Pathogen Recognition (MT1)

Explain innate target recognition

The innate immune system does not recognize specific pathogens, rather it makes use of PAMPs to differentiate between self and non-self

What detects PAMPs?

Pattern Recognition Receptors (PRRs)

What are the four families of PRRs?

TOLL-like receptors (TLRs)
Nucleotide-binding domain leucine-rich repeat receptors (NLRs and NOD-like receptors)
Formylated-Peptide Receptors (FPRs)
Retinoic-acid inducible gene-like (RLRs)

Define: TOLL

A protein known to play a role in dorsal-ventral patterning, found that flies who lacked TOLL were more prone to fungal infections

Outline the sequence of TOLL in flies

1. GNBP and PGRP recognize gram-positive cell walls, beginning a proteolytic cascade culminating in the
cleavage of spätzle.
2. Cleaved spätzle binds 2 TOLL molecules (dimerization)
3. TOLL dimer recruits 2 dMyD88, 2 Pelle, and 1 TRAF3
4. TRAF3 binds Cactus kinase which phosphorylates Cactus, resulting in the release of the transcription factor DIF
5. Translation + synthesis of antimicrobial peptides

Outline the sequence of TLR Signaling using myD88 in humans

1. TLR binds to MyD88 and MAL, IRAK binds to MyD88
2. IRAK binds TRAF-6 and TRICA1, which recruitment and activate TAK1
3. TAK1 phosphorylates IKK (inhibitor of NFκB kinase)
4. IKK phosphorylates IκB (inhibitor of NFκB)
5. IκB is degraded and releases NFκB
6. NFκB translocates to the nucleus and transcribes target
genes

Outline the sequence of TLR Signaling using TRIF in humans

1. TRIF binds to TLR3 or TLR4, allows kinases TBK1 and IκKε to bind
2. Kinases become phosphorylated, resulting in their
activation
3. Activated kinases then phosphorylate IRF3 (interferon
responses factor 3)
4. IRF3 translocates to the nucleus and transcribes target
genes

Outline the sequence of NOD Signaling

1. PAMPs bind to NOD1 or NOD2, NOD dimerizes
2. Dimerization facilitates the recruitment of the cytoplasmic kinase RIPK2 to the CARD domain of the NOD
3. RIPK2 phosphorylates TAK1, activating it
4. TAK1 phosphorylates IKK (inhibitor of NFκB kinase)
5. IKK phosphorylates IκB (inhibitor of NFκB)
6. IκB is degraded releasing NFκB
7. NFκB translocates to the nucleus and transcribes target
genes

Define: TOLL-like receptors (TLRs), how many are there?

Receptors in humans with sequence similar to that present in fly TOLL, there are 13 mammalian TLRs.

What is the lipopolysaccharide (LPS) sensing molecule (receptor)?

TLR4

Which receptor(s) are only found in pairs/bound together? (heterodimers)

TLR-2/TLR-6, TLR-2/TLR-1

Which receptors are found on the plasma membrane (outside of cell)?

TLR-2/TLR-6, TLR-2/TLR-1, TLR-5, TLR-4

Which receptors are found within cells?

TLR-3, TLR-7, TLR-9

What is the major difference between mammalian and fly TLRs?

Mammalian TLRs bind directly to the PAMP, unlike flies which need adapters like spätzle

True or False: Mammalian TLRs play a role in developmental tissue patterning like in flies

False, they play no role in tissue patterning. As a result we have no need for extracellular molecules to bridge between the PAMP and TLR.

How are heterodimers bound?

The convex (outward curved) surfaces of heterodimers (TLR 1,2, and 6) have binding sites for side chains of triacyl lipopeptides. These lipopeptides induce dimerization bringing the cytoplasmic TIR (cytoplasmic tails) domains to close proximity

Which molecules use MyD88, which use TRIF?

TLR - 1,2,4,5,6,7,8,9 use MyD88
TLR - 3,4 use TRIF

Note that TLR-4 makes use of BOTH MyD88 and TRIF

If almost all TLRs use MyD88, how do you get different immune responses (viral, bacterial, fungal)?

Depends on 3 factors:
Subcellular location of molecules
Signal duration
Additional signaling molecules

Explain how subcellular location affects immune responses

TLR-3,7,8,9 are not on the cell surface but rather are located in the endosome. These receptors tend to recognize PAMPs from within the pathogen (like RNA and DNA). Endosomal localization can influence the recruitment of signaling molecules, the strength and duration of the resulting signal. These differences can influence which target genes are transcribed.

Explain how signal duration affects immune responses

Depending on the magnitude of the signal and the number of receptors involved, the duration of the TLR signal can vary. Additionally, negative regulators of the TLR (Tollip) can inhibit signaling. These changes in TLR signal duration have been shown to modify the resulting gene transcription profile

Explain how additional signaling molecules affect immune responses

Different combinations of signaling molecules result in different cellular responses
Although most TLRs us MyD88 – it is not the only signaling adapter
• TLR-3 uses TRIF
• TLR4 – can use MyD88, MyD88 and TRIF, or just TRIF
(TRIF is not negatively regulated by Tollip)

Define: Tollip

Toll interacting protein (TOLLIP) is an inhibitory adaptor protein (negative regulatory of TOLL-like receptors (TLRs).

Define: NOD-like receptors (NLRs)

Cytoplasmic receptors that can be grouped into a
number of families depending on their amino-terminal
structural motif
Upon binding to a cytoplasmic PAMP, NLRs oligomerize and recruit additional molecules

What are the two families of NLRs?

- NOD family has an n-terminal CARD (caspase
recruitment domain) motif
- NLRP family has an n-terminal Pyrin domain

What is the Serpentine Receptor (or G-coupled/7 trans-membrane receptor)

Present in Formylated peptide receptors (FPRs).
A large group of cell surface receptors that detect molecules outside the cell and activate cellular responses.

Explain the process of Formylated peptide receptors (FPRs)

Formylated peptide receptors (FPRs) leave a gradient of formylated peptides as a sort of trail of breadcrumbs that neutrophils follow to areas with high bacterial density.