required
for the immune response?
• It means you are not necessarily testing the
appropriate conditions.
Pattern recognition receptors were defined by Janeway
as genome-encoded non-clonally distributed receptors
that recognize certain molecular patterns found in
microbes but not on self tissues. The best documented
examples are the various Toll-like receptors present on
mammalian immune responsive cells,which bind distinct
microbial patterns to activate NF-kB.
Nature 414, 756-758
There is no physical evidence
that Toll binds a ligand
• Has never been shown in the fly.
• Papers quoted as demonstrating this in
vertebrate cells merely show that receptor is
required for signaling.
• Don’t believe the simple models yet.
Tons of Tolls
• 9 in the fly
• 10 in humans
• Many in plants
Toll acts as a bridge to the
adaptive immune response
• Medzitov and Janeway created a dominant
allele of Toll in Jurkat cells.
• Found it induced the production of
cytokines.
• Suggest that this is the bridge between
innate and adaptive immunity.
Vaccines require an adjuvant
• Must inject an irritant along with the
antigen.
• Explanation is that this informs the body a
pathogenic event is occurring.
• Only under these conditions will the
adaptive immune response turn on.
Danger hypothesis - Matzinger
• Immune response is stimulated by “danger”
• Immune system is responding to signs of
pathogenesis - release of intracellular
molecules.
• Suggest that bacteria are not being
recognized by host rather they are revealing
themselves to the host.
Both extremes are ridiculous
• We can learn from both models.
• Pattern recognition functionally appears to
inform the adaptive immune response that a
pathogenic event is occurring.
• Pattern recognition receptors do recognize
damage to the body causing the release of
intracellular components.
• Most bacteria are not pathogenic.
• In general, these bacteria have been
interacting with innate, not adaptive
immune systems over the course of history.
• Answers may lie in how our bodies deal
with commensals not how they deal with
pathogens.