Lecture 1 - Innate recognition Flashcards
Three main ways of defending against pathogens
- Barriers - antimicrobial enzymes, antimicrobial peptides, complement systems, physical barrier
- Innate immune effector mechanisms
- Adaptive immune response
Inflammation: is it good or bad
Good - used to aid destruction of pathogens
Bad - chronic inflammation may damage its own body
Innate vs adaptive immunity: the molecules involved
Innate:
* Macrophages
* Neutrophils
* Dendritic cells
* Innate lymphoid cells
* NK cells
Adaptive:
* NK-T
* B cell
* T cell
PAMPs: what are they and what examples of them are there?
Pathogen-associated molecular patterns
- Bacterial component: LPS, Peptidoglycan,Flagellin, etc
- Single stranded viral RNA (e.g. SARS-Cov-2)
- Double stranded viral DNA (e.g. Herpes virus)
DAMPs: what are they, how are they formed, and what examples are there?
Damage associated molecular patterns
Motifs that are shielded from the immune system until the cell is injured - they then act as a signal for the immune system
- Heat Shock Proteins (HSPs),
- Uric acid crystals (Gout),
- ATP,
- DNA,
- β-Amyloid (Alzheimer),
- Some cytokines (e.g. IL-1α, HMGB1)
PRRs: what are they, what do they do, where are they present, and what do they result in?
Pattern recognition receptors
Recognise PAMPs/DAMPs
Cell surface (TLR, C-lectin, etc) or intracellular (RLRs/NLRs/cGAS/STING)
- Phagocytosis/ROS production
- Chemokine/cytokine production
Phagocytosis: what is it, what receptors are on phagocytes for them to recognise pathogens, what do they detect, and which are not PRRs?
The endocytosis of unwanted substances (bacteria, dead cells, debris, etc) that results in their breakdown and destruction
- Mannose receptor - mannosylated ligands (fungi, bacteria, and viruses)
- Dectin-1 - bind β-1,3 linked glycans - fungi
- Complement - bind complement coated microbes (all)
- Lipid - CD36, recognises lipids (bacteria)
- Scavenger - two classes, class A (bacteria) and class B (bacteria and fungi)
- Fc receptors - bind marked pathogens (all)
Fc and complement - they recognise the marking on pathogens that have been identified already, not the pathogens themselves
Mannose receptor: what does it detect and what pathogens does it consequently detect?
Mannosylated ligands
- Fungi
- Bacteria
- Viruses
Dectin-1 receptor: what does it detect and what pathogens does it consequently detect?
Bind β-1,3 linked glycans
- Fungi
Complement receptor: what does it detect and what pathogens does it consequently detect?
Bind complement coated microbes
- All
Lipid receptor: what does it detect and what pathogens does it consequently detect?
Recognises lipids and lipoproteins (such as LPS) using CD36
- Bacteria
Scavenger receptor: what does it detect and what pathogens does it consequently detect?
Two classes:
* Class A - bind anionic polymers and acylated low density lipoproteins using SR-A I, SR-A II, and MARCO - bind bacteria
* Class B - bind lipids and lipoproteins using CD36 - bind bacteria
Fc receptor: what does it detect and what pathogens does it consequently detect?
Bind marked pathogens
- All
Cytokines: what are they, do they only promote inflammation, and what may happen if balance isn’t maintained?
Chemical messengers
Can be pro or anti-inflammatory
Imbalance - disease likely caused
Cytokines: what examples are there?
- IL-1β
- IL-6
- IL12
- TNF-alpha
- CXCL8 (IL8)
TLRs: what are they, where does their name come from, and what do they have?
Toll-like receptors
Receptors that are similar to Toll receptors in drosophila
- Extracellular/endosomic domain - contains LRR (leucine rich repeats)
- Transmembrane domain
- Intracellular domain
TLRs: are they all the same?
No, different TLRs bind different pathogens:
* TLR-2/6, -1/2, 5, and 4 all detect bacterial/fungal PAMPS - these are located on the plasma membrane
* TLR-3, -7, and -9 all detect viral PAMPS - these are located in the endosome
TLRs: how does their signalling work in those in the plasma membrane?
- TLRs have a highly conserved cytoplasmic domain (the toll/IL-1R (TIR) domain)
- Ligand binding causes recruitment of an adaptor molecule - MyD88
- MyD88 recruits IL-1R associated kinases (IRAKs), forming an IRAK1/IRAK4 complex
- IRAK1/IRAK4 complex phosphorylates TNF receptor associated factor-6 (TRAF-6)
- TRAF-6 builds a scaffold allowing TGF-β activated kinase-1 (TAK-1) to bind to IRAKs allowing it to be phosphorylated
- TAK-1 binds the inhibitor of nuclear factor-κB kinase (IKK) complex, phosphorylating it
- IKK complex phosphorylates and inactivates IκB
- NFκB is now active and can be translocated into the nucleus and cause the production of cytokines and antibacterial molecules
TLR-3: how does its signalling work?
- TLRs have a highly conserved cytoplasmic domain (the toll/IL-1R (TIR) domain)
- Ligand binding in the endosome causes recruitment of an adaptor molecule - TRIF
- TRIF activates TRAF-3 which builds a scaffold allowing a complex to bind containing IKKε and TBK1 kinases
- The IKKε and TBK1 complex phosphorylates IRF3
- Interferon regulatory factor 3 (IRF3) is then activated and translocated to the nucleus where it acts as a TF for innate antiviral and antibacterial genes
Endosomal TLRs: how does their signalling work?
- TLRs have a highly conserved cytoplasmic domain (the toll/IL-1R (TIR) domain)
- Ligand binding in the endosome causes recruitment of an an adaptor protein - MyD88
- MyD88 recruits IRAK1/IRAK4 complex
- IRAK1 phosphorylates IRF7, activating it
- Interferon regulatory factor 7 (IRF7) is then activated and translocated to the nucleus where it acts as a TF for innate antiviral and antibacterial genes
TIR domain: what is it and what does it do?
Toll/interleukin-1 receptor - a conserved cytoplasmic domain
Responsible for continuing the signal after a ligand binds the extracellular (or endosomic) domain
MyD88: what is it, what is it activated by, and what does it do?
Myeloid differentiation primary-response protein 88, the main adaptor protein, involved with all TLRs except TLR-3
TLR activation
An adaptor protein that recruits to activated TLRs and activates IRAKs to continue signalling
TRIF: what is it, what is it activated by, and what does it do?
TIR-domain-containing adaptor protein inducing interferon- β, the only adaptor protein for TLR-3 and an adaptor protein along with MyD88 in TLR-4
TLR activation
An adaptor protein that recruits to activated TLRs and activates IRAKs to continue signalling
IRAKs: what is it, what is it activated by, and what does it do?
Interleukin-1 receptor (IL-1R) associated kinases
Adaptor proteins (mostly MyD88)
Phosphorylate TRAFs
TRAF-6: what is it, what is it activated by, and what does it do?
Tumour necrosis factor (TNF) receptor associated factor-6 (TRAF-6)
IRAKs
TRAF-6 builds a scaffold allowing TGF-β activated kinase-1 (TAK-1) to bind to IRAKs allowing it to be phosphorylated
TAK-1: what is it, what is it activated by, and what does it do?
Transforming growth factor-β (TGF-β) activated kinase-1
TRAF-6
TAK-1 binds the inhibitor of nuclear factor-κB kinase (IKK) complex, through the IKKγ subunit (NEMO (NFKB essential modifier)), and then phosphorylates and activates the IKKβ subunit, phosphorylating it and allowing it to interact with cytoplasmic IkB and target it for degradation, allowing NF-KB to translocate to the nucleus and transcribe genes
IKK-complex: what is it, what is it activated by, what is its structure, and what does it do?
Inhibitor of nuclear factor-κB kinase (IKK) complex
TAK-1
- Two kinase subunits - IKKα and IKKβ
- Regulatory subunit - NEMO/IKKγ
NEMO binds to scaffold and brings IKK-complex to TAK1 where IKKβ gets activated and phosphorylates IκB, resulting in NFκB activation
IκB: what is it, what is it inactivated by, and what does it do?
The cytoplasmic inhibitor of nuclear factor-κB
IKK complex phosphorylates and inactivates IκB
NFκB is now active and can be translocated into the nucleus and cause the production of cytokines and antibacterial molecules
NFκB: what is it and what does it do?
Nuclear factor-kappa B - a transcription factor
Promotes the production of cytokines and other antibacterial molecules:
* Pro-inflammatory cytokines
* IL-6/1beta promoting Th17 differentiation
TLR-2/6: what is its functional shape, where is it located, what cells are they found in, what does it detect, and what pathogens does it consequently detect?
Dimer consisting of TLR-2 and TLR-6
Plasma membrane
- Monocytes
- Dendritic cells
- Mast cells
- Eosinophils
- Basophils
Diacyl lipopeptides
- Bacteria
- Fungi
TLR-1/2: what is its functional shape, where is it located, what cells are they found in, what does it detect, and what pathogens does it consequently detect?
Dimer consisting of TLR-1 and TLR-2
Plasma membrane
- Monocytes
- Dendritic cells
- Mast cells
- Eosinophils
- Basophils
Triacyl lipoproteins
- Bacteria
- Fungi
TLR-3: where is it located, what cells are they found in, what does it detect, and what pathogens does it consequently detect?
Endosome membrane
- NK cells
dsRNA
- Viruses
TLR-4: what is its functional shape, where is it located, what cells are they found in, what does it detect, and what pathogens does it consequently detect?
TLR-4 along with MD-2 and CD-14
Plasma membrane (and also in the endosomal membrane, utilising the same signalling pathway as TLR-3 but to a weaker effect)
- Monocytes
- Dendritic cells
- Mast cells
- Eosinophils
LPS
- Bacteria
- Fungi
TLR-5: where is it located, what cells are they found in, what does it detect, and what pathogens does it consequently detect?
Plasma membrane
Intestinal epithelium
Flagellin
- Bacteria
TLR-7: where is it located, what cells are they found in, what does it detect, and what pathogens does it consequently detect?
Endosome membrane
- Plasmocytoid dendritic cells
- NK cells
- Eosinophils
- B cells
ssRNA
- Viruses
TLR-9: where is it located, what cells are they found in, what does it detect, and what pathogens does it consequently detect?
Endosome membrane
- Plasmocytoid dendritic cells
- NK cells
- Eosinophils
- B cells
CpG DNA
- Viruses
- Bacteria
TLRs: what do they do and how do they do what they do?
Activate transcription factors (NF-kB, AP-1 and IRF) to induce expression of cytokines and interferons
They can stimulate anti-viral or anti-bacterial responses
IRFs: what are they, what are the types, what are they produced by, and what do they do?
Interferon regulatory factors
- IFR3 - TLR-3 signalling - act as a TF for antibacterial and antiviral innate immunity genes
- IRF7 - TLR-7/9 signalling - act as a TF for innate antiviral immunity genes
- IRF9 - (ER/i cba)
RLRs: what are they, what immune function do they do, how do they do this detection, what types are there, what do they each specifically recognise, and how does RLR signalling work?
RIG-I-like receptors - intracellular PRRs
Cytoplasm viral RNA recognition - using sn RNA helicase-like domain
RIG-1 - recognizes mainly ssRNA/uncapped 5’-triphosphate dsRNA
MDA-5 - Mainly recognizes longer dsRNA, doesn’t require the uncapped 5’-triphosphate DNA for detection
- RLR binds viral RNA leading to the assembly of RIG-1 proteins along the dsRNA
- Riplet and TRIM25 are recruited to to the complex to form a K63 protein scaffold to allow RIG-1 to form filaments along the dsRNA and allow the aggregation of CARDs from different RIG-1 and MDA-5 molecules as well as MAVS
- This aggregation allows the proline-rich region of MAVS to associate with TRAFs
- MAVS, an adaptor protein in the mitochondrial membrane, generates K63 from Riplet and TRIM25
- This aggregation allows the proline-rich region of MAVS to associate with TRAFs
- TRAF activation forms a scaffold allowing both TBK1/IRF3 and IKK/IκB pathways to be activated
- IRF3/NFκB act as TFs, producing type-1 interferons and cytokines to induce antiviral/antibacterial innate immune responses
RIG-1: what is it, where is it expressed, what is its signalling, how do these receptors detect viral RNA and not self RNA, and are there examples of viruses that evade this detection?
Retinoic acid inducible gene-1 - a type of RLR
Widely expressed across tissues and cell types
- RIG-1 binds viral 5’ uncapped tri-phosphate dsRNA, leading to the assembly of RIG-1 proteins along the dsRNA
- Riplet and TRIM25 are recruited to to the complex to form a K63 protein scaffold to allow RIG-1 to form filaments along the dsRNA and allow the aggregation of CARDs from different RIG-1 and MDA-5 molecules as well as MAVS
- This aggregation allows the proline-rich region of MAVS to associate with TRAFs
- MAVS, an adaptor protein in the mitochondrial membrane, generates K63 from Riplet and TRIM25
- This aggregation allows the proline-rich region of MAVS to associate with TRAFs
- TRAF activation forms a scaffold allowing both TBK1/IRF3 and IKK/IκB pathways to be activated
- IRF3/NFκB act as TFs, producing type-1 interferons and cytokines to induce antiviral/antibacterial innate immune responses
Eukaryotic RNA undergoes capping in the nucleus which viral RNA does not, allowing the 5’ uncapped end to be recognised as foreign
Picornaviruses (Hepatitis A and Poliovirus) undergo covalent attachment of a protein to the 5’ uncapped end, allowing them to evade RLR recognition
MDA-5: what is it, where is it expressed, what is its signalling, and how is it different to RIG-1 signalling?
Melanoma differentiation-associated 5
Widely expressed across tissues and cell types
- MDA-5 binds viral dsRNA, leading to the assembly of RIG-1 proteins along the dsRNA, forming filaments along the dsRNA
- These filaments allow the aggregation of CARDs from different RIG-1 and MDA-5 molecules as well as MAVS
- This aggregation allows the proline-rich region of MAVS to associate with TRAFs
- TRAF activation forms a scaffold allowing both TBK1/IRF3 and IKK/IκB pathways to be activated
- IRF3/NFκB act as TFs, producing type-1 interferons and cytokines to induce antiviral/antibacterial innate immune responses
Doesn’t require Riplet and TRIM25 to form a scaffold for CARD domain binding - potentially because MDA-5 readily forms filaments along the dsRNA
RLR activation: does only one RLR interact with one viral RNA?
No, once RIG-1 or MAD-5 is bound, they bind into filaments (with the help of TRIM25 and Riplet in RIG-1) along the viral RNA which promotes aggregation of CARDs from multiple different RIG-1 and MDA-5 molecules and the continuation of the signal
cGAS: what is it, what does it do, what is its structure, and what is its signalling pathway?
cGAS (cyclic-CMP-GMP synthase) - an intracellular PRR
- Sensor of intracellular infection (dsDNA)
- Self-recognition (self DNA)
Contains a nucleotidyltransferase domain and two major DNA-binding domains
- Recognises viral dsDNA
- cGAS activated - produces cyclic GMP-AMP (cGAMP) from GTP/ATP
- cGAMP activates STING
STING: what is it, what does it do, where is it present, and what is its signalling pathway?
Stimulator of interferon genes
Senses cyclic dinucleotides (cGAMP, c-di-GMP/c-di-AMP, etc)
Present on ER membrane
- Activated by cGAMP and/or bacterial dinucletides which are similar to cGAMP (c-di-GMP/c-di-AMP)
- Activates TBK1
- TBK1 phosphorylates IRF3
- IRF3 enters nucleus and induces type I interferon gene expression
NLRs: what are they, what do they do, what are the types, how do they differ, and what is traditional NLR signalling?
Nod-like receptors
- Recognise PAMPS and DAMPs (self and non-self)
- Inflammasome forming
Differing N-terminus (structural not functional):
* NLR-A: acidic transactivating domain (not a PRR!)
* NLR-B: BIR-domain containing
* NLR-C: CARD-domain containing
* NLR-P: Pyrin-domain containing
Type:
NOD 1 - recognise iE-DAP, a peptide present in the peptidoglycan of all Gram-negative and some Gram-positive bacteria
NOD 2 - MDP, bioactive peptidoglycan motif common to all bacteria
- Bacteria ligands are broken down into fragments able to be recognised by NLRs
- Bacterial ligand binding, activating NLRs
- Aggregation is induced, promoting CARD-dependent recruitment of RIP2
- RIP2 associates with cIAP1, cIAP2, and XIAP which forms a scaffold recruiting TAK1 and IKK
- IKK complex phosphorylates and inactivates IκB
- NFκB is now active and can be translocated into the nucleus and cause the production of cytokines and antibacterial molecules
NLRs: what diseases may be caused as a result of their mutation
NOD2 mutations:
* Chron’s disease - inflammatory bowel disease affecting any part of the GI tract from the mouth to anus
* Blau syndrome - spontaneous inflammation of eyes, joints, and skin
Cytokine secretion: what are the types of secretion, how do they each work, and what cytokines are secreted by each pathway?
Classical secretion pathway - cytokines move through the ER to the Golgi and then get secreted from secretory vesicles out of the cell (IL-6/IL-12, etc)
Unconventional protein secretion pathway - ER/Golgi independent, requires processing of molecules followed by secretion via membrane pores/microvesicles/endosomes/lysosomes(IL-1β/IL-18, etc)
Inflammasome: what does it do, what is the most studied NLR inflammasome, what is it activated by, and what are the steps of its formation and activation?
Act as platforms for the activation of inflammatory caspases (ie caspase 1) which cleave pro-cytokines into cytokines for secretion (ie pro-IL-1β and pro-IL-18 into IL-1β and IL-18)
NLRP3 (ER):
* Reduced intracellular K⁺
* Generation of ROS
* Disruption of lysosomes (ie by phagocytosis of crystalline material)
* The presence of extracellular ATP (may be formed from the death of nearby cells)
- NLRs become activated either by pathogens directly or as a response to pathogen activity
- Activated NLRs form an inflammasome
- inflammasome forms a platform for inactive caspase 1 and results in its activation
- Caspase 1 undergoes its effects: pro-cytokine Gasdermin D cleaving
Caspase 1: what does it do?
- Cleaves pro-IL-1β into IL-1β
- Cleaves pro-IL-18 into IL-18
- Cleaves Gasdermin D, which then enters the plasma membrane and creates a pore for the exit of IL-1β and IL-18 as well as promoting pyroptosis (lytic form of cell death)
Why does caspase 1 cause cell death?
Allows for the exposing of pathogens that were hidden within the cell, allowing for neutrophils/activated macrophages to kill the exposed pathogens
CAPS: what are they, what are they caused by, what do they lead to, and what is the treatment for these conditions?
Cryopyrin periodic associated syndromes
Autosomal dominant GoF mutation of NLRP3
Spectrum of inflammatory syndromes:
* Familial cold autoinflammatory syndrome (FCAS)
* Muckle-Wells syndrome (MWS)
* Neonatal-onset multisystem inflammatory disease (NOMID)
Canakinumab (a monoclonal antibody) administered by injection under the skin every 8 weeks
TLRs in autoimmunity (ER)
To reach endosomes, TLR-3, TLR-7, TLR-8, TLR-9, TLR-11, TLR-12, and TLR-13 must interact with a specific protein, UNC93B1. UNC93B1, which is composed of 12 transmembrane domains, binds TLRs in the endoplasmic reticulum and shuttles them to endosomes.
Rare human mutations in UNC93B1 have been identified as causing susceptibility to herpes simplex encephalitis, similarly to TLR-3 deficiency, but they do not impair immunity to many other viral pathogens, presumably because of the existence of other viral sensors
Sensing of nucleic acids enables broad recognition of diverse pathogens, but, in abnormal
settings, TLRs may be activated by self-derived nucleic acids, leading to autoimmunity
In healthy cells, RNA and DNA are normally confined to the nucleus and cytoplasm and are
not present in endosomes; however, nucleic acids can be released into the extracellular space
during tissue damage or inflammation
E3 ligases: what are they, what do they do, and what are some examples, and what signalling pathways are they involved in?
Enzymes that form protein scaffolds to allow for binding of molecules used in signalling pathways
- XIAP (X-linked inhibitor of apoptosis protein) - used in the NLR signalling pathway
- cIAP1 (cellular inhibitor of apoptosis 1) - used in the NLR signalling pathway
- cIAP2 (cellular inhibitor of apoptosis 2) - used in the NLR signalling pathway
- Riplet - used in the TLR signalling pathway
- TRIM25 (Tripartite Motif Containing 25) - used in the TLR signalling pathway
