Lecture 29 - Innate Immunity 2

Question 1

What is the Danger-Stranger model?

Answer 1

It is not just foreign antigen that is recognised by PRRs, but also Danger signals:

DAMPs: Danger associated molecular patterns
+ DAMP receptors

DAMPs in their normal location (e.g. DNA) will not trigger the pathways, but when it is found in the cytosol. This indicates that severe damage has occurred and that the best things now for the cell is death.

Question 2

What are PAMPs?

Give examples

Answer 2

Pathogen associated molecular patterns

Highly conserved molecules or molecular patterns in microbes

Enable discrimination between self and foreign

Examples:
 • viral DNA
 • viral RNA
 • Peptidoglycan
 • Flagellin
 • LPS
 • Mannan
 • Protozoan DNA

Question 3

What are the various cellular sites of pathogen encounter?

Which types of pathogens can be encountered in each?

Answer 3

Cell membrane
 • Bacteria
 • Viruses
 • Parasites
 • Worms

Phagosomes / endosomes
• Bacteria
• Viruses
• Parasites

Cytosol
• Bacteria
• Viruses

Question 4

What are Toll-like receptors?

What encodes them?

Where are they located?

Answer 4

Pattern recognition receptors; specifically recognise PAMPs

Germline encoded receptors (i.e. there is no rearrangement and education, as with BCRs and TCRs)

Location:
• Always associated with membranes

Question 5

Which TLRs are found in the following locations:
• Cell membrane
• Phago-/endosome?

Answer 5

Cell membrane:
 • TLR 4
 • TLR 2
 • TLR 5
 • TLR 11

Phago-/endosomes:
• TLR 3
• TLR 7
• TLR 9

Question 6

Which interleukin is produced due to TLR 4 engagement?

Answer 6

IL-6

Question 7

What leads to the release of IL-6?

What does it bring about?

Answer 7

Release:
 • Engagment of TLR or RLR
 • Transduction pathway
 • Activation of NFκB
 • Transcription of IL-6

Function:
• Induction of acute phase, CRP production in liver
• Th17 differentiation
• B cell differentiation
• Increased co-stimulatory molecule expression
• Systemic inflammatory responses (fever, fatigue)

Question 8

Which cytokine is produced with TLR 3 stimulation?

Answer 8

IFN-α / IFN-β

i.e. type I IFN

Question 9

What leads to the release of IFN-α / IFN-β?

Answer 9

• Engagement of TLR3 or RLRs
• Transduction pathways
• Activation of NFκB (TLR3) or other stuff (RLRs)
• Gene transcription of IFN-α / IFN-β

Question 10

Which pathogens can be encountered in the cytosol?

Answer 10

Viruses
Some bacteria (Listeria)

Question 11

What are RLRs?

What do they recognise?

Describe signalling through RLRs

Answer 11

RIG-I-like receptors

Recognise viral RNA present in cytosol

Function:

1. Viral RNA in cytosol binds to RIG-1
2. RIG-1 associates with MAVS on mitochondrion
3. MAVS dimerise
4. MAVS activate transduction pathway
5. Activation of NFκB and TRAF
6. NFκB turns on gene transcription of type I interferon and IL-6

Question 12

What are NLRs?

Where are they often found?

What is their structure?

Answer 12

NOD-like receptors

aka Inflammasomes

Structure:
• NLRs associate with additional molecules to form large protein complexes in the cytosol
• Including pro-caspase-1

Question 13

What are inflammasomes?

Where are they found?

Answer 13

A multi protein oligomer

Containing:
• Caspases
• ASC
• NLRs

Responsible for activation of inflammatory processes and pyroptosis

Found in the cytosol

Question 14

What are the ligands for the following receptors:
 • TLR 3
 • TLR 4
 • TLR 5
 • TLR 9
 • TLR 11
 • RIG-I
 • MDA5
 • NLRC4?

Answer 14

TLR 3: dsRNA

TLR 4: LPS

TLR 5: Flagellin

TLR 9: CpG-DNA

TLR 11: Flagellin

RIG-I: dsRNA

MDA5: dsRNA

NLRC4: Flagellin

Question 15

What are the sources of the ligands of the following PRRs:
 • TLR 3
 • TLR 4
 • TLR 5
 • TLR 9
 • TLR 11
 • RIG-I
 • MDA5
 • NLRC4?

Answer 15

TLR 3: Viruses, Bacteria (dsRNA)

TLR 4: Bacteria (LPS)

TLR 5: Bacteria (Flagellin)

TLR 9: Virus, bacteria (CpG-DNA)

TLR 11: Bacteria (Flagellin)

RIG-I: Virus, Bacteria (dsRNA)

MDA5: Virus, Bacteria (dsRNA)

NLRC4: Bacteria (Flagellin)

Question 16

Which sorts of PRR are membrane bound, and which are cytosolic?

Answer 16

TLRs: membrane bound

RLRs & NLRs: cytosolic

Question 17

What are the effects of engagement of the following receptors:
 • TLR 3
 • TLR 4
 • TLR 5
 • TLR 9
 • TLR 11
 • RIG-I
 • MDA5
 • NLRC4?

Answer 17

TLR 3: IFN-α / IFN-β

TLR 4: IL-6

TLR 5: IL-6

TLR 9: IL-12, IFN-α / IFN-β

TLR 11: IL-6

RIG-I: IL-6, IFN-α / IFN-β

MDA5: IL-6, IFN-α / IFN-β

NLRC4: IL-1β, IL-18, IL-33

Question 18

Which TLRs detect flagellin?

Answer 18

TLR 5
TLR 11
NLRC4

Question 19

PRR engagement leading to IFN-α / IFN-β release indicates…

Answer 19

Viral infection

IL-6 generally upregulated in response to bacteria

Question 20

Which sorts of pathogen do NLRs generally recognise?

Answer 20

Bacteria that have escaped the endosome

For example: Salmonella, Listeria

Question 21

Why are the receptors called Toll-‘like’ receptors?

Answer 21

Because the ones found in humans are ‘like’ the ones found in Drosophila, which is the animal in which they were initially studied.

Question 22

What is MyD88?

Answer 22

Molecule required for the transduction of TLR activation

All TLR signals with it, apart from TLR3

Question 23

Which receptor activates both MyD88 and TRIF?

Answer 23

TLR 4

Question 24

Which cytokine is good at stimulating the acute phase response?

Answer 24

IL-6

Question 25

What is CRP?

Discuss its importance

Answer 25

C-reactive protein

Useful marker for the acute phase response

Only bacterial infectious elicit the acute phase response and thus CRP

In a clinical setting, CRP levels in serum can be used to determine whether a common cold is due to a viral or bacterial infection, and thus inform prudent use of antibiotics.

Question 26

Which are the Type 1 IFNs, and which are type 2?

Answer 26

Type 1: IFN-α / IFN-β

Type 2: IFN-gamma

Question 27

What do IFN-α and IFN-β bring about?

Answer 27

Induction of ‘antiviral state’:
Transcription of around 2000 genes

• Inhibition of viral protein synthesis
• Degradation of viral RNA
• Inhibition of virus assembly
• Increased co-stimulatory molecule expression

Question 28

What is observed in mice that lack the IFN-α/IFN-β system?

Answer 28

Unable to control viruses

Herpes becomes lethal

Question 29

Which organelle are RLRs always associated with?

Answer 29

Mitochondria

Question 30

Describe what happens when there is damage to mitochondria

Answer 30

1. Damage to mitochondria
2. Mitochondrial DNA leaks into cytosol
3. Activation of cytosolic receptors
4. Induction of apoptosis

Question 31

Describe inflammasome signalling

Answer 31

1. Binding of complex with danger signals / Flagellin:NLRC interaction
2. Activation of caspase 1
3. Caspase 1 cleavage of pro-cytokines:
   • Pro-IL-1β → bioactive IL-1β
   • Pro-IL-18 → IL-18

Question 32

Which receptors are very good are recognising DAMPs?

Answer 32

Inflammasomes

Ways in which they do this:
• Monitoring of the membrane
• Crystal recognition (e.g. Gout)

Question 33

Which TLR brings about a response w/o the use of MyD88?

Answer 33

TLR3

Question 34

What are the two different outcomes of TLR signalling?

Answer 34

1. IL-6 transcription
   • Acute phase response in liver → CRP → Fever, Fatigue
   • Th17 induction
   • B cell differentiation
   • Increased co-stimulatory molecule expression

2. Type I IFN transcription
 • Induction of Anti-viral state
 • Upregulation of proteasome subunits
 • Degradation of viral RNA
 • Inhibition of viral protein synthesis
 • Inhibition of viral assembly
 • Increased co-stimulatory molecule expression

Question 35

Compare the signal transduction in TLRs resulting in the following:
• IL-6
• IFNα/β

Answer 35

IL-6:
 • Engagement of TLR at surface
 • Association of MyD88
 • MyD88 activates a phosphorylation cascade
 • Activation of NFκB
 • Transcription of IL-6

IFNα/β
 • Engagement of TLR (3)
 • Association of adaptor molecules
 • Signalling cascade
 • Activation of NFκB
 • Transcription of IFNα/β