Quail_Innate Immunity Flashcards
From which common progenitor do most innate immune cells come from?
Myeloid progenitor cell
What is the difference between innate and adaptative immunity?
It’s not necessarily the cells - It’s how they recognize pathogens
- Adaptive immune cells recognize specific antigens
- Innate immune cells recognize molecular patterns (non-specific) → First cells to defend your body!
What are the molecular patterns recognized by the innate immune cells?
DAMP = damage-associated molecular patterns (self and non-self)
- Necrosis
PAMP = pathogen-associated molecular patterns (non-self)
- Molecules that are found in pathogens that can be recognized by the immune system (not found in eukaryotic organisms)
- ex: peptidoglycans, RNA/DNA, Flagella, Lipopolysaccharide (LPS)
What are the molecular patterns considered as DAMPs?
- Necrosis
Ex: Psuedopalisading necrosis in glioblastoma → pink swirly region (contents of necrotic cells) surrounded by dense region of nuclei (cells recognizing the DAMPs) - Mitochondrial DNA
- HSP released duing necrosis
What is Necrosis vs Apoptosis?
Necrosis leads to spilling of the cell contents to the microenvironement. The body recognizes this as a sign of danger (e.g. proteins, ATP, nucleic acids, etc.)
Apoptosis, on the other hand, is more strategic. Cells become fragmented (including DNA); contents do not spill out. The body recognizes this as normal.
How are PAMPs and DAMPs (molecular patterns) recognized by cells of the innate immune system?
Recognized by Pattern Recognition Receptors (PRR) → 2 main types:
1. Toll-like receptors (TLR) → membrane bound → found on plasma membrane and within endosomes
2. NOD-like receptors (NLR) → found in the cytoplasm
Both lead to production of cytokines that trigger a RAPID & ROBUST immune response
What are important features of TLRs?
- Membrane boud → can be found in plasma membrane ou within endosomes
- Can act alone of in dimers → different combination can recognize different PAMPs/DAMPs
- Signalling leads to production of pro-inflammatory cytokines (NFkB pathway) and Type 1 IFN (IRF7 pathway)
- Common/similar downstream signaling pathways involving NFkB for plasma membrane TLRs and IRF7 for phagosomes TLRs
- In endosomes, they mainly recognize dsRNA, ssRNA CpG (intracellular contents)
- Plasma membrane TLRs recognize mainly extracellular features
What is the main advantage of having TLR dimerization?
It can create many different combinations → with different intracellular domains → recruit different adaptor proteins for downstream signaling
- They all converge to similar pathways
- Allow multiple signals to be sent simultaneously
- Allows cross-talk between pathways to have the strongest possible signals
*Ex: of adaptor protein → MyD88
What are the main features/what is known about NOD-like receptors? How do they work?
- 23 proteins
- 2 broad categories: NLRP and NLRC → all cytosolic receptors
- They are inactive as monomers → regulated by chaperone proteins which need to be cleaved to form active inflammasome complex
- Ligand binding
- Conformational change → oligomerzation/inflammasome formation
- Recruitment of adator protein with Pyrine and CARD domains
- pro-caspase-1 binds to the assembled NLRP (CARD adaptor proteins) → NLRPs with pro-caspase-1 form a ring-like structure
- This close proximity of multiple pro-caspase-1 → self-cleavage of caspase-1 → active caspase-1
- Active caspase-1 → activation of pro-proteins (pro-IL 1b, pro-IL 18) + pro-inflammatory cell death (pyroptosis)
What are inflammasome complexes and what are they induced by?
Inflammasomes = cytosolic multiprotein complexes of the innate immune system responsible for the activation caspase-1, inflammatory responses and cell death
Induced by:
- Phagocytosed PAMPs/DAMPs
- Changes in ion gradients
- ROS
- proteases leaked from lysosomes
- Inflammatory cytokines from TLRs
What are the main differences between NLRP and NLRC? What do these letters stand for?
NLRP = NOD-like receptor with Pyrine domain
NLRC = NLR with caspase-recruitment domain
*They need to assemble/dimerize to be active
What is Pyroptosis?
How is it different from Apoptosis?
Inflammation-associated cell death
- Voluntary process (“voluntary necrosis”)
Inflammatory method of programmed cell death: In response to a foreign molecular pattern, cells will voluntarily burst and die to elicit a strong and fast immune response. (generates DAMPs and PAMPs)
→ Cell death from pyroptosis results in membrane rupture, spilling contents of the cell. This releases DAMPs into the environment.
*Unlike apoptosis, pyroptosis requires
Caspase-1
What determines which PRRs a PAMP/DAMP will encounter?
Its location!!
Extracellular recognition → TLR on plasma membrane
Cytosolic reognition → NLR
Endosomal recognition → TLRs in endosomes
Important note:
Innate immune cells express multiple PRRs → allows them to recognize diverse pathogens
*Different from lymphocytes which have 1 type of antigen receptor
→ Multiple PRRs can be activated by one pathogen to amplify the response
Give an exmaple of how multiple PRRs can be activated by one pathogen to amplify the response.
Dendritic cells can recognize LPS or gram+ bacteria via TLR4 on their plasma membrane. This causes phagocytosis and degradation within the endosomes. TLR9 within the endosomes then binds to unmethylated dsDNA (CpG DNA) from the bacteria to trigger a second signal.
What are different types of PRRs innate immune cells can express? (Other than TLRs and NLRs)
- Receptor for advanced glycation end products (RAGE) → binds endogenous ligands
- G-protein coupled receptors (GPCRs) → ex: for extracellular nucleotides, calcium
- Membrane channels → activates by ROS and contributes to calcium influx
- Triggering receptors expressed on myeloid cells (TREM) → binds lipids and certain proteins like extracellular actin
Regardless of which receptor is engaged, similar downstream effect:
1. Expression of inflammatory cytokines (w/ pro-domains)
2. Activation of NLRs and caspase-1
What are some other consequences of PRR activation?
Causes trained immunity → to the bone marrow for progenitors and in the mature experienced cell
- Epigenetic changes (long lasting, open chromatin of inflammatory response genes)
- Metabolic changes (more efficient growth and division)
What is the difference between trained immunity and memory?
Memory is antigen-specific (adaptative)
Trained immunity is not antigen specific, the cell will be better at responding at all PAMPs/DAMPs it will later encounter
How do vaccines contribute to immunity?
What happens to the innate immune system when we encounter a pathogen?
Ex of a skin wound
Ex of a skin wound:
1. Inflammatory Response
- From tissues damage (DAMPs) + bacteria coming in (PAMPs)
- Activation of tissue-resident macrophages/DCs → signal to recruit more immune cells
- Immune Recruitment
- Innate immune cells are recruited by chemokine gradient
- Vascular inflammation causes increased permeability of the blood vessel for infiltration of immune cells - Pathogen Removal (innate + adaptative)
*DC makes the link between innate and adaptative immunity
How are innate immune responses translated to the adaptative immune system?
In peripheral tissue:
1. Immature DC recognizes PAMPs and becomes activated
- Pathogen is internalized in phagocytic vesicle
- Phagocytic vesicle is acidic and has high levels of proteases → fragmentation of the foreign antigens
- TLR signaling induces CCR7 and enhances processing of pathogen-derived antigens
- CCR7 is essential for DC maturation and homing of immune cells to 2nd lymphoid organs
- Antigen presented to adaptative immune cells through MHC II + expression of costimulatory molecules (CD80/CD86)
In the lymph node:
DC migrated to draining lymph nodes via CCR7
- Mature DC present antigenic peptide on MHC + 2 othe signals
Which 3 signals have to be given from DCs in the lymph nodes to T cell to activate them?
3 signal are required for naive T cell activation:
1. MHC II - TCR → Activation
2. CD80/86 - CD28 → Survival
3. Inflammation/cytokines → Differentiation
Depending on which cytokines are released, different T cells phenotypes are favoured
What is the importance of CXCL8 (IL-8)?
Produced by macrophages and dendritic cells
- Acts on phagocytes
- Strong chemoattractant for neutrophils (sworm to the site of pathogenesis)
What is the importance of TNF-a?
Produced by macrophages and dendritic cells
Acts up the Vascular endothelium (induces changes):
- Expression of cell-adhesion molecules (E- and P-selectin)
- Changes in cell-cell junction with increased fluid loss (swelling), local blood clotting
What happens at the level of the endothelium when cytokines and chemokines are released in the tissue (ex: skin)?
Vascular inflammation → Weakened junctional adhesions + Upregulation of luminal adhesions
- Caused by PRR engagement: cytokines, neutrophils, NETosis, ROS, histamine, etc.
Leukocyte Extravasation (from blood → tissues)
1. Chemoattraction by chemokine gradient
2. Tethering and Rolling → neutrophils bind adhesion molecules on vascular endothelium near sites of infection and receive chemokine signal
3. Migration → Neutrophils transmigrate across the endothelium into tissue
4. Activation of neutrophils (ex:NETosis, degranulation, phagocytosis, cytokine production)
What is the difference between the interaction of Leukocytes with the endothelium and the interaction of lymphocytes with the endothelium?
Leukocyte - Endothelium:
Sialyl lewis + X glycan + epitope - E-/P-Selectin
Lymphocytes - Endothelium:
L-Selectin - Sialyl lewis + X glycan + epitope
*Selectin is on the endothelium for leukocytes, but on the lymphocytes
What can explain the pain caused during an innate immune response?
- Irritation of nerves when inflammatory cells migrate into tissue, releasing inflammatory mediators that cause pain
- Fluid influx into the tissue with increased vascular permeability causes increase in pressure
What are the key step of the initiation of an immune response?
- Pathogen is encountered
- Recognized by PRRs (e.g. NOD-like receptors, Toll-like receptors)
- Intracellular signalling cascade is initiated
- Three consequences: 1. Production of pro- inflammatory cytokines 2. Presentation of antigen 3. Initiation of co-stimulation
- This engages the adaptive immune system, and stimulates an immune response against the pathogen
How can obesity lead to chronic inflammation?
- Adipocytes become large, and undergo hypertrophy.
- Hypertropic adipocytes release DAMPs into the microenvironment (when they dye, they spill their contents)
- Causes recruitment of macrophages and formation of «Crown-Like Structures» (highly inflammatory)
- CLS produce pro-inflammatory cytokines, creating chronic inflammation in obese individuals.
*Obesity-Associated inflammation leads to adverse health conditions (e.g. cancer)
How is chronic inflammation associated with severe COVID?
Viral genome entering the cells → disruption in ion balance and other factors → engage different sets of PRRs
Chronic inflammation → higher baseline levels of pro-inflammatory cytokines already present in the cells (IL-1b)
When caspase-1 is activated, many more pro-domains are present which induces a stronger response when all these cytokines are activated
*higher levels of IL-1b are associated with severe COVID
How can organ transplant & ischaemia-reperfusion injury induce innate immune responses?
GRAFT side:
1. Transplantation → cell lysis → release of DAMPs
2. DAMPs → inflammation and activation of TLRs
3. Inflammation → Changes in vasculature (adhesion molecules)
4. Inflammation → Activation of DCs
5. Inflammation → Cytokine production
HOST side:
- Activated DCs from the host → host T cell activation in the Lymph nodes
- Cytokines from the graft donor → inflammation amplification in the host
Can lead to increase risks of graft rejection
What are different ways vaccines induce immunity?
Vaccines contain virus or virus particles (PAMPs), or more recently, mRNA encoding parts of a virus. These are recognized by innate immune cells at the site of injection, which undergo pyroptosis.
Spilling of the cell contents releases more PAMPs and DAMPs into the microenvironment to amplify the innate immune response.
Interactions with PRRs on innate immune cells leads to 3 signals: production of pro-inflammatory cytokines, antigen presentation, and co-stimulation.
This stimulates T cells, which interact with B cells to stimulate production of antibodies specific to the viral antigen → MEMORY
*The insertion of a needle also creates tissue dammage (DAMPs)
Innate immune cells undergo some for of trained immunity following a vaccine as well as the memory effect
What are the 3 branches of innate immunity?
- Pattern recognition
- Antigen presentation
- Complement
How is the complement system related to innate and adaptative immunity?
- Enhances the ability of the immune system to clear pathogens and damaged cells
- Plays a role in both innate and adaptative
Pathogen exposure for the 1st time = Complement is activated by the pathogen in a non-specific manner
Repeated pathogen exposure = Complement is activated by Ab-Ag complexes (recognizes Ab-Ag complexes)
What is complement?
The complement system is made up of a large number of different plasma proteins that interact with one another to opsonize pathogens and to induce inflammation.
*Found in the fluid that comes into the tissue
Complement proteins are produced by the liver as pro-enzymes. Exist as inactive enzymes in the blood and interstitial fluid at all times, and need to be cleaved in order to become activated. These cleavage events potentiate an immune response.
Cleavage → a and b components
- a component will lead to an anaphylaxic reaction
- b component will continue to potentiate the complement cascade
What are C5a and C5b?
C5a and C5b are key components of the complement system, each with distinct roles in immune responses.
C5a → Anaphylatoxins, Chemotaxis
- Inflammatory mediator, chemoattractant
C5b → Continue to potentiate the pathway
- Initiates MAC formation
What is the core complement system?
The core complement proteins are named C1 to C9 and function as part of the complement cascade:
1) C1 complex – Composed of C1q, C1r, and C1s, which initiate the classical pathway.
2) C2, C3, C4 – Key components in all pathways.
- C3 is the central protein of the system, with C3b playing a major role in opsonization.
3) C5, C6, C7, C8, C9 – These contribute to forming the membrane attack complex (MAC), leading to direct cell lysis.
What are some exception of complements that do not act like the others?
- C1 and C6-9 are NOT cleaved
- C6-9 are not enzymes
*These are the end points of the complement cascade, make the whole thing relevant
Which complement proteins are part of the membrane attack complex? What conformation?
C5b → initiates MAC formation → causes recruitment of C6 → assembles and causes recruitment of C7 → assembles and causes recruitment of C8 → goes into the plasma membrane → all the C9s form a ring
The active MAC is made up of:
4 complement proteins, including C5b, C6, C7 and C8. These bind to the outisde of the plasma membrane.
Multiple copies of C9, which form a ring like structure within the plasma membrane.
- Hydrophobic external face to facilitate association with the plasma membrane.
- Hydrophilic internal face to allow fluid to pass through.
- C6 - C9 are all NOT cleaved
- b subunit of C5 potentiates the response by engaging additional proteins, while C5a is free to act as a chemoattractant
- contents of the cells getting out of the cell causes an inflammatory response
What is the purpose/consequence of the membrane attack complex?
The MAC creates pores in the plasma membrane of an invading pathogen.
This leads to exchange of fluids between the cell and the extracellular space - this is cytotoxic to the cell.
This spills the contents of the cell into the microenvironment to potentiate the innate immune response (inflammatory response as the contents are full of PAMPs/DAMPs).
What is the central player in the complement system?
C3
The most important event is C3 activation (cleaved from pro-enzyme → C3a + C3b)
What happens when C3 becomes active?
There are three complement pathways, which are are named based on how C3 cleavage is triggered (i.e. they are not fully distinct pathways). All complement pathways converge on C3 cleavage and the events that follow are common to all pathways.
- Classical pathway (Antigen:Ab complexes)
- Mb-lectin pathway (Lectin binding to pathogen surface)
- Alternative pathway (pathogen surface)
*ALL 3 cause C3 activation → C3 activation leads to the following (common to the 3 pathways)
1. Recruitment of inflammatory cells
2. Opsonization of pathogens
3. Killing of pathogens
*After C3 everything is the same!!
What are the 3 complement pathways?
Complement proteins patrol the blood and the tissues as inactive pro- enzymes. They become activated when they encounter a pathogen. → 3 pathways of complement activation
- Classical pathway (Antigen:Ab complexes)
- Mb-lectin pathway (Lectin binding to pathogen surface)
- Alternative pathway (pathogen surface)
How does the alternate pathway of complement activation function?
- When a pathogen is encountered, C3 that is surveilling the tissue becomes activated (cleaved).
- C3b binds/is deposited to the cell surface (starts opsinising the pathogen) of the pathogen —> flags to the rest of the body that this is a pthogen
- Along with another factor called Bb, they form C3bBb = C3 convertase.
- This cleaves more C3, providing more C3b. **C3bBbC3b = C5 convertase.
- C5 convertase cleaves C5 into C5a and C5b.
- C5b initiates formation of the membrane attack complex in the pathogen
C3b will also facilitate clearance through a process called opsonization.
- This basically ‘flags’ the pathogen for phagocytosis by innate immune cells such as macrophages, which express complement receptors (help PRRs!)
In the alternate pathway of complement activation, when happens to C3a and C5a?
These are anaphylatoxins!!
2 functions:
1. Activation of mast cells, to release histamine through degranulation
- This causes the bronchioles to constrict, and the endothelium to open up.
2. Attraction of neutrophils and other immune cells to the site of infection
What is C3bBb vs C3bBbC3b?
C3bBb = C3 convertase
C3bBbC3b = C5 convertase.
How does the Lectin pathway of complement activation function?
Most pathogens have mannose on their surfaces.
This is bound by mannose binding lectin (MBL).
Activation of the MBL occurs through binding with mannose, and associated proteins (MASPs)
Activation of this structure causes cleavage:
- C4 into C4a and C4b. C4b attaches to the cell surface of the pathogen
- C2 into C2a and C2b. C2a binds to C4b.
The C4bC2a complex is another C3 convertase.
This activates C3 cleavage
*C3b feeds into the amplification loop to generate C5 convertase, and initiate the membrane attack complex
What is C4bC2a?
It is a complex formed in the Lectin pathway of complement activation
C3 convertase → activates C3 cleavage → C3b feeds into the amplification loop to generate C5 convertase, and initiate the membrane attack complex
How does the Classical pathway of complement activation function?
The classical pathway involves adaptive immunity(!!).
After prolonged infection, the body starts to develop antibodies against specific antigens.
When Ab bind Ag, complement proteins can help expedite the response. → The C1 complex binds to Ab-Ag pairs - this causes activation
Initiated by the binding of the C1 complex (C1q, C1r and C1s) to bound antibody. C1r activates C1s which first cleaves C4 and then cleaves C2 leading to the formation of the classical pathway C3 convertase (C4bC2a).
What is similar between the classical and the lectin pathways of complement activation?
- C4 into C4a and C4b. C4b attaches to the cell surface of pathogen (ex: bacterial surface)
- C2 into C2a and C2b. C2a binds to C4b
The C4bC2a complex is another C3 convertase. This activates C3 cleavage.
*C3b feeds into the amplification loop to generate C5 convertase, and C5 initiate the membrane attack complex
Can the lectin pathway and the alternative pathway occur simultaneously?
The complement pathways have different names based on how C3 cleavage is triggered (i.e. they are not fully distinct pathways).
- Alternative pathway: C3 is cleaved in response to the pathogen.
- Lectin pathway: C3 is cleaved in response to upstream events that follow MBL-
mannose interactions.
- Classical pathway: C3 is cleaved in response to upstream events that follow Ab-
Ag interactions.
What determines the type of C3 cleavage?
The initial triggering event depends entirely on the composition of the cell surface of the pathogen. The alternative pathway is always active, and constantly deposits C3b onto all cell surfaces. Host cells deactivate these C3b molecules to avoid complement-mediated destruction (“don’t eat me” signal). However, there are also “eat me” signals (i.e. MBL-mannose or Ab-Ag, for the lectin and classical pathways, respectively). These pathways are only activated when those specific patterns are detected on the cell surface of the target cell.
Ex: CD47
- Expressed on cells that are ‘self’
- Elevated in cancer
- Not expressed once the cell dies
What are the consequences of complement activation?
- Lysis (membrane attack complex)
- Opsonization (binding to pathogen surface to help phagocytes)
- Activation of inflammatory response (Chemoattraction + Anaphylaxis)
- Clearance of immune complexes(assist with adaptative immunity)
What does anaphylaxis involve?
- Histamine release
- Bronchiole constriction
- Vasodilation
*Part of the activatio of inflammatory response
What are 2 effects of antigen presentation?
- Activation of the complement classical pathway
- Mediates T cell activation in response to Pattern Recognition
How does the innate response trigger the adaptative response?
In peripheral tissue:
1. Immature DCs recognizes PAMPs and beocme activated
2. TLR signaling indces CCR7 and enhances processing of pathogen-derived antigens
In Lymph Nodes
3. DCs migrate to the draining lymph nnodes via CCR7
4. DCs present antigenic peptides on MHC to naive T cells in the lymph node
*3 signals are required
How do APCs «present antigens»?
Major Histocompatibility Complex (MHC) molecules → determine compatibility between individuals.
- Peptide-binding glycoprotein molecules
- Interaction via the TCR
Expressed on the cell surface:
- Class I = expressed by all cells except RBCs
- Class II = expressed by ‘professional’ APCs
*Class II expression is induced by Pattern Recognition Receptors
What are the steps for APCs to present and antigen through the MHC?
- Antigen processing (need to phagocytose it)
- Antigen presentation (via MHC)
What are the 3 major classes of molecules encoded by the MHC genes?
Class I: Glycoproteins expressed on the surface of nearly all nucleated cells.
Major function is to present endogenous (intracellular) antigens to cytotoxic T cells (CD8+)
Class II: glycoproteins expressed primarily on APC (Dendritic cells, Macrophages and B cells).
Major function is to present exogenous (extracellular, phagocytosed) antigens to helper T cells (CD4+).
Class III: proteins that have immune functions including components of complement (C2, C4) and inflammation
What are the MHC-I equivalent in humans? (MHC is in mice)
HLA (Human Leukocyte Antigens):
HLA-A, HLA-B, HLA-C (found on all nucleated cells)
- Intracellular antigens are processed for presentation by MHC-I (in the cytosol)
*location of the antigen matters for what type of MHC will present it
What is the intracellular pathway of MHC-I antigen presentation?
1.MHC will encounter an antigen in the ER where MHC it is synthesized and held open by calnexin
2. Antigen is brought to the ER (from cytosol) via TAP
3. MHC-Antigen complexe are transporter via a secretory vesicle from the ER to the plasma membrane
How do cytosolic proteins get into the ER to bind MHC-I?
They do so through a transporter (TAP1) which delivers cytosolic proteins to the ER
TAP = Transporter associated with antigen processing
What is the difference between binding of MHC-I vs MHC-II to antigens for presentation?
(Focus on MHC-II)
MHC-II is only expressed on professional APCs (ex: DCs)
MHC-I = intracellular antigens
MHC-II = Extracellular antigens → internalized/phagocytosed and processed for presentation by MHC-II
Meanwhile, MHCII is synthesized in the ER.
Class II-associated invariant chain peptide (CLIP) binds MHCII and remains bound until it is exchanged for Ag. This prevents binding of self-Ag
Unlike MHCI, MHCII encounters the antigen in the lysosome (not the ER, APCs degradepathogens within lysosomes).
After Ag binding, they are transported to the plasma membrane to present antigen to CD4+ T cells (not CD8+ T cells)
What are the differences between the structure of MHC-I and MHC-II?
MHC-I = alpha 1, apha 2, alpha 3, b2
- alpha 3 = single TM domain
- alpha 1 and alpha 2 = peptide-binding cleft
MHC-II = alpha 1, alpha 2, beta 1 beta 2
- 2x TM domains = alpha2 + beta2
- peptide-binding cleft = alpha1 + beta1
Which are the human equivalents of MHC-II?
In humans, the HLAs corresponding to MHCII are HLA- DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR.
How do cells choose which antigen presentation method to use?
- What type of cell they are? (if they aren’t a professional APC, they have no other choice than MHC-I)
- If professional APC, have both options - Where is the pathogen encountered?
Intracellular → MHC-I
Extracellualr → MHC-II - Exceptions → cross presentation of exogenous antigens when you get phagocytosis, but that pathogen gets into the cytoplasm and presented by MHC-I to naive CD8+ T cells
What is the role of C2, C3, C4 and C5?
Pro-enzymes that are cleaved into an a and a b component. Generally, the a component causes a «allergic»-like reaction and the b component potentiates complement cascade ultimately leading to cleavage of C5 and formation of the MAC
What PAMPs are expressed by gram+ vs gram- bacteria?
gram+ bacteria → Peptidoglycans
gram- bacteria → LPS
