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.

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Gastrulation in Drosophila

Dorsal group

nudel

pipe

easter

snake

windbeutel

spaetzle

Toll pelle tube cactus dorsal

Gastrulation defective

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

TlDeaster spaetzle pelle dorsal

V V D D

eaD V D D D

cac V V V D

- ---W.T.

V

V

V

Dorsal group

nudel

pipe

easter

snake

windbeutel

spaetzle

Toll pelle tube cactus dorsal

Gastrulation defective

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

Intracellular domain

Extracellulardomain

Some Interesting Toll Mutants

C Y

Dominantactivated

Dominant Negative

DominantActivated - but Requires wt allele and ligand

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 PhagocytoseParticles Trypan Blue QuenchesExtracellular Fluorescence

3 minutes post E.coli injectionNo trypan blue

3 minutes post E. coli injectionPlus trypan blue

30 minutes post E.coli injectionPlus trypan blue

3 minutes post E.coli injectionNo trypan blue

3 minutes post E. coli injectionPlus trypan blue

30 minutes post E.coli injectionPlus trypan blue

30 minutes post E.coli injectionPlus trypan bluePre-injected with plastic beads

Beads

Bacteria

Bacteria and beads

Day post infection

Wild type survival curve

020406080100immune defective (imd) survival curve

Beads

Bacteria

Bacteria and beads

Day post infection

BacteriaBacteria DeathDeath

Humoral immunityHumoral immunity

Cellular immunityCellular immunity

BeadBeadTreatmentTreatment

BacteriaBacteria DeathDeath

Humoral immunityHumoral immunity

Cellular immunityCellular immunity

imd

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

Spaetzle

Toll

Protease “X”Necrotic(serpin)

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 Janewayas genome-encoded non-clonally distributed receptorsthat recognize certain molecular patterns found in microbes but not on self tissues. The best documentedexamples are the various Toll-like receptors present on mammalian immune responsive cells,which bind distinctmicrobial 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.

spaetzleproteaseTollpelletubedorsal cactus

LPSToll

NFkBikB

MyD88IRAKTRAF 6NIKIKK

Fly Human

Tak1JNKKJNK

AP1

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.