innate immunity
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
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Innate immunity
Toll signaling and related topics
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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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QuickTime™ and a decompressor
are needed to see this picture.
Gastrulation in Drosophila
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Dorsal group
nudelpipe
eastersnake
windbeutel
spaetzle
Toll pelle tube cactus dorsal
Gastrulation defective
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Injection into the perivitelline space to monitor
“polarizing activity”
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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
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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
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Dorsal group
nudelpipe
eastersnake
windbeutel
spaetzle
Toll pelle tube cactus dorsal
Gastrulation defective
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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”.
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Toll protein structure
Intracellular domain
Extracellulardomain
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Some Interesting Toll Mutants
C Y
Dominantactivated
Dominant Negative
DominantActivated - but Requires wt allele and ligand
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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.
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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.
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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
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Identification of imd
• While testing a mutation called black cell, Lemaitre found a closely associated mutant which made flies sensitive to bacterial infections.
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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
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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
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Second generation screen
• Survival of a bacterial infection• Found mutations in Dredd - a caspase and dTAK1
an Map kkk
• Enhancing an immune phenotype
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Particles are taken up by hemocytes
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Phagocytosis Assay
Cells PhagocytoseParticles Trypan Blue QuenchesExtracellular Fluorescence
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3 minutes post E.coli injectionNo trypan blue
3 minutes post E. coli injectionPlus trypan blue
30 minutes post E.coli injectionPlus trypan blue
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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
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Beads
Bacteria
Bacteria and beads
Day post infection
Wild type survival curve
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020406080100immune defective (imd) survival curve
Beads
Bacteria
Bacteria and beads
Day post infection
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BacteriaBacteria DeathDeath
Humoral immunityHumoral immunity
Cellular immunityCellular immunity
BeadBeadTreatmentTreatment
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BacteriaBacteria DeathDeath
Humoral immunityHumoral immunity
Cellular immunityCellular immunity
imd
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Main points
• Demonstrate a number of forward genetic approaches to identifying genes involved in immunity.
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Recent findings upstream of Toll
• Seml: Semmelweiss• Sensitivity to bacterial infection• Blocks drosomycin expression from gram
positive bacteria but not fungi.
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Pathway upstream of Toll in flies
Fungi Gram positive bacteria
Semmelweiss
Spaetzle
Toll
Protease “X”Necrotic(serpin)
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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.
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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
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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.
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spaetzleproteaseTollpelletubedorsal cactus
LPSToll
NFkBikB
MyD88IRAKTRAF 6NIKIKK
Fly Human
Tak1JNKKJNK
AP1
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Tons of Tolls
• 9 in the fly• 10 in humans• Many in plants
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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.
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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.
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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.
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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.
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• 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.