Lecture 25 - Evolutionary Immunology - Innate Flashcards
1
Q
Compare the organisms in which innate and adaptive immunity are found
A
Innate:
- Lower and higher organisms
- Lower: invertebrates
- Higher: vertebrates
Adaptive:
- Higher organisms (only)
2
Q
When did adaptive immunity first appear?
In which organisms did it appear?
A
- ~450 million years ago
- Appeared in agnathans (hagfish, lampreys)
3
Q
Describe immunity in drosophila
A
- Production of anti-microbial peptides
- Epidermis
- Fat body / lymph
- Phagocytosis
- Haemocytes
- Phagocytose and destroy
- Coagulate surrounding haemolymph
- Proteolytic cascades
- Blood coagulation
- Complement like components
4
Q
Phylogenetically, what are the oldest mediators of innate immunity?
A
Phagocytes
5
Q
Describe the function of haemocytes in Drosophila melanogaster
A
-
Phagocytosis
- ROS
- Oxidants
- NO
- Lysozyme
- Protease
- Acidification
-
Proteolytic cascade
- Release of C’-like components
- Results in blood coagulation and killing of pathogens
- Release of inflammatory mediators
6
Q
Describe proteolytic cascades
A
eg Complement
- Many enzymes present in serum (10% of globulin fraction)
- Zymogens
Activation:
- Trigger provided by the pathogen
- Proteolysis of the proteins
- Products of proteolysis are involved in the desruction of the pathogen
Effects:
- Lysis of microbe
- Opsonisation of microbe
- Inflammation
- Recruitment of inflammatory cells
7
Q
Describe the alternative pathway in C’ activation
A
- C3 ‘tickover’ in steady state
- C3b binds pathogen when present
- Thioester group on C3b binds pathogen surface
- Factor B binds C3b and is cleaved into Bb and Ba by Factor D
- Bb joins with C3b on pathogen surface to form the C3 convertase
- Cleavage of C3
- etc
8
Q
Describe activation of the lectin pathway
A
- MBL binds mannans on pathogen surface
- Activation of MASPs associated with MBL
- C4 binds pathogen surface covalently
- MASPs convert C4 → C4a & C4b
- C4b binds C2
- C2 → C2a & C2b by MASPs
- C2a joins with C4b
- C4bC2a is C3 convertase
- C3 → C3a & C3b
9
Q
Describe the evolutionary homologues of the C’ cascade
A
- Activation
Alternate pathway
- Echinoderms (sea urchins)
- Homologues:
- C3 ⇔ SpC3
- Factor B ⇔ SpBf
- Factor D homologue?
Lectin pathway
- Urochordate (sea squirts)
- Homologues
- MBL ⇔ Ficolin
- Effector function
- Deposition of C3b on the pathogen surface
- Opsonisation by coelomocytes
10
Q
Describe evolutionary homologues of anti-microbial peptides
A
Small cationic molecules
- eg Defensins
- Phylogenetically conserved:
- Humans
- Plants
- D. melanogaster
- Phylogenetically conserved:
Features
- Over 400 identified
- Some show microbe specificity
Structure
- α-helix and β-sheet
- This structure is conserved through from plants to humans
Function:
- Permeabilise pathogen membrane
- Results in lysis of the pathogen cell
11
Q
Compare the location of release of defensins in humans and D. melanogaster
A
Humans
- α-defensins: paneth cells
- β-defensins: epithelial cells in skin
D. melanogaster
- Fat body cells (liver equivalent) → systemic response
- Epithelial cells → local response
12
Q
Briefly describe defensins in D. melanogaster
A
- *Drosophila *make many different defensins
- Expression is induced by Toll signalling following PAMP recognition
13
Q
What happens to Drosophila if Toll receptors are knocked out?
A
Susceptible to fungal infections
14
Q
Describe the structure of TLRs
A
LRR: leucine rich repeats
- 25 aa long
TIR
- Toll/IL-1R domain
15
Q
What is the function of TLRs?
A
Recognise PAMPS
Examples:
- TLR3 - dsRNA
- TLR4 - LPS
- TLR5 - Flagellin
- TLR9 - CpG DNA
16
Q
List some other types of PRRs
A
- TLRs
- NLRs (NOD-like receptors)
- RLRs (RIG-like receptors)
- CLR (C-type lectin like receptors)
17
Q
In which organisms did innate immunity arise?
A
Metazoans
eg Cnidarians, arthropods etc.
18
Q
Describe the components of the innate immune system in the following:
- Protozoa
- Sponges
- Worms
A
All have phagocytic cells
Worms also have NK cells
19
Q
Describe the emergence of LNs
A
Seen in mammals (+/- birds)
Not found in sharks or fish
20
Q
Describe the evolution of Ig
A
Increasing n° of classes seen as we move up the phylogentic tree
21
Q
What type of organism are the following:
- D. melanogaster
- C. elegans
- Sea urchins
A
- D. melanogaster - Arthropod
- C. elegans - Nematode
- Sea urchins - Echinoderm
22
Q
What is the basis of TLR diversity in humans?
A
Variation in LRR sequences
23
Q
Compare the location of the various PRRs in humans
A
Cell surface
- TLR4 ⇔ LPS
- TLR5 ⇔ Flagellin
- TLR6-TLR2 ⇔ Peptidoglycan etc.
- TLR1-TLR2 ⇔ Peptidoglycan etc.
Endosomes
- TLR3 ⇔ dsRNA
- TLR7 ⇔ ssRNA
- TLR9 ⇔ CpG DNA
Cytosol
- RIG-I ⇔ dsRNA
- NOD-1 ⇔ Degraded Gram -ve peptidoglycan
- NOD-2 ⇔ Muramyl dipeptide
24
Q
Describe signalling through TLRs in mammals and Drosophila
What is evolutionarily interesting about this?
A
The sigalling cascades are conserved through from Drosophila to mammals
Mammalian
- PAMP ligates TLR
- MyD88 recruitment to TIR domain of TLR
- IRAK1 and IRAK4 recruitment to Death domain of MyD88
- IRAK1/4 are kinases, and result in a phosphorylation cascade
- TRAF6
- TAK1
- Degradation of IKK
- Release of NFKB
- NFKB drives transcription of pro-inflammatory cytokines and co-stimulatory molecules
Drosophila
- Gram +ve bacteria
- Peptidoglycan binds PGRP-SA and GNBP-1
- These activate Grass
- Fungi
- PR1 released by fungi
- This activates Persephone
- *Persephone *and Grass cleave and activate Späztle
- Späzle dimerises Toll receptors on cell surface
- dMyd88 is recruited to TIR domains
- Recruitment of Pelle and Tube to dMyD88
- Phorphorylation cascade?
- dTRAF?
- Cactus kinase?
- Phosphorylation and degradation of Cactus by Pelle (equivalent of IκB)
- Release of DIF (equivalent of NFKB)
- DIF activates gene transcription
- Drosomycin
- Immune response proteins
25
Describe TNF signalling in mammals and *Drosophila*
What are the potential outcomes of this signalling?
The pathways are shared in these organisms
Mammals
1. TNF binds TNFR1
2. Recruitment of RIP
3. Recruitment of TRAF2 to RIP
4. TRAF triggers a phosphorylation cascade
5. Activation of IKK (IκB kinase complex)
6. Phosphorylation and degradation of IκB
7. Release of NFκB
8. Activation of transcription
* Pro-inflammatory cytokines
* Co-stimulatory molecules
*Drosophila*
1. PGN products from Gram -ve bacteria activate PGRP-LC receptor on surface
2. Recruitment of Imd
3. Recruitment of dFADD
4. dFADD triggers phosphorylation cascade
* dTAK1
5. Phosphorylation and degradation of C-terminal of Relish (equivalent of IκB)
6. Cleaved Relish moves into nucleus and activates gene transcription
* Diptericin
**Triggering of apoptosis**
Mammals
1. Receptor recruits FADD
2. Activaiton of caspase-8
3. Apoptosis
Drosophila
1. PGN products from Gram -ve bacteria activate PGRP-LC on cell surface
2. Recruitment of Imd
3. Recruitment dFADD
4. Recruitment of *Dredd* (Caspase-8 equivalent)
5. Apoptosis
26
Describe expanision of innate immunity in echinoderms
Huge expansion of SRCR (Scavenger receptors)
* 1000 compared to human 81
Protects them from the many viruses that it experiences every day
Though sea urchins lack adaptive immunity, they are working with what they do have
27
Explain the funciton of the following molecules
* PGRP-SA
* TRAF
* DIF
* Cactus
* IKK
* *Grass*
* Pelle
* GNBP-1
* IRAK1/4
* *Persephone*
* MyD88
* Spätzle
* NFκB
PGRP-SA
* Proteoglycan recognition protein SA
* Binds peptidoglycan of Gram +ve bacteria
TRAF
* Part of phosphorylation cascade after TLR activation in mammals
DIF
* *Drosophila* equivalent of NFKB
* Turns on transcription of immune response proteins
Cactus
* *Drosophila* equivalent of IKK
IKK
* IκB kinase
* Phosphorylates the inhibitory component of NFκB, causing its degradation
* NFκB is free to translocate into the nucleus
*Grass*
* Serine protease
* Activated by GNBP and PGRP that have recognised peptidoglycan extracellularly
* Activates Späztle complex
Pelle
* *Drosophila *equivalent of IRAK1/4
* Serine kinase
* Phosphorylates cactus (IκB equivalent), resulting in its degradation and release of DIF (NFκB equivalent)
GNBP-1
* Gram -ve binding protein
* Binds peptidoglycan of Gram +ve bacteria
IRAK1/4
* Serine/threonine kinases
* Recruited to death domain of MyD88
* Trigger a phosphorylation cascade, resulting in activation of IKK → release of active NFκB
*Persephone*
* Serine protease
* Directly activated by PR1, a virulence factor from fungi
* Acts on Späztle complex
MyD88
* Adaptor protein
* Recruited to (intracellular) TIR domain of activated TLR
* Recruits IRAK1/4 to death domain
Späztle
* Cleaved by activated *Grass *and *Persephone*
* Once cleaved, acts on Toll receptor
NFκB
* Transcription factor
* Turns on transcription of pro-inflammatory cytokines, co-stimulatory molecules
28
Describe the domains of MyD88 and why these are important
* Domain that binds intracellular TIR domain of TLR
* Death domain
* Recruits IRAK1/4
29
Describe the role of the following molecules:
* Imd
* FADD
* Caspase-8
* dFADD
* TRAF
* RIP
* PGRP-LC
* Dredd
Imd
* 'Immune deficiency'
* Adaptor protein for PGRP-LC
FADD
* Adaptor protein recruited to TNFRI
* Leads to apoptosis through recruitment of Caspase-8
Caspase-8
* Recruited by FADD
* Leads to formation of apoptosome
dFADD
* *Drosophila* equivalent of FADD
* Recruits Dredd
TRAF2
* Recruited to TNFRI
* Triggers phosphorylation cascade, leading to degradation of IκB and release of NFκB
RIP
* Recruited to TNFRI
* Recruits TRAF2
PGRP-LC
* Peptidoglycan recognition protein LC
* Recognises peptidoglycan products from Gram -ve bacteria
Dredd
* *Drosophila* equivalent of Caspase-8
* Recruited by dFADD
30
Compare detection of Gram +ve and -ve bacteria by *Drosophila*
Gram +ve:
* Detected by PGRP-SA
* Activates Toll pathway
Gram -ve:
* Detected by PGRP-LC
* Activates Imd pathway
31
Compare the effects of recognition of Gram positive and negative bacteria in *Drosophila*
**Gram positive**:
* Activation of Toll pathway
* Transcription of Drosomycin
**Gram negative**
* Activation of Imd pathway
* Transcription of Diptericin or apoptosis
32
Compare the expression of the following:
* Diptericin
* Drosomycin
Diptericin: Gram -ve bacteria
Drosomycin: Gram +ve bacteria & fungi
33
Where are defensins produced in *Drosophila?*
* **Fat body cells** (liver equivalent) → systemic response
* **Epithelial cells** → local response
34
Compare recognition of pathogens in mammals and *Drosophila*
Humans:
* Many receptors that are capable of recognising specific pathogen markers
* Receptors are at the cell surface, cytosol, or endosome
*Drosophila*
* Much of the recognition occurs extracellularly
* **Pathogen detection proteins:**
* PGRP-SA
* GNBP1
* The binding of these proteins to pathogen products brings about cleavage and activation of proteases, which cleave and activate **Spätzle**.
* Spätzle can then activate Toll, triggering the signalling cascade
35
Describe, in detail, how Gram +ve bacteria and Fungi activate Toll receptors
Gram +ve bacteria:
* Peptidoglycan binds **PGRP-SA** and **GNBP1**
* This complex activates *Grass*
* *Grass* cleaves and activates Späztle into a dimer
Fungi:
* Fungal cell wall components bind **PR1**
* This complex activates *Persephone*
* *Persephone* cleaves and activates Späztle into a dimer
Spätzle dimerises Toll receptors on the surface of the cell, triggering a signalling pathway
36
What are **PGRP-SA, GNBP1, **and **PGRP-LC**?
Pathogen detection proteins:
* PGRP-SA
* GNBP1
* Recognise peptidoglycan from Gram +ve bacteria extracellularly
* This complex is now capable of cleaving and activating *Grass*
PGRP-LC:
* Binds Gram -ve peptidoglycan products
* Triggers the Imd pathway
37
How are Fungi recognised by *Drosophila*?
* Fungal cell wall components bind PR-1
* This complex then activates *Persephone*
* etc. (Toll pathway)
