1
Q
Innate vs adaptive immunity
A
- innate: rapid but non specific
- adaptive: long term and specific
2
Q
The innate immune system acts through
A
- surface barriers: skin hair, mucous membranes, endothelium tight junctions etc.
- Chemical barriers: defensins, complement system, stomach acid, sweat, urine, tears, fatty acids of the skin
- Microbial barriers: microbiome of epithelium
- Inflammation: triggered by cytokines and histamines, increased vascular permeability (edema)
- Complement system: killing of pathogens by antibodies
- Cellular responses: Phagocytes and NK cells
3
Q
Functions of the epithelia
A
- Physical barrier to infection
- Killing of microbes by locally produced antibodies
- Killing of microbes and infected cells by intraepithelial lymphocytes
4
Q
PRRs (Pattern Recognition Receptors)
A
- germ line encoded receptors on cells of the innate immune system.
They recognize: - molecules that are broadly shared by pathogens, but distinguishable from host
molecules. - signals from damaged, injured, or stressed
cells (DAMPs-Danger Associated Molecular Patterns).
5
Q
defensins
A
anti-microbial peptides, cationic peptides that disrupt cell wall integrity
6
Q
opsonins
A
proteins that bind to bacteria?
7
Q
Purposes of inflammation
A
- Establish a physical barrier to prevent spread of the infection
- Eliminate the initial cause of cel injury
- clear out dead cells and damaged tissues
- initiate tissue repair
8
Q
3 steps of inflammation
A
- Inflammation is triggered by cytokines and histamines
- Increased vascular permeability
- Leukocyte migration and pathogen destruction
9
Q
4 Steps of Leukocyte trafficking to the site of inflammation
A
- Rolling adhesion: selectins bind to glycosylated ligands
- Tight binding integrins bind to adhesion molecules
- Diapedesis (leukocyte extravasation)
- Migration: chemokine gradient
10
Q
Complement System and how is it activated
A
- alerts the phagocytic cells of the immune
system to uptake and destroy the pathogens and facilitates clearance of debri - can be activated directly by pathogens or
indirectly by pathogen-bound antibody (assistance with humoral immunity)
11
Q
Complement proteins
A
- Plasma proteins synthesized in the liver that are part of the complement system
- exist in the plasma and on cell surfaces as
inactive precursors (zymogens) that are activated at the site of inflammation. - act as proteases and cleave one another in
the cascade - activation leads to a cascade of reactions that occurs on the surface of pathogens and generates active components with various effector functions
12
Q
Effects of the complement cascade
A
- Opsonization of invading pathogens mediated by coating of pathogens by C3b and IgG activation of phagocytosis
- Clearance of immune complexes and cellular debris
- Chemotaxis mediated by release of C5a into bloodstream
- Direct cytolysis of pathogens through formation of the membrane attack complex (MAC)
- Vascular changes such as dilation caused by immune reactions to C3a, C4a and C5a (Anaphylaxis if released systemically)
13
Q
3 pathways that activate and are a result of the complement system
A
Pathways to activate:
- Classical: Antigen-antibody complexes
- MBL: Lectin binding to pathogen surfaces
- Alternate: Pathogen surfaces
Results of activation:
- Recruitment of inflammatory cells
- Opsonization of pathogens
- Killing of pathogens
14
Q
C1 complex
A
- C1 activation initiates the Classical Pathway
- C1q links the adaptive humoral immune response to the complement system by binding to antibodies complexed with antigens
15
Q
Classical Pathway of Complement Activation slide 19
A
- Activated C1s cleaves C4 to C4a and C4b
- C4b then binds C2 which is cleaved by C1s, to C2a and C2b, forming the C4b,2b complex
- C4b,2b cleaves C3 into C3a and C3b
- C3b binds to the C4b,2b complex or to the microbial surface
- 1 molecules of C4b,2b can cleave up to 1000 C3s, many C3b molecules bind to the microbial surface
16
Q
MBL pathway of complement activation
slide 20
A
- MBL forms clusters of 2 to 6 carbohydrate-binding heads around a central collagen-like stalk
- Associated with this complex are 2 serine proteases (MASP-1 and 2)
- On binding of MBL to bacterial surfaces these serine protease become activated and can then activate the complement system by cleaving and activating C4 and C2
17
Q
Ficolin vs MBL
A
- similar in structure to MBL but bind to oligosaccharides containing acetylated sugars rather than mannose and fructose residues
18
Q
Alternative pathway of complement activation
Slide 22
A
- C3 undergoes spontaneous hydrolysis to C3(H2O), which binds to factor B, allowing it to be cleaved by factor D into Ba and Bb
- The C3(H2O)Bb complex is a C3 convertase, cleaving more C3 into C3a and Cb. C3b is rapidly inactivated unless it binds to a cell surface
- Factor B binds noncovalently to C3b on a cell surface and is cleaved to Bb by factor D
19
Q
Activation of complement effectors is triggered by
A
- PAMP, Antigen-Ig, and spontaneous C3 activation
20
Q
Effector function of C3a and C3b
A
- Covalent “complement fixation of” debris and cells
– Clears serum debris through blood cell (RBC) “garbage truck” mechanism (via CR1, CR3)
– “Co-stimulates” B cells (via CR2)
– Activates DC, macrophages and neutrophils (CR3,CR4)
– C3a,C5a: WBC activation through GPCR
– C5b,6,7,8,9 : Bacteria and mammalian cell lysis through membrane attack complex (MAC)
21
Q
Formation of membrane attack complex
A
- C5b binds C6 and C7
- C5b67 complexes bind to membrane via C7
- C8 binds to the complex and inserts into the cell membrane
- C9 molecules bind to the complex and polymerize
- 1-16 molecules of C9 bind to form a pore in the membrane
22
Q
Opsonization
A
- Improves phagocytosis
1. Bacterium is coated with complement and IgG antibodies
2. When C3b binds to CR1 and antibody binds to Fc receptor, bacteria are phagocytosed
3. Macrophage membranes fuse, creating a membrane-bounded vesicle, the phagosome
4. Lysosomes fuse with these vesicles, delivering enzymes that degrade the bacterium
23
Q
C5a mediated phagocytosis of opsonized bacteria
A
- Bacterium is coated with C3b
- When only C3b binds to CR1, bacteria are not phagocytosed
- C5a can activate macrophages to phagocytose via CR1
24
Q
Know the complement system summary slide (slide 27)
A
25
Cells involved in innate cellular immunity
Professional Phagocytes: Neutrophils, Dendritic cells, Macrophage
Non-professional phagocytes: Epithelial cells etc.
26
Role of Neutrophils phagocytosis in innate cellular immunity
Elimination of invading microorganisms by intracellular degradation (Host defense against exogenous pathogens)
27
Role of Dendritic cells in innate cellular immunity
Induction of adaptive immunity by presenting antigens to T cells (Host defense against exogenous pathogens)
28
Roles of Macrophages in innate cellular immunity
Elimination of exogenous pathogens as well as host-derived molecules including dead cells (Host defense against exogenous pathogens + Maintenance of tissue homeostasis)
29
Role of non-professional phagocytes in innate cellular immunity
Elimination of neighboring dead cells (Maintenance of tissue homeostasis)
30
Phagocytosis
- Ingestion of particles larger than 0.5 um in diameter
- Specialized form of endocytosis
- Rapid changes in microtubules, actin filaments, membrane geometry, formation of phagocytic cup
- Leads to endosome fusion, acidification
31
Pinocytosis
Endocytosis of small fluid particles
32
Autophagy
conserved degradation of a biological cell that removes unnecessary or dysfunctional components through a lysosome-dependent regulated mechanism. It allows the orderly degradation and recycling of cellular components
33
Process of Phagocytosis
- Recognition and attachment of microbe by phagocyte
- Pathogen engulfed
- Internalized, endocytosed - in the phagosome
- Microbe destroyed - phagosome fuses with lysosomes to form phago-lysosome
- Process may damage host
- Reactive oxygen species kill
bacteria
34
Functions of professional phagocytes
- Respond to stranger and/or danger signals
- Chemotaxis
- Homing to sites of inflammation & lymph nodes
- Tissue-specific “fixed” location (eg. Kupffer cells in liver)
- Cytokine production IL-1; IL-6; IL-8; IL-12; TNF, IFN
- Cell killing: ADCC; FasL-dependent killing, tumor-destruction
- Splenic clearing RBC of C3b-linked debris
- Antigen processing and presentation to T cells
- Activation and inhibition of T cells
35
What methods do phagocytes use to kill bacteria?
- Cationic Proteins –⍺ and defensins active at neutral pH,
abundant in phagocytic and immune cells.
- Hydrogen ion – pH drops below 4.0 in the phagolysosome
- Acid-activated proteases and other digestive enzymes
- Reactive molecules O2-, OCl-, NO and others, generated by oxidative burst through NADPH oxidase
- H2O2 triggers release of elastase and NETS from neutrophils.
- NETS –Neutrophil Extracellular Trap (DNA + modified histones)
- Nutritional competition - lactoferrin and
vitamin B12 binding protein sequester critical nutrients
36
How can bacteria subvert phagocytosis
Polysaccharide Capsule to alter PAMPs : Neisseria, Pseudomonas
- Chemical modification of PAMPs to make them undetectable by
the host: Helicobacter, Salmonella
- Avoidance/Scavenging of Opsonization/Opsonins: Ig proteases ex
Streptococcus, Neisseria, Staphylococcus
- Stimulate production of a “floppy” phagosome, block phago-
lysosome fusion & acidification
- Dissolve phagosome membrane and migrate into cytoplasm: Listeria, Shigella
37
Two paths of NETosis - know the figure on slide 35
Non-lytic path (rapid release from live cells):
1. Expulsion of nuclear chromatin and degranulation
2. Extracellular assembly of NET
3. You are left with a phagocytic cytoplast
Other path:
1. Nuclear delobulation and disassembly of the nuclear envelope
2. Cellular depolarization and chromatin decondensation
3. Plasma membrane rupture and release of NETs
38
NETosis
- NETs: Neutrophil Extracellular Traps
- Neutrophils can expel their nuclear DNA to trap microbial pathogens
- It inhibits bacterial movement and facilitates macrophage phagocytosis and killing
39
Pattern Recognition Receptors (PRRs) activate
- Phagocytosis
- Phagolysosomal fusion and microbial killing
- Cytokinesis and chemotaxis
- Cytokine release
- these exist on phagocytes so they can recognize pathogens
40
Neutrophils
- Origin
- Life Span in the Tissues
- Responses to activating stimuli
- Reactive oxygen species
- Nitric Oxide
- Degranulation
- Cytokine production
- Extracellular Traps
- Origin: HSCs in the bone marrow
- Life Span in the Tissues: 1-2 days
- Responses to activating stimuli: Rapid, short-lived, enzymatic activity
- Reactive oxygen species: Rapidly induced by assembly of phagocyte oxidase (respiratory burst)
- Nitric Oxide: low levels to none
- Degranulation: Major response; induced by cytoskeletal rearrangement
- Cytokine production: Low levels per cell
- Extracellular Traps: Rapidly induced, by extrusion of nuclear contents
41
Macrophages
- Origin
- Life Span in the Tissues
- Responses to activating stimuli
- Reactive oxygen species
- Nitric Oxide
- Degranulation
- Cytokine production
- Extracellular Traps
- Origin: HSCs in bone marrow (in inflammatory reactions) but there are also many tissue resident macrophages
- Life Span in the Tissues: Inflammatory: days to weeks, Tissue resident: years
- Responses to activating stimuli: Prolonged, slow, often dependent on new gene transcription
- Reactive oxygen species: Less prominent
- Nitric Oxide: Induced following transcriptional activation of iNOS
- Degranulation: not prominent
- Cytokine production: Major functional activity, large amounts per cell, requires transcriptional activation of cytokine genes
- Extracellular Traps: Little
42
3 major cellular locations of innate receptors
- cell membrane
- endosomal membrane
- cytoplasm
43
Toll-like receptors (TLRs)
- largest class of PRRs
- bind to extracellular PAMPs or intracellular DNA or RNA
Has 3 domains
- ligand binding domain
- trans membrane domain
- signal transducing domain
44
TLR-1:TLR-2 bind to
Bacterial lipopeptides (gram +)
45
TLR-2:TLR-6 binds to
Bacterial lipopeptides (Gram +)
46
TLR-2 bind to
Bacterial peptidoglycan (Gram +)
47
TLR-4 binds to
LPS (Gram -)
48
TLR-5 binds to
- Bacterial flagellin
49
TLR-3 binds to
dsRNA (viruses)
50
TLR-7 binds to
ssRNA (viruses)
51
TLR-8 binds to
ssRNA (Viruses)
52
TLR-9 binds to
CpG DNA (viruses, bacteria, fungi)
53
TLR signaling during bacterial infection
1. Acute inflammation
2. Stimulation of adaptive immunity; number of neutrophils increase
54
TLR signaling during viral infection
- Interferon production and development of
antiviral state; number of lymphocytes increase
55
What can trigger the formation of the inflammasome (multiprotein complex)
- Bacterial products
- Extracellular ATP
- Crystals (uric acid crystals as in gout)
- K+ efflux
- Reactive oxygen species (ROS)
- Viral DNA
56
Scavenger Receptors
A group of large and diverse family of cell surface receptors that bind and remove modified lipoproteins.
57
Ligands of scavenger receptors
polyanionic ligands including
- lipoproteins
- cholesterol ester
- phospholipids
- proteoglycans,
- ferritin
- carbohydrates
- apoptotic cells
58
NK (Natural Killer) Cells
- Innate (or, maybe not!) lymphocytes that can destroy some cancer, allogeneic, and virally infected cells
- Distinguish both self from non-self and DANGER
- Some evidence of memory function!
- An early component of the host response to virus infection
- kill host cells infected by intracellular microbes, thus eliminating reservoirs of infection
- respond to IL-12 produced by macrophages and secrete IFN-gamma which activates the macrophages to kill phagocytosed microbes
- mostly innate cells with some adaptive capability
59
Know graph on slide 46
60
NK cell killing depends on
- the balance between activating and inhibitory signals
- Activating: 2ndary stress ligands
- Inhibitory: MHC class 1
61
ADCC - antibody dependent cell mediated cytotoxicity by NK cell
1. Antibody binds antigens on the surface of target cells
2. Fc receptors on NK cells recognize bound antibody
3. Cross-linking of Fc receptors signals the NK cell to kill the target
4. Target cell dies by apoptosis
62
Innate and Adaptive characteristics of NK cells
Innate:
- Rapid response to infections
- Germline encoded activating and inhibitory receptors rather than rearranged antigen-specific receptors
- Cytotoxic function without prior exposure to the antigen
Adaptive:
- Memory function
- Some antigen specific response
63
Eosinophils
- Upon activation by antibody-coated parasites, eosinophils can release granule proteins (Arg-rich) and free radicals that can “sting” and kill microorganisms and worms.
- part of innate immunity to large parasites
64
Mast cell
- Residents of mucosa and epithelial tissues near capillaries guard against invasion.
- Release granules (acidic) containing many inflammatory compounds.
- Example: Leukotrienes and histamine cause epithelial cell contraction and vascular permeability.
- functions in innate immunity to large parasites
65
When bacteria penetrate the skin or mucous membranes after damage, they are then ...
taken up by resident phagocytes such as macrophages and DCs
66
pathogens and/or damaged barrier cells release ____ or ____ recognized by _____ on ______
PAMPs or DAMPs recognized by PRRs on phagocytes. Phagocytes then become activated
67
Activated phagocytes release....
Soluble factors (e.g. cytokines) that recruit neutrophils and macrophages
- These soluble factors can also increase the permeability of vessels, expression of adhesion molecules and chemical barriers
68
What is the bridge between innate and adaptive immunity
- Phagocytes break down the pathogen proteins into short peptides. DCs are the
primary antigen-presenting cells (APC) in the body.
- APCs travel through the lymphatic system to peripheral lymphoid organs, like
the lymph nodes, where they meet T cells initiate adaptive responses.
69
Examples of signs of inflammation
- Redness
- Pain
- Swelling
- Warmth
- Some loss of function
70
How does the complement system recruit phagocytic cells to the site of infection and promote inflammation
Through C5a and C3a
71
All complement pathways generate
- a C3 convertase, which cleaves C3, leaving C3b bound to the microbial surface and releasing C3a
72
Activation and assembly of the inflammasome promotes the activation of ___ that does ___
- Caspase-1 which cleaves precursor IL-1Beta so that mature IL-1Beta is secreted
- mature IL-1Beta causes acute inflammation which helps fight infection
73
What is the sensor for the inflammasome
NLRP3
74
Missing Self Hypothesis
The idea that NK cells can distinguish aberrant cells by recognizing 'absence of the expected
GS-IY-6200
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