3- Innate Immunity: Induced Response

What are the steps for initiating signaling?

1. receptor binds ligand

2. perpetuation of signal

3. transcription factor

4. gene expression

What does the innate induced response entail?

signaling

Where are receptors found?

generally expressed on cell surface

How is the signaling pathway activated?

ligands bind to receptors

What happens after ligands bind to receptors?

signaling molecules carry signal downstream until reach transcription factor

What induces gene expression?

transcription factor

What can gene expression cause?

effector functions determined by cell type, activated receptor, etc

ex → AMP production, increased phagocytosis, cytokine production, induce proliferation, activate other cells

How does perpetuation of the signal occur?

kinases, phosphatases, ubiquitin ligases

Which ubiquitin ligase is used in signaling?

K63ub

Which ubiquitin ligase is used to degrade proteins?

K48ub

Self

healthy human cells, want them to be there, won’t initiate a response

What happens if there is a response to self?

failure of self, autoimmune/self attacking

What can a receptor distinguish?

self vs microbe, w/in self = non-self, altered, true self

Non-Self

microbial cells, receptors can distinguish between our cells & microbial cells, immune system activated

Altered Self

infected or cancerous cells, normal proteins in us modified to tell immune system we’re infected or cancerous, immune response activated but NO MICROBE

Tissues Resident Macrophages

all tissues have macrophages

• long lived

• able to self renew → can make new macrophages

• scavengers → breakdown dead/dying cells, microbes in tissues, recycling janitors

• phagocytosis

Why are immune cells unique in comparison to other terminally differentiated cells?

terminally differentiated cells → lose capacity to make new cells, not long lived

immune cells are

Macrophage receptors

scavenger receptor or others (CD14, C3/C4, TLRs)

Scavenger Receptors

macrophage receptor

• no infxn → recognize dead/dying cells

• infxn → recognize components on microbes

What microbial components can scavenger receptors detect?

LPS, CPG DNA (methylated DNA diff from ours), sugars

SR-E3

scavenger receptor, mannose receptor, CD206

• does phagocytosis, no signaling

• targets bacteria

• LIGANDS → LPS, CPs, ManLam

What other receptors are macrophage receptors?

• CD14 → lipopolysaccharide receptor

• complement receptors C3 & C4 → phagocytose things w/ C3b tags

• toll-like receptors (TLRs)

What is a mannose receptor?

scavenger receptor on macrophage, SRE3

What is the process of SRE3?

1. receptor on cell surface of mphage, recognizes sugars on bacteria

2. receptor interacts w/ bacteria/ligand

3. binding = signaling → mphage starts endocytosis

4. endosome starts degrading microbe

5. fuse w/ lysosome → kill bact

6. receptors recycled to mphage surface

Toll-Like Receptors

have distinct domains, recognize PAMPs & initiate immune responses

• leucine-rich repeats

• membrane-spanning domain

• TIR domain

Leucine-Rich Repeats

C-shaped region, binds to PAMPS, outside cell/inside endosome

• external domain, recognize & bind to microbes

• pathogen recognition domain (PRR)

• in TLR4

Membrane-Spanning Domain

inserted into membranes, PM or endosomal, in TLR4

TIR Domain

• signaling domain of TL4

• always in cytoplasm

PAMPs

pathogen associate molecular patterns, recognized by PRR/TLR4

How is TLR4 activated?

1. bacteria releases LPS bound to binding protein (LBP)

2. LBP-LPS complex taken into cell w/ phagocytosis by macrophage

3. LPS transfer to CD14, goes to cell surface

4. LPS binds TLR4 on surface

5. TIR domains bind M4D88

6. IRAK4 phosphorylates TRAF6

7. TRAF6 signals IKK

8. IKK signals NF-KB, transcription factor

9. NF-KB induces gene expression in nucleus

10. cell secretes cytokines & adhesion molecules

What does TLR4 recognize?

TLR4 binds & recognizes LPS, toxic & unique to gram (-) bacteria

What is M4D88?

adaptor connecting TIR domains to kinase IRAK4

T/F: LPS is not a PAMP.

False, LPS is a pathogen associated molecular pattern, unique to gram (-) bacteria

What is TLR4?

toll-like receptor 4, recognizes LPS of gram (-) bacteria, macrophage receptor

What is IRAK4?

protein complex, signaling molecule, kinase

• activated by → MyD88 binding TLR4

• phosphorylates TRAF6

What is TRAF6 (TLR)?

protein complex, signaling molecule

• activated by → IRAK4 phosphorylating it

• signals → IKK

What is IKK (TLR)?

protein complex, signaling molecule

• activated by → TRAF6

• signals → NF-KB

What is NF-KB?

transcription factor, does gene expression to:

• make cytokines

• increase adhesion

What are cytokines?

small proteins, inflame & recruit immune cells

What leads to IKK activation?

MyD88 binds TLR4, activates IRAK4 to phosphorylate TRAF6, leads to IKK activation

How are cytokines released in the NF-KB pathway?

NF-KB induces transcription of cytokine genes, made in cytoplasm & secreted using ER

What is the ultimate goal of the NF-KB pathway?

producing & releasing cytokines for inflaming & recruiting other immune cells

Plasma Membrane Receptors

most TLRs are on plasma membrane, on many cell types

• LIGANDS → carbs, lipids, proteins (LPS, flagellin), surface of microbe

• recognize extracellular infxns (fungi, bacteria, parasites)

Endosomal Receptors

w/in endosomes

• LIGANDS → nucleic acids (DNA/RNA), ours is methylated, bacteria isn’t = easy target

• recognize intracellular infxns = viruses

• signaling domain always faces cytoplasm

What kinds of ligands can TLR4 detect?

LPS & any kinds of product from pathogen

NOD Receptors

intracellular cytoplasmic receptors, non membrane-bound, soluble

• NOD 1

• NOD 2

What do NOD receptors recognize?

breakdown products of peptidoglycan, make up bacterial cell wall

What does NOD 1 recognize?

y-glutamyl diaminopimelic acid, in gram (-) bacteria only

What does NOD 2 recognize?

muramyl dipeptide, breakdown product of gram (+)/(-) bacteria

How is the NOD receptor like RTK?

NOD receptor binds to ligand, dimerizes (RTK callback), causes phosphorylation cascade, cause gene transcription & expression

How are NOD receptors & TLRs similar/different?

Both are pattern recognition receptors that detect microbial components and initiate immune responses

• NOD is soluble though!

• both can activate NF-KB

What unique domain does a NOD receptor have?

leucine-rich domain, binds ligands & signaling domain

How must NOD receptors be recognized?

must be taken up in phagosome/endosome & breakdown components released into cytoplasm

What is the process of activation for NOD receptors?

1. microbes broken down in phagolysosome

2. PG product moved to cytoplasm (muramyl dipeptide), binds to & dimerizes NOD2 proteins

3. NOD binds peptidoglycan

4. binds & activates RIPK2

5. IPK binds to & phosphorylates TAK

6. TAK signals IKK

7. IKK activates NF-KB

8. NF-KB induces gene expression activating mphage

What does activating the macrophage cause?

increase killing → use enzymes for degradation

increase phagocytosis → more receptors on cell surface

are the same proteins used in the complement pathway being used to activate NF-KB/macrophage pathway?

No, they are distinct pathways.

Does the complement pathway get immediately activated alongside NF-KB, or does one happen before the other?

The complement pathway is activated independently of the NF-KB pathway, meaning one does not necessarily occur before the other.

What is RIPK2?

signaling molecule, NOD receptor activation pathway

• activated by → NOD-PG binding

• signals → TAK

What is TAK?

signaling molecule/kinase, NOD receptor activation pathway

• activated by → RIPK2

• signals → IKK

What is IKK (NOD)?

signaling molecule, NOD receptor pathway

• activated by → TAK

• signals → activating NF-KB

Are NOD receptors or TLRs more favorable to use?

NOD receptors are generally more favorable for detecting intracellular pathogens, while TLRs are key for sensing extracellular pathogens. Both play critical roles in innate immunity.

How are NOD receptors & TLRs different?

NOD receptors detect intracellular pathogens, while TLRs are activated by extracellular stimuli.

Which signaling molecule is involved in 2 major pathways discussed?

IKK, protein complex, signaling molecule

• activated by → TRAF6 (TLR), TAK (NOD)

• signals → NF-KB for both pathways

What is an interferon?

IFN, cytokine, secretes IFN α/β

What do interferons target?

viral infxns, induction happens in virally-infected cells

How do virally infected cells help neighboring cells?

signal to neighboring cells to prepare them

How does IFN induction occur?

recognizes infxn & signal transcription factors (IRF 3/7)

• induces gene expression of type 1 interferons (IFNs)

• IFN α/β secreted

How does the IFN response occur?

in neighboring cells, IFN α/β binds receptors

• induce expression of antiviral genes/ISGs

• get antiviral signal = make antiviral state = cells getting ready to prevent infxn

What are ISGs?

interferon stimulated genes, enhance antiviral responses, induced by IFN α/β

What is the purpose of IFN β?

paul revere, warns cells to make antiviral state/get ready to prevent infxn

How is an antiviral state useful?

prevent viruses from infecting cells

• breakdown viral genome

• shutdown protein production/translation

• stop viral entry

• target intermediates of replication

What are RIG-1-like receptors?

proteins detecting viral infxns → dsRNA

• receptors → RIG-1, MDA-5

• CARD, helicase, CTD domains

Where are RIG-1-like receptors located?

cytoplasm, since RNA viruses replicate there

What unique conformational occurs w/ RIG-1-like receptors?

proteins are open when binding virus, usually closed

What are CARD domains?

in RIG-1-like receptors, signaling domains

• in RIG-1 & MDA-5

What are helicase domains?

in RIG-1-like receptors, bind nucleic acids (dsRNA)

• in RIG-1 & MDA-5

What is a CTD?

in RIG-1-like receptors, C-terminus, regulatory domain, holds mol in inactive form

• in RIG-1 & MDA-5

What 3 domains do RIG-1 & MDA-5 have?

CARD 1 & 2, Hel 1 & 2, CTD

What is the signaling pathway for RIG-1/MDA-5?

RLR signaling

1. RIF-1 binds dsRNA = cause signaling cascade

2. signals to MAVS, ADAPTOR

3. MAVS signals TRAF6

4. TRAF6 activates TBK1/IKK

5. TBK1 activates IRF 3/7

6. induce expression of IFN α/β

What is IRF 3/7?

transcription factors, in signaling pathway for RIG-1/MDA-5

• induce expression of IFN α/β

• activated by TBKI

• immune cell specific

What is the difference between ID3 & IRF7?

cell type specific

What is TBKI?

a kinase involved in RLR signaling that activates IRF3 and IRF7, leading to the production of interferons, signaling molecule

What is TRAF6 (RLR)?

signaling molecule involved in RLR (& TLR)

• RLR → activates TBKI/IKK

How is the IFN response autocrine or paracrine?

IFN α/β made in IFN induction

• interact w/ receptors on same cell secreting it (autocrine), boosting response, or on neighboring cell

How does the IFN response occur on neighboring cells?

1. IFN α/β activates STAT1 (transcription factor)

2. STAT1 causes gene expression of ISGs

3. ISGs make antiviral state in cell

What does IFN β bind to?

IFN β binds to the type I interferon receptor (IFNAR), triggering downstream signaling that activates antiviral responses in cells/neighbors

What are examples of ISGs?

100s of ISGs

• OAS1 → degrade viral RNA (RNA virus only)

• PKR → shut down translation, cell eventually dies, self destruct our infected cell, binds vRNA

What is the IFN response pathway?

1. IFN α/β binds to IFNAR

2. JAK

3. STAT

4. gene expression → ISGs made

What are plasmacutoid dendritic cells?

circulate in blood/lymph looking for viral infxns

Are plasmacutoid dendritic cells in tissue?

no, circulate in blood & lymph

What do plasmacutoid dendritic cells do?

activate TLR7 & TLR9 → systemic IFN response, express specific TLRs for viruses

• TLR7/9/3 → IRF 3/7 → IFN a/B (IFN response)

• RLR but in endosome

What is an inflammasome?

giant proteasome, specifically degrade pro-I1-a/B → active forms

What is IL-1B?

cytokine, signals to no one, has to get out to activate/do job, pro-inflammatory marker, increase/tons of inflammation

What are IL-1a/B?

cytokines, effector proteins activated by inflammasome, pre-formed & stored inside cells as active form

What is the process of the inflammasome?

1. NLRP3 receptor bound

2. oligomerization of ASC, procaspacce 1 & NEK7 = inflammasome, make nonactive form & store in cytoplasm

3. breakdown pro-IL-1a/B to IL-1a & B (inactive → active), cytokines released

What is NLRP3?

PRR, no transcription factor

If the inflammasome is activating/breaking down into active from inactive, is it just in an inactive form/making itself, then pulls its own trigger?

The inflammasome can recognize danger signals and undergoes a process of oligomerization, which leads to the activation of pro-inflammatory cytokines like IL-1a and IL-1b from their inactive precursors. It serves as a regulatory complex that, once formed, initiates its own activation under specific stimuli.

When does pyroptosis happen?

result of inflammation, happens after inflammasome’s function

What is pyroptosis?

inflammatory cell death, how IL-1B/cytokine is released

• makes pore/complex

• LOTS of inflammation

How does pyroptosis happen?

1. caspase 4 activtes gastrodermin D

2. active portion of gastroermin D inserts N terminus into PM = make pore

3. IL-1B released from cell → cause TONS of inflammation