Prelim 1 Flashcards
(211 cards)
1
Q
What is the purpose of the immune system?
A
The purpose of the immune system is to maintain the healthy state of homeostasis
2
Q
Vaccines
A
Deliberately stimulate the immune system
Vaccinated people make long-lived B cells and T cells to recognize the smallpox virus when it infects. These memory cells activate, VIGOROUSLY cloning themselves into 1000’s of defenders and antibodies. Antibodies and immune cells tag and eliminate the virus quickly
3
Q
Most vaccines prevent ___ not __
A
Disease not infection
4
Q
Infection
A
Invasion of barriers and usually replication of microbes within the host
5
Q
Infectious disease
A
Unhealthy state caused by infection with a pathogen. It may be due to damage from the microbe and a vigorous immune response.
6
Q
Pathogens
A
- Cause infectious disease
- Bacteria, viruses, fungi, and parasites.
- They typically infect hosts through mouths, eyes, noses, gut, reproductive tract, open wounds, or insect bites
7
Q
Immunity
A
The ability to resist disease caused by a pathogen using pathogen-specific defenses (antibodies, memory B cells and memory T cells)
8
Q
Why don’t vaccines work great?
A
Because they mutate so frequently.
ex. antibodies that are specific to an antigen won’t be able to bind super well to a mutated version of the virus so they won’t be able to neutralize as great
9
Q
What is the immune system’s strategy for dealing with threats?
A
- Recognize and tag
- Activate
- Eliminate by any means necessary
10
Q
How are fungi and parasites taken care of since they are too large to be phagocytosed?
A
- They are expelled from the body (nose, mouth, diarrhea) so toxins are secreted
11
Q
Commensal microbe
A
co-exists on the outer barriers. Doesn’t infect because it is successfully repelled by immune system. Does not cause disease; some are beneficial. Low threat.
12
Q
Opportunistic pathogen
A
A microbe that is usually harmless but causes disease in some situations (opportunities). Commensal microbes can be opportunistic, like Pneumocystis jiroveci which infects and kills AIDs patients who have lost T cell immunity.
13
Q
What are the names of immune cells?
A
Leukocytes
14
Q
Leukocytes
A
General term for an immune cell (aka white blood cell). Lymphocytes, monocytes, and granulocytes are all leukocytes
15
Q
Where are leukocytes derived from?
A
A pluripotent multipotent hematopoietic stem cell made in the bone marrow
16
Q
Hematopoiesis
A
Generation of leukocytes, erythrocytes (red blood cells), & platelets from a common pluripotent stem cell by step-wise, irreversible development changes. As a stem cell differentiates, it proliferates… making many clones of the new cell type that migrate to specific parts of the body
17
Q
Where do B cells, T cells, NK cells, and ILC cells come from?
A
Common lymphoid progenitor
18
Q
Where do Dendritic cells come from?
A
Common myeloid progenitor
19
Q
Where do neutrophils, eosinophils, basophils, and monocytes come from?
A
Granulocyte/macrophage progenitor which come from the common myeloid progenitor
20
Q
Where do macrophages come from?
A
Monocytes
21
Q
Where do platelets come from?
A
Megakaryocyte/ megakaryocyte erythrocyte progenitor
22
Q
Where do erythrocytes (red blood cells) come from?
A
Erythroblast / megakaryocyte erythrocyte progenitor
23
Q
What are the phagocytes?
A
- Neutrophils
- Macrophages
- Dendritic Cells
24
Q
What are the granulocytes?
A
- Neutrophils
- Eosinophils
- Basophils
- Mast cells (allergic response)
25
Eosinophil
Killing of antibody-coated parasites
26
Basophil
Promotion of allergic responses and augmentation of anti-parasitic immunity
27
Mast Cell
Release of granules containing histamine and active agents (allergic response)
28
Primary Lymphoid Tissues
Thymus and bone marrow
29
Secondary Lymphoid Tissues
Spleen, lymph nodes, Peyer's Patches
30
Where does the T and B Cell Response start?
- In the secondary lymphoid tissue. Lymph nodes are immune cell gathering places.
- To alert (activate) resting T cells and B cells, Dendritic cells bring microbes and antigens for T cells.
- More antigens and microbes flow into the lymph node for B cells`
31
What are antibodies?
- Immune molecules that bind to threats like toxins or bacteria
- Antibodies block the actions of (neutralize) microbes and toxins
- Antibodies tag threats for elimination by several ways. One way is phagocytosis by a macrophage
32
Cytokines and Chemokines
Proteins that immune cells and tissues use to communicate. Cytokines send a message, chemokines call for help
33
Epithelial barriers
Skin, mucosal barriers. The skin has to be waterproof and have a low pH. The epithelium of the respiratory system and gut have to be thinner because there needs to be an exchange of nutrients/oxygen.
34
Mucosal epithelial (slide 38)
Contain multiple parts and multiple defenses like bricks
- Goblet cells secrete mucus
- Paneth cells secrete antimicrobial defensins and lysozyme
- Epithelial cells start inflammation and transport antibodies
35
Leukocytes at the epithelial barrier
- Macrophages defend at barrier
- Dendritic cells go to lymphoid tissue to summon T help
- Plasma cells secrete antibodies
*mucus and microbes are also a defense
36
What are the two chemicals that the gut has?
- Antimicrobial enzymes
- Antimicrobial peptides
These work together, defensin and lysozyme
37
Lysozyme
Is an enzyme that digest outer cell wall peptidoglycan linkages and exposes lipid membrane of microbe
38
Defensin
- Antimicrobial peptides
- Defensins attack highly negatively charged surfaces (most microbes)
- The positive end of the defensin is pointing out of the membrane
- Defensins are amphipathic.
- Intercalates into membrane and forms pores. Microbes burst.
- Deployed on the outer surface of barriers, within phagolysosomes, and released from paneth cells into the mucus
39
Outer side of barriers
- Mucus w/ defensins, lysozyme and antibodies
40
Inner side of barriers
- Complement proteins
- More pattern recognition molecules (MBL)
- Leukocytes
- Inflammatory response
- Acute-phase
- Interferon response
- T Cells and B Cells
41
Pathogen recognition mechanisms
Pattern recognition molecules (like opsonization with C3b) & recognition receptors on immune cells (like complement receptors)
42
Effector mechanisms
Defensive immune responses (like phagocytosis and destruction) that slow or eliminate threats
43
Why must we have immediate responses to the immune system instead of uniquely generated receptors?
Microbes take many forms and mutate often. It would be impractical to encode a ready-to-use unique receptor or tag for each microbe. Additionally, making this receptor/tag takes time and it might be too late by the time it is created.
44
3 Immediate Strategies for Attacking Microbes
1. Attack highly negatively charged surfaces. If it's from the host, it's not a healthy cell (since cells are positively charged on the surface but negatively inside)
2. Tag all surfaces where a threat is detected. Healthy host cells will inactivate tags. Unhealthy cells that can't remove tags will be eliminated.
3. Tag the tell-tale molecular patterns on microbes, like their unique cell walls. Healthy host cells don't have these patterns or cell walls.
45
How do defensins deal with amphipathic toxins?
- Many toxins are also amphipathic so that they can fold and unfold to exert their toxic effects
- Amphipathic defensins binds and unfold toxins which stops toxin activities, especially at higher body temperatures during fever
- Poorly folded toxins clump together (aggregate). Clumped toxins are destroyed by proteases and the whole thing is ingested by a phagocyte
46
Defensins are kept in an __ state because...
- Kept in an inactive state until needed.
- The pro-region blocks the positively charged domain from inserting into membranes
- Proteases made by alarmed epithelial cells and leukocytes cut at a specific amino acid sequence (cut site). Now, the defeinsin is active. It inserts into the nearest exposed membrane or unfolds a toxin
47
What are the two things defensin can do?
- Tear holes in microbes
- Unfold toxins
*there is also alpha and beta defensin that do the same thing but are just at different sites
48
Mannose-binding lectin (MBL)
- An immune pattern recognition molecule (PRM) that circulates in the blood. When it reaches infected sites, it binds to a mannose pattern. Mannose is common on the surface of bacteria and fungi. MBL can tag many microbes since they typically build cell walls covered in repeated arrays of molecles like sufars that terminate in mannose.
- Has MASP 1,2, and 3. MASP stands for Mannose-binding lectin (MBL)-associated serine proteases that cut C2 and C4 apart
49
Mannose
Is a microbe associated molecular pattern (MAMP) found on the terminal of microbe cell walls
50
Pattern Recognition Molecule (PRM)
Innate immune molecule that has a free-floating (soluble) and binds to microbial patterns or damage-induced patterns
51
Microbe-Associated Molecular Pattern (MAMPS)
A tell-tale molecular feature of a microbe that's not usually present in a vertebrae host.
Many microbes rely on rigid cell walls and outer cell envelopes that vertebrates don't use
- Unique molecular features that are often present in a repeating pattern
52
Is Lysozyme a Pattern Recognition Molecule?
No, it just cuts wherever without specificity
53
Explain slide 52 on week 1
The diagrams show that gram-positive and negative bacteria have unique markers such as the peptidoglycan cell wall or more specificlaly, gram-negative bacteria having lipopolysaccharide in it's cell wall. Additionally, vertebrae have sialic acid or fucose as our terminal sugar while yeast have mannose
54
Complement proteins
Are a family of innate molecules that are devoted to tagging threats (microbes, damaged host cells, aggregated proteins) for elimination by several means
55
C3
- A central complement protein that tags all surfaces spontaneously or with help from other immune molecules
- Exists as an inactive form with 2 domains, C3a and C3b
- C3 is then cleaved to expose the binding site. The thioester bond is exposed and can either be quenched by water and recycled in the liver or C3b covalently binds to a molecule or a surface.
- All surfaces have hydroxyl or amino groups that can covalently bind to C3b (including pathogens and our own cells). THis is called complement fixation.
- This is a spontaneous process
56
Different roles of complement proteins (what can each kind of complement protein do)
- Many bind to and act on surfaces or large protein complexes (like MBL)
- Many are inactive enzymes (zymogens) until they are cleaved or their shape is changed (activated) by a preceding complement enzyme in a cascade (like MASP 1,2,3,)
- Many amplify a step in a pathway (C4b C3b amplifying the signal)
- Some are receptors on immune cells (CR1)
- Some slow or inactive a step (CD59)
57
Zymogen
A functionally inactive form of a protease which requires cleavage by another protease to become active
58
Protease
An enzyme that specifically cleaves another protein
59
Important characteristics of the complement cascade
- Most complement proteins circulate in blood as inactive proteases that are activated by cleavage. Water or proteases perform the cleavage. Multiple, sequential cleavages cause a complement cascade.
60
Steps of the Alternative Complement Pathway
- Can start spontaneously
- C3 flows freely until it interacts with water to create iC3 which changes the conformation of C3
- This conformational change allows factor B to bind iC3 and open it's cleavage site (since binding to iC3 changes its conformation as well)
- Factor D can bind to the cleavage site in factor B and cut it into Bb and Ba. Factor D leaves and so does Ba but Bb stays attached to the iC3 creating a molecule called iC3Bb
- iC3Bb is a C3 convertase which cleaves C3 into C3a (anaphylatoxin) and C3b which might covalently bond its thioester bond with a hydroxyl group/amino group on a nearby cell or microbe
61
Convertase
Complex of several complement proteins with protease enzyme activity; cleaves an inactive complement protein to activate its activity.
62
How does C3b also act as a convertase?
-Once C3b is bound to the surface, it can then bind factor B and D again to create the C3bBb complex (similar to the iC3Bb complex)
- This complex is a C3 convertase
- The C3b is able to amplify the amount of C3b to the surface since it is already so close to the surface, so any C3 it cleaves will be in close enough proximity to attach it's C3b.
- Results in a lot of soluble C3a and a lot of fixed C3b
63
3 outcomes of Complement Activation
1. Opsonization for phagocytosis
2. Lyse targets with pores
3. Calling for help (inflammation)
64
Opsonization and phagocytosis
Opsonization, tagging something for phagocytosis, allows macrophages to use a set of phagocytic receptors to ingest (phagocytose) any microbe tagged with complement
65
Lyse target with pores in lipid membranes
- C3bBb can be attached to another C3b to create C3b2Bb which is a C5 convertase
- This complex will cleave C5 into C5a and C5b.
- C5a is a anaphylatoxin but C5b will bind C6 and C7 first
- Once these are attached, C8 will attached to form C5b678 into the membrane. C8 is what's anchoring it to the membrane.
- Once attached, many C9s will attached to form a pore called the Membrane Attack Complex (MAC)
- Water and sodium rush into the cell & bursts or destructive molecules in a phagolysosome rush in and kill the microbe
66
Why doesn't complement lyse our own cells? (DRAW)
Regulatory complement proteins like protectin (CD59) are on healthy host cells to prevent assembly of C9 and the Membrane Attack Complex
- Human cells have a lot of CD59
67
Calling for help (inflammation)
- C5a, C4a, and C3a are powerful anaphylatoxins that change blood vessel cells (endothelial cells)
- Induce cells to detach partially from each other (leaky)
- Induce cells to express adhesion molecules to catch blood leukocytes
- Promote local coagulation to wall off the infection
Arriving neutrophils and monocytes use receptors for anaphylatoxins (like the C5a receptor) to activate and chase microbes (chemotaxis once it's slows down from adhesion molecules)
- Arriving monocytes become destructive macrophages
- Activated neutrophils chase microbes
- Phagocytes engulf C3b tagged microbes
68
Anaphylatoxin
Early name for small complement peptides C3a, C4a, C5a because when they are erroneously produced throughout the entire body at once, they are deadly toxins causing anaphylactic shock
69
What are the three ways to start a complement cascade?
1. Lectin Pathway
2. Classical Pathway
3. Alternative Pathway
70
Which complement cascades start by pattern recognition?
The Lectin Pathway and Classical Pathway. These pathways are more efficient because they are not spontaneous
71
What convertase do all the complement cascades generate? What are the outcomes of the cascades?
- All pathways generate a C3 convertase
- The three outcomes are still opsonization, lysis of cells with pores, and calling for help (inflammation)
72
Lectin Pathway
- You can have either MBL or Ficolin as the pattern recognition molecules. They both have 3 MASP associated with them
- If you have MBL, it will bind to mannose and the change in conformation will activate the serine proteases
- MASP-2 cleaves C4 into C4a and C4b. C4b will attach to the microbial surface (similarly to C3b)
- MASP-2 will then cleave C2 into C2a and C2b
- In this case, C2b is the bigger piece and will bind onto C4b to create the C4bC2a convertase for C3
- This convertase will split C3 into C3a and C3b the same way that we see other C3 convertases in the alternative pathway
73
Classical Pathway
- C-reactive protein binds phosphorylcholine in lipid membranes which is common on microbes or damaged host cells
- C-RP provides a landing pad for complement protein C1q that carries serine proteases C1rs. The whole structure is called C1qrs
- Proteases (C1r and C1s) are activated when C1q binds. Then they cleave C4 and C2 to make C3 convertase (C4bC2a)
* Instead of having C-reactive protein as a landing pad, a C1q can also land on the FC portion of an antibody
74
Name all the C3 convertases and draw how they form
- iC3Bb convertase aka C3(H20)Bb (fluid phase)
- C3bBb convertase (alternative)
- C4bC2a convertase (Lectin and classical)
75
Name all the C5 convertases and draw how they form
- C3b2Bb (alternative)
- C4bC2aC3b or C4b2a3b
76
Does the classical & lectin pathways add a C4b to C4b2a to also create another C5 convertase?
No, you can only have a C4b2a3b
77
What are some ways complement is regulated (including the molecules) ?
1. Soluble C3b is rapidly inactivated by water and proteases (proteases chew them up)
2. Complement proteins are continually degraded
3. Most complement proteins act only when bound to a surface
4. Some regulatory complement proteins INACTIVATE surface-bound convertases. Host make them; microbes don't... Like protectin (CD59)
5. Some regulatory complement proteins bidn to microbes and stabilize complement one microbes... like Properdin (factor P)
78
Properdin (Factor P)
-Up-regulates complement action on microbes
- Binds to a microbe surface and stabilizes the short-lived C3 convertase since it degrades in minutes (C3bBb)
- Factor P is a pattern recognition molecule, so once it finds C3bBb, it stabilizes it by binding to the pathogen surface
- Factor P is secreted by neutrophils
79
C1 INH
- C1 INH permanently inactivates the serine proteases (C1r and C1s) on C1q (by dissociating/removing them)
80
Which complement proteins down-regulate complement activity?
- C1 INH
- Factor I
- CD59
81
Factor I
- Healthy host cells use complement regulatory proteins to deactivate convertases
- Factor I cleaves C4b with the help of other complement regulatory proteins to inactivate the C3 convertase
- DAF, C4BP, and CR1 displace C2a from the complex so that C4b can be bound by C4BP, MCPm or CR1 so that Factor I can come in and cleave it into C4d and C4c
82
How does a healthy cell protect itself against complement proteins?
- Protectin
- Factor I
83
Microbes Smaller than ______ are readily phagocytosed by macrophages
70 microns (everything except fungi and parasites)
84
Microbes Smaller than ____ are readily phagocytised by neutrophils
30 microns
85
What do you need for efficient phagocytosis?
Different types of receptors transmit signals simultaneously for efficient phagocytosis
ex.
When C3b binds to only CR1, the bacteria are not phagocytosed. C5a must bind to C5aR, a G-protein coupled receptor, to actually move the exoskeleton
86
What are some phagocytic receptors that recognize microbes?
- Complement Receptor
- Lipid Receptor
- Scavenger Receptor (binds sugars)
- Dectin-1 (binds sugars)
- Fc receptor (antibodies)
- Mannose receptor (binds sugar)
*neutrophils have most of these too
87
What are receptors that can just bind microbes or damage called? (on phagocytes)
Pattern Recognition Receptors (PRRs)
*examples are mannose receptor, dectin-1 etc. This is different from just having receptors that bind to opsonins
88
Damage Associated Molecular Patterns
A molecule that is formed during the infection process, inflammatory response or cell damage. DAMPS can be by-products of microbes or host-derived molecules (like cyclic dinucleotides or reactive radicals)
89
Which pathogens does dectin-1 target?
- Bacteria
- Fungi
90
What are the ligands of dectin-1 (SR-E2) ?
- Mycobacterial ligand
- Beta-glucans
91
Which pathogens does C3 and C4 target?
- Bacteria
- Fungi
92
What are the ligands for C3 and C4
Oligosaccharides, proteins, Beta-glucans
93
Chemotaxis
Cellular movement in response to chemicals in the environment like chemokines or microbial patterns
94
How does a neutrophil recognize and chase bacteria like Staphylococcus aureus bacteria?
- Many bacteria use a formulated form of methionine (f-Met) to make their proteins. Vertebrates don't use f-Met
- Bacteria shed a LOT of tripeptide, f-MLF which is a MAMP
- Neutrophils and macrophages use the PRR, N-formyl-Met receptor to recgonize f-MLF
- F-MLF receptor allows the cell to alter its cytoskeleton and move towards (chemotaxis) f-MLF
95
How does a neutrophil bind and remove bacteria like Staphylococcus aureus bacteria? (Respiratory Burst)
- Multiple receptors cooperate such as the CR1, f-MLF R, and C5aR or C3aR
- fMLF receptor with bound f-MLF activates Rac2 with the C5a receptor, which is a signal GTPase that will associate subunits of the NADPH oxidase in the cytosol (p67, p40, p47) and (b558 subunits in the plasma membrane p22, gp91)
- A phagosome will form from the CR1 binding the C3b and fuse with the primary and secondary granules, and the lysosome to form the phagolysosome
- The complete NADPH oxidase will be in the membrane of the phagolysosome
- Active NADPH oxidase transfers electrons from NADPH to molecular oxygen, forming the superoxide ion O2- and the other free oxygen reactive radicals (OCl-) in the lumen of the phagolysosome.
- Potassium and hydrogen ions are then drawn into the phagolysosome to neutralize the charged superoxide ion, increasing acidification of the vesicle.
- Association dissociates granule enzymes such as cathepsin G and elastase from the proteoglycan matrix, leading to their cleavage and activation by lysosomal proteases (pac-mans)
- O2 is converted by superoxide dismutase (SOD) to hydrogen peroxide (H2O2) which can kill microorganisms. Superoxide dismutase is only activated in low pH.
96
Respiratory Burst
- Effector mechanism of phagocytes that uses enzymes to form oxygen-based toxic radicals that, in turn, oxidize and disrupt the function of most organic molecules
- The radicals damage ingested microbes and the surrounding cells and tissues
97
How does respiratory burst kill microbes?
- Radicals like the release of hydrogen peroxide unfold a protein, inactivating it and causing serious damage.
- Unfolded proteins tend to aggregate so that they can be chopped up by proteases
- Or the unfolded protein itself will kill the bacteria without having to be chopped up
98
Compare and contrast the abilities of macrophages and neutrophils when it comes to attacks and lifespan
- Macrophages can phagocytose larger microbes, but they have less diverse antimicrobial effectors than neutrophils (they also don't have granules)
- Neutrophils are more destructive killers with so many types of granules. But they are short lived
99
What are granules loaded with? And when can they be activated?
- They are loaded with lysozymes, proteases, defensins, elastase, lactoferrin, NADPH oxidase subunits, iron-sequestering proteases, matrix proteases (LPDELIM)
- They act in the altered pH of the phagolysosome
100
What is another way granules can kill things (not intracellularly)
They can be released (degranulated) at an inflammed site to kill microbes
101
What engulfs a neutrophil?
Macrophages. Neutrophils die within a day.
102
Netosis
Neutrophil extracellular Traps (NETs)
- One form of programmed cell death mainly used by neutrophils to trap and kill extracellular microbes
- Charged NETS of DNA carry defensins and proteases that degrade cell walls
- Nets trap and prevent microbes from spreading through tissues
- This occurs when neutrophils sense a DAMP through their PRR in the cytoplasm of the cell or the cell membrane, like sensing super-oxide
- Also, a build-up of cytokines can induce the neutrophils to reach this net state
103
What are the signaling PRRs that start inflammation?
- TLRs
- NOD and NOD-like receptors
- Other CARD domain receptors (like NLRP3)
104
How do macrophages generally raise the alarm of danger?
- There are resident macrophages that recognize MAMPs and DAMPs of infection or damage
- Their pattern recognition receptors transmit signals to activate cytokine gene expression
- They release pro-inflammatory cytokines
- The cytokines instruct neighboring cells and call help... neutrophils are the first wave of blood leukocytes to arrive
105
Membrane associated PRRs that signal
TLRs
106
Cytosolic PRRs that signal
- NOD proteins
- CARD domain family such as NLRs/inflammasomes, RLRrs, and cGAS-STING
107
Signaling pattern recognition receptors
Include Toll-like receptors, NODs, NOD-like receptors and RLRs. They activate transcription factors for expressing cytokine responses (as well as other genes)
108
What do signaling PRRs induce?
Cytokines
109
TLRs
- Toll-like receptors are membrane-associated
- Different ones on the plasma membrane (like TLR4) or endosomal membrane (like TLR3)
- Bind MAMPS/DAMPS in the extracellular environment or endosomes and phagosomes
- Activate transcription factors (NFkB and IRFs) for genes
- Induce inflammatory cytokines and some interferons
110
What do all pattern-recognition molecules bind?
MAMPS AND DAMPS
111
How many domains do TLRs have and what are they?
- TLRs have 2 functional domains to send signals
1. Leucine-rich repeat (LRR) domain - binds MAMP or DAMP and then dimerizes
2. Intracellular TIR domains gather adapter proteins (or kinases) , either MyD88 or TRIF
112
Protein domain
Structural part/area with a particular conserved function, like binding to another protein or shielding a hydrophobic site
113
When do TLRs transmit signals?
-When they dimerize because their TIR domains are together to send a signal
114
When do TLRs dimerize?
When they bind a ligand
115
How does TLR1/2 dimerize?
- The convex surfaces of TLR-1 and TLR-2 have binding sites for lipid side chains of triacyl lipopeptides. The TIR domains are far apart at this point and can't bind adapters
- Binding of each TLR to the same lipopeptide induces dimerization, bringing together their cytoplasmic TIR domains so it can bind adapters and transmit a signal
116
How does TLR4 dimerize?
- Lipopolysaccharide has multiple fatty-acyl chains linked to a glycan head. Five acyl chains can bind to a pocket within MD-2, but one acyl chain is free
- The free acyl chain of an LPS molecule then binds to the outer convex surface of another TLR-4 molecule, inducing a dimer
- Then a signal can be transmitted
117
Are most TLRs homodimers or heterodimers?
Most TLRs will form homodimers
118
Which TLRs are heterodimers?
TLR-1 and TLR-2 (triacyl lipopeptides)
TLR-2 and TLR-6 (diacyl lipopeptides)
119
Which TLR has MD-2 attached?
TLR-4
120
Which TLRs are plasma membrane bound?
- TLR-1
-TLR-2
-TLR-6
- TLR-5 (flagellin)
- TLR-4 (LPS)
121
Which TLRs are endosomal membrane bound?
- TLR-3 (dsRNA)
- TLR-7 (ssRNA)
- TLR-8 (ssRNA)
- TLR-9 (CpG DNA)
122
Important Facts about TLR4
- Chromosome: 9
- Ligand: Lipopolysaccharide (LPS)
- Microorganism recognized: Gram-negative bacteria
- Cells carrying receptor: macrophages, dendritic cells, mast cells, eosinophils
- Location: Plasma membrane
123
MD-2
An accessory protein that helps TLR4 bind LPS
124
Describe the Transmission signal of TLR4
- When 2 TLR chains dimerize (with the help of MD-2), 2 MyD88 adapters and 2 IRAK4 kinases are brought together
- The 2 bound IRAK4s activate one another (kinases)
- Then, they phosphorylate ligases (TRAF6) and start a kinase cascade leading to IKK activation
- IKK phosphorylates IkB inducing its degradation and the passage of NFkB to the nucleus
- NFkB (transcription factor) induces transcription of cytokine genes. The inflammatory cytokines are synthesized in the cytoplasm and secreted via the ER
125
Transcription Factor (TF)
Like NFkB, AP-1, and Interferon response factors (IRFs) are proteins that enter the nucleus and bind promoters or enhancers on genes. They increase the frequency of gene transcription, More than one type of TF is required, and hundreds of the same TF are required. A TF, like NF kB drives any gene that has the element to bind NFkB
126
MyD88
Adapter protein
127
What is the general scheme for TLR
1. PRR that binds MAMP
2. Adapter protein
3. Signaling enzymes/kinases
4. Inhibitor transcription factor
5. Active transcription factor
6. Gene expression
128
NOD2 Protein
- Binds MAMPs or DAMPs (viruses) that are in the cell cytosol
- Transmit signals
- Activate key transcription factors (NFkB and IRFs) for genes
- Express pro-inflammatory cytokines, interferons and enzymes like nitric oxide synthase
129
Give me an example of 3 DAMPs
- Reactive Oxygen Radicals
- NLRP1
- NLRP3
| lowkey idek what i put this it dont seem right
130
How many functional domains do NOD PRRs have and what are they?
They have 3 domains
1. Leucine-rich receptor - binds MAMPs
2. Nucleotide-binding and oligomerization domain (NOD) - gathers other NOD-carrying PRRs
3. Caspase-recruitment domain (CARD) binds other molecules, like caspases and kinases that have CARDS
(when RIP2 kinase binds, it unfolds and becomes an active serine/threonine kinase)
131
What is the general scheme for NOD proteins?
- PRR is NOD2
- There is no adapter protein
- Signaling enzymes
- Inhibited transcription factor
- Active transcription factor
- Proinflammatory cytokine genes secreted or stored as inactive form
132
Describe the transmission signal for NOD2
- Bacteria degraded in the macrophage phagolysosome release muramyl dipeptide that binds to and dimerizes NOD2 proteins
- Dimerization of NOD2
- RIP2 kinase binds, unfolding and becoming an active serine/threonine kinase
- RIP2 phosphorylates TAK1 which phosphorylates IKK
- IKK phosphorylates IkB, degrading it from NFkB so it can bind to the DNA in the nucleus and start transcribing genes for cytokines or interferons
133
NLRP3 forms an inflammasome
- Family of NOD-Like Receptors (NLRCs and NLRPs)
- In the cytosol
- Some recognize MAMPs, many recognize DAMPs (superoxide, ion flunctuation)
- Assemble a structure with active caspase enzymes (inflammasome) that results in the release of powerful pro-inflammatory IL-1B and IL-18. Often involves cell death (pyroptosis)
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What are the two types of inflammasomes and what are the differences?
- Caspase-1 inflammasome (allows for the activation of IL-1B and IL-18)
- Caspase-4 inflammasome (allow for the release of cytokines)
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Inflammasome
Protein complex that assembles and activates caspase enzymes (a big wheel of enzyme activity) for potent inflammatory actions
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How many functional domains do NLRPs have?
3 functional domains
- LRR
- NOD -gathers other NOD PRRs
- Pryine domain - binds other proteins with pyrin domains
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How does the caspase-1 inflammasome work?
- A priming step (TLR stimulation induces expression of the pro-forms of IL-1B and IL-18
- Some NLRs detect pathogens directly (PAMP) while others response to pathogen activities or the change in potassium efflux. This activation triggers a conformational change (the HS90 leaves) that enables oligomerization (having the NLRP come together instead of monomers) and recruit the inflammatory protease caspase 1
- First, the PYCARD domain binds to the pryin domain of the inflammasome. It brings caspase 1 in its inactive form
- In unstimulated cells, caspase 1 is inactive, but binding to the assembled inflammasome (at the pryin) activates its protease activity.
- Active caspase 1 cleaves pro-IL-1B and pro-IL-18
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Caspase
a type of enzyme that cleaves inactive forms of proteins to their inactive forms. Caspases exist in inactive pro-enzyme forms until cleaved by a kinase in the inflammasome. Then, the active caspases cleave their targets, like pro-IL-1B
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Different ways inflammasome is activated
- Directly by NLR that recognizes MAMPS, like bacterial flagellin (then the individual NRLs form an inflammasome)
- By combined signals from TLRs and NLR that recognize MAMPs
- Infection-associated damage (DAMPS), like reactive oxygen intermediates or phagolysosomal contents being spilled into the cytoplasm
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Pyroptosis
- A form of programmed cell death that releases stored cytokines and disrupts the replication of any obligate intracellular pathogens, like viruses
- Occurs during inflammation in heavily infected macrophages, and their released contents recruit more immune cells (inflammation)
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Which caspase cleaved gasdermin D
Caspase 4. It works similarly to other inflammasomes but its sole purpose is to cleave the pro-region of gasdermin D (C) so it can go into the plasma membrane (amphipathic)
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What does NOD2 bind?
Muramyl dipeptide (MDP)
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Which effector mechanisms act extracellularly?
Degranulation & Netosis
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What are the 4 cardinal signs of inflammation?
- Heat
- Redness
- Swelling
- Pain
*loss of function
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Inflammation
Innate response to injury or infection. It involves many simultaneous responses, locally (site of infection) & systematically (body wide)
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What are the first wave of cells to arrive to a inflammation signal?
Neutrophils. Then monocytes and lymphocytes
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Endothelial cells
- Blood vessels are composed of endothelial cells
- Endothelial cells have MANY cytokines and chemokine receptors
- Endothelial cells are activated by cytokines, like TNF-alpha. Then, they express new adhesion molecules and chemokines
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Local Inflammation Response
- Endothelial cells are activated by cytokine , like TNF-alpha. Then, they express new adhesion molecules and chemokines
- Professional phagocytes arrive in response to TNFa and chemokines, like CXCL8 and CCL2, become activated by the cytokines, chemokines and MAMPs and act against microbes
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Cytokines
- IL-1B
- TNF-a
- IL-6
- CXCL8
- IL-12
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IL-1B
- Master cytokine for inflammation is very potent and exerts local and systemic effects. Made and stored in cytosol of macrophages in an inactive form. Must be activated by caspases and released from the cell to act
- Activates vascular endothelium
- Activates lymphocytes
- Local tissue destruction
- Increases access of effector cells
Systemic EFfects:
Fever
Production of IL-6
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TNF-a
- Activates vascular endothelial and increases vascular permeability, which leads to increased entry of IgG, complement, and cells to tissues
- Also leads to increased fluid drainage to lymph nodes
Systemic Effects:
- Fever
- Mobilization of metabolites
- Shock
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IL-6
- Activation of Lymphocyte
- Increased antibody production
Systemic Effects:
- Fever
- Induces acute-phase protein production
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CXCL8
- Chemotactic factor recruits neutrophils, basophils, and T cells to the site of infection
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IL-12
- Activates NK cells
- Induces the differentiation of CD4 cells into TH1 cells
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What receptors do cytokines act by?
JAK-STAT Receptors
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JAK-STAT receptor and cytokines pathway
- Cytokine receptors (on endothelial cell) consist of at least two chains, the cytoplasmic domains of which bind Janus Kinases (JAKs)
- The cytokine binds and dimerizes the receptor chains, bringing together the cytoplasmic JAKs, which activate each other and phosphorylate the tail of the receptor itself
- The phosphorylation of the receptor produces a docking site for inactive STAT transcription factor chains. Once they bind, they are phosphorylated
- Phosphorylated STATs form dimers that translocate into the nucleus to initiate gene transcription
(STATs are transcription factors)
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What do chemokines start with? What does the TNF family usually have?
- CC or CX
- an alpha
- IFN for inteferons
- IL-1 family is IL-1B and IL-18
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What are some of the actions of cytokines
- Cytokines exert multiple effects (they are pleiotropic)
- Autocrine action (when they act on the same cell that made it)
- Paracrine action (when they work on other cells)
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CCL2
Key pro-inflammatory chemokines
- Calls monocytes and T cells, activates cells to
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4 Steps of Extravasation
- IL-1B, TNFa, C3a and C5a activate endothelial cells
- Activated endothelial cells near inflamed sites express adhesion molecules like E-selectin, ICAM-1, and CD31 and chemokines (like CXCL8 & CCL2)
Steps:
1. Rolling Adhesion
2. Tight Binding
3. Diapedesis
4. Migration
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Which cytokines are being expressed on the endothelial surface
CXCL8 and CCL2
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Diapedesis
Act of breaking through the basement membrane into the tissue
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What is the ligand for ICAM-1?
LFA-1
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What is the ligand for E-selectin?
s-Lex
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Extravasation
The process of leaving the bloodstream.
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Types of Adhesion Molecules
- Selectin (sugar binding interactions) (e-selectin)
- Integrins (think of integrins like a claw that grabs onto ligands and holds) (LFA)
- Immunoglobulin super family (ICAMs) (the ligand for ICAMS are integrins like LFA)
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Acute-phase Response
Inflammatory change in the immune proteins in the bloodstream during the early phase of an infection. The acute-phase response is due to pro-inflammatory cytokines activating liver cells to produce acute-phase proteins with immune functions (IL-6)
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How do cytokines act distantly in the body?
-TNFa, IL-1B, and IL-6 act on the liver, bone marrow endothelium, hypothalamus, and fat/muscle
- Liver: crates acute phase proteins (C-reactive protein, mannose-binding lectin) which eventually leads to activation of complement and opsonization
- Bone marrow endothelium: neutrophil mobilization which leads to phagocytosis
- Hypothalamus: increased body temperature which leads to decreased viral and bacterial replication, increased antigen processing, increased specific immune response
- Fat, muscle: protein and energy mobilization to allow increased body temperature which leads to leads to decreased viral and bacterial replication, increased antigen processing, increased specific immune response
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What are all the important parts of the immune system made in the liver during the acute-phase response?
- C-reactive protein
- Mannose binding lectin
- LBP (binds LPS so it can give it to MD-2 in TLR4 easier)
- Complement components C3, C4, C9, factor B
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Give example of 3 opsonins
- C3b
- C-reactive protein
- MBL
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Can receptors in the cytosol recognize viruses?
Yes like NLR and RIG-1
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What are the unique challenges that viruses present?
- Obligate intracellular microbes hide from antibodies and complement
- Hijack host cellular machinery
- Look much like us to innate immune cells
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Obligate intracellular microbe
Must invade a cell to replicate. Viruses are examples that must invade the cell cytosol. Some bacteria too
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What interferons can all cells make?
IFNa and IFNb
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TLR7 pathway
- TLR7 in the endosome binds ssRNA and signals MyD88 to activate IRAK1 and IRAK4
- IRAK1 and IRAK4 activate (TBK1 also a signaling kinase)
- Phosphorylate the transcription factor IRF7 which drives gene expression to make Type 1 interferons like IFN alpa and beta
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What does TLR7 produce?
Interferons (b and a)
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Type 1 Interferon
They instruct any cell to resist intracellular infection
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What are the first types of cells to be infected with virus?
EPITHELIAL cells (not endothelial). They resist.
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Cytosolic PRRs (these are not the same as signaling cytosolic PRRs)
They are freely floating in the cytosol similar to how PRMs like MBL
- Rig-I which binds dsRNA
- MDA-5 which binds dsRNA
- cGAS which binds DNA
All cells use these receptors
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RLRs (RIG-I like receptors)
are a family of cytosolic PRRs in most cells that have CARD domains ad bind nucleic acid patterns
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How many functional domains do RLRs?
1. C-terminal domain- stabilizes
2. Helicase domain - bind nucleic acid MAMPs
3. CARD binds CARDs of other molecules (link together)
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What are the 3 general strategies for interferon response?
1. Warn neighbors
2. Resist - make it difficult for viruses to replicate/assemble
3. Call for help from Natural Killer (NK) cells
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Interferons warn neighbors
- Virus infects an epithelial cell that responds by secreting the cytokine interferon (IFN-B)
- IFN-B binds type I IFN receptors on adjacent uninfected cell, giving a paracrine IFN-B response
*IFN-B is the easiest to induce
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Resist Viral Replication/The Antiviral State
- Type-1 IFNs act on the cells that made them... induce transcription factors (IRFs) that resist viral replication, assembly, and budding (GOAL)
THE ANTIVIRAL STATE:
- Cells slow synthesis of ALL RNA, DNA and proteins
- Degrades mRNA more quickly
- Expresses protein kinase R that prevents protein synthesis
- Expresses more MHC I on the surface to show T-cells what's happening
- Prepares to undergo programmed cell death if instructed to by NK cells
-Uses tetherin protein to block enveloped viruses from budding from cell and spreading
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What does TBK1 always actiate
IRF7
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What does IKK always activate
NFkB
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How does RIG-I work?
- RIG-I will recognize a very long piece of dsRNA in the helicase domain and hold it in place in its C terminal domain.
- The CARD domain on RIG-I will bind to MAVS on other organelles to anchor the entire trascription process
- TBK1 route activates IRF7
- TRAF, IKK route activates NFkB
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Which IFN is released first in the interferon response?
First IFN-beta then alpha
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Call for help from NK cells
- Infected cells use Type I interferons to call for help from NK cells
- Large quantities of IFNa and IFNb recruit NK cells by binding to the NK cell's JAK-STAT IFN-alpha receptor (instead of a cytokine binding to the JAK, the IFN does)
- NK cells patrol and migrate into inflamed sites then activate and kill infected cells
- Some NK cells are specialized to patrol certain tissues like the uterus
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Natural Killer Cells
First to kill cells that show signs of stress
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Innate Lymphoid Cell (ILC)
-Lymphocytes sense MAMPS/DAMPS or altered self and provide very early cytokine warnings at an infecte site or in a secondary lymphoid tissue. Notice that ILC1, ILC2, and ILC3, make different cytokines (effector molecules) so they drive different responses
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Which family are NKs apart of?
ILC family
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Which cytokines induce NK cells and which ones do they produce?
Induced by: Type I IFN, IL-12, IL-18
Create: IFN-y, perforin, granzyme
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Which cytokines induce IL 1 cells and which ones do they produce?
Induced by: IL-12, IL-18
Create: IFN-y
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What types of pathogens to NK cells take care of?
Viruses and intracellular pathogens
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How do NK cells kill cells?
- Immature (resting) NK cells can't kill.
- The IFN response will bind to a NK cell, driving the proliferation of multiple NK cells
- Type I interferon drives differentiation of NK cells into cytotoxic effector cells (they not contain molecules that will kill cells)
- Effector NK cells kill virus-infected cells by inducing apoptosis (NK cells must be very close and can only kill one cell at a time)
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Apoptosis
All DNA (and microbial DNA/RNA) is destroyed and contents are trapped in vesicles (apoptotic bodies) so that a macrophage can engulf them
*granzymes in NK cells cause this by activating caspases in the target cell that drive the programmed cell death
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What exactly do NK cells recognize when they bind to infected cells?
- They do not recognize MAMPs. They recognize changing molecules on infected host cells that are stressed. THis is known as altered self
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Altered self
Ligands made and expressed by host cells to intentionally activate host defenses. For example, stressed cells express stress molecules that are recognized by activating receptors on NK cells
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How is killing by an NK cell regulated?
1. NK cells can only kill after they differentiate into an activated effector cell
2. NK cell can only kill one at a time, so healthy bystanders are safe
3. NK cell is prevented from killing if there are many inhibitory ligands (i.e., HLA-E)
4. NK cell can only kill if multiple activating ligands are present (i.e. MIC and CD48)
5. Only kill in close proximity
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What do infected cells express that NK cells recognize?
MIC and CD48
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Why doesn't a NK infect a cell with HLA-E
Because the HLA-E is binded to NKG2A which inhibits the release of granules
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If there are more activating signals, than inhibitory ones...
NK cell kills
- The cytotoxins are granzymes and perforin in the granules
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HLA-E
- A healthy cell expresses LOTS of inhibitory ligands like HLA-E to prove the cell is making lots of other MHC I molecules
- THe only way that HLA-E can go to the surface is by carrying a piece from another type of HLA molecule (from the endoplasmic reticulum) that can show if the cell is infected
- Inhibitory receptor NKG2A binds HLA-E and blocks any activating signals (that are received by the NK cell)
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How did ELISAs help Dolly?
You can do an ELISA for anti-C8
- Have IgM which can be bound by C1qrs.
- C1qrs will cleave C4 and create the membrane attack protein
- You'll have an antibody detect for C9
- If there is color, you know that there is C8
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Double Diffusion Agar Assay
Dolly's serum was in the center and you could have an anti-C8 antibody to detect C8. If there was any, you'd see streaks
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CH50 Assay to measure lysis by MAC
- Sheep red blood cell with antigen that rabbit antibodies bind
- Add the patients serum at different dilutions to figure out when red blood cells are lysed (because remember the complement proteins are contained in this so the C1qrs can start up the pathway by binding to the antibody)
- If the RBC is lysed, then all complement proteins are there and work
- If the RBC are not lysed, then it does not tell you specifically which part of the MAC doesn't work
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Name the anitmicrobial mechanisms
- Acidification: low pH
- Toxic-oxygen derived products: superoxide, hydrogen perozide
- Toxic Nitrogen oxides: NO
- Antimicrobial peptides: defensins
- Enzymes: lysozyme
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Which receptors do NK cells use to recognize activating ligands?
NKG2D for MIC and 2B4 for CD48
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Which receptors do NK cells use to recognize HLA-E
NKG2A
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