Immunology II: Innate Immunity & Inflammation

Question 1

Who has an innate immune defenses? How long does it take for it to act?

Answer 1

Innate immune defenses exist in all individuals and act within minutes - hours after an encounter with infectious agent

Question 2

Does innate immunity discriminate effectively between host cells and pathogens?

Answer 2

Mechanisms of innate immunity discriminate very effectively between host cells and pathogens

Question 3

When is an adaptive immune response required?

Answer 3

Only when innate defenses are overwhelmed/bypassed/evaded is an adaptive immune response required

Question 4

Barriers chart: What are the 3 types and the 5 places they exsist?

Answer 4

Question 5

What are the 2 chemical barriers?

Answer 5

Lysozyme and antimicrobial peptide - defensins

Question 6

Lysozymes: where do they present? How do they function?

Answer 6

Present in secretions (mucus, tears, milk, saliva)

Uses hydrolysis to break apart the peptidoglycan wall  lysis of bacterial cell wall

Question 7

Antimicrobial peptide - defensins: what are they? What do they kill? What are their effects?

Answer 7

Small, heterogeneous, cationic peptides

killGram-negativeandGram-positivebacteria, some enveloped viruses, fungi

Multiple antimicrobial effects:
Destabilize membranes and Pore formation in bacterial cell walls
Proteolytic degradation of bacterial proteins
Inhibit viral binding and entry
Inhibit virus particle assembly

Question 8

What roles aside from chemical barrier do defensins perform?

Answer 8

Defensins can act as a chemical barrier when they are secreted by epithelial cells in a variety of mucosal surfaces

Defensins and other AMPs (i.e. cathelicidins) are also stored in neutrophil granules and can be released within tissues in response to inflammation

Can kill microbes extracellularly  released when neutrophils die during inflammation

Can kill microbes intracellularly after a cell (i.e. neutrophil) phagocytoses a pathogen
Just like many molecules and cells of the immune system, defensins perform a number of roles – not just a chemical barrier

Question 9

Describe phagocytosis and phagocytes. What is their role? What do they do? Innate or adaptive?

Answer 9

One of the first lines of defense if microbes do invade tissue

Engulf and destroy microorganisms, especially bacteria

Key role in innate immunity as they can recognize, ingest and destroy many pathogens without aid of an adaptive immune response

Phagocytosis can also occur after an antibody has bound to an antigen – the antibody can act as a “signal” that triggers efficient phagocytosis

Question 10

What are the major phagocytes in the body?

Answer 10

Macrophages and neutrophils are the major phagocytes in the body

Question 11

Monocytes & Macrophages:

Answer 11

Pro-monocytes (bone marrow)–>monocyte (blood)–> macrophage/macrophage-like cells (tissues)

Long-lived cells resident within the tissues

Question 12

Neutrophils

Answer 12

Derived from hematopoietic precursors in the bone marrow

Non-dividing, short-lived cell type in blood (dominant WBC)

Question 13

What does PRR stand for?
(chart of Phagocytosis - the basics)

Answer 13

PRR = Pattern Recognition Receptor

Question 14

Pattern recognition- What is it? When does it occur? What does it activate? How does it happen?

Answer 14

Evolutionarily conserved mechanism for recognizing common, conserved ‘signs’ of microbial infection, physiological stress, or other damage
Recognition is immediate, does not require prior recognition, and activates several arms of the innate (and adaptive) immune response
Responses are elicited via the engagement of Pattern Recognition Receptors (PRRs) found on phagocytes, in response to:
Pathogen Associated Molecular Patterns (PAMPs)
Danger Associated Molecular Patterns (DAMPs)

Question 15

Why does the function of macrophages rely on PRRs?

Answer 15

The function of Macrophages relies on (PRRs) which allow a limited range of immune cells to detect and respond rapidly to a wide range of pathogens that share common structures, known as pathogen associated molecular patterns (PAMPs). Examples of these include bacterial cell wall components such as lipopolysaccharides (LPS) and double-stranded ribonucleic acid (RNA) produced during viral infection.

Question 16

Examples of PRRs? What responses do they elicit?

Answer 16

Examples of PRRs:
Toll-like receptors
Nod-like receptors
Lectins
Elicit responses such as:
Phagocytosis
Cytokine secretion

Question 17

Step 1 of phagocytosis?

Answer 17

A pattern-recognition-receptor (PRR) binds to a microbe or bit of debris, OR an opsonin created by another cell binds to the microbe
A microbe
A bit of debris
An opsonin
An opsonin is basically a soluble, secreted PRR that enhances the effectiveness of phagocytosis
An opsonin coats a microbe, the phagocyte has receptors for parts of that opsonin

Question 18

Step 2 of phagocytosis?

Answer 18

2. The microbe is engulfed – the PRR receptors signal the cell membrane to approach, coat and then surround the sites where the receptor is bound
   Forms a phagosome
   Mediated by intracellular signalling events and actin polymerization – see diagram
   PI3 kinase seems to be important here

Question 19

PRRs that trigger phagocytosis

Answer 19

Question 20

What is an example of a lectin receptor?

Answer 20

Mannose receptor

Lectin receptors
(lectin receptors recognize “carbohydrate patterns”)

Question 21

What are 2 examples of scavenger receptors?

Answer 21

SR-A and SR-B

Question 22

What is an example of a complement receptor?

Answer 22

CR3

Question 23

What is the receptor for the constant region of an antibody?

Answer 23

Fc receptors

Question 24

Steps 3 and 4 phagocytosis?

Answer 24

3 & 4. Microbe killing – phagosomes fuse with lysosomes as well as (in neutrophils) primary and secondary granules
Phagosomes have many molecules that are effective at cellular killing – a little more later
Major groups include:
Reactive oxygen species
“pore”-forming proteins or peptides
Hydrolytic enzymes
pH changes – i.e. acidic environment of the lysozyme

Question 25

Step 5 phagocytosis?

Answer 25

The microbe remnants are either digested and used, or can be excreted from the phagocyte

Question 26

What are the options for microbe killing?

Answer 26

After the microbe has been phagocytosed, the phagosome will dock with a lysosome and/or neutrophil granules Lysosomes can pretty much break down anything (acid hydrolases) The low pH of a lysosome is also unpleasant for many bacteria This complex becomes associated with the membrane of the phagolysosome Uses a large amount of oxygen (respiratory burst) If a particle is too large to phagocytose, macrophages will surround it and “place” their NADPH oxidases close to it to try to kill it Macrophages in particular are also capable of killing cells by inducing the synthesis of nitric oxide at high concentrations Neutrophils have a multitude of pore-forming molecules within their granules – these granules will fuse with the phagosome

Question 27

What do macrophages place near to a particle when it is too large to phagocytose?

Answer 27

NADPH oxidase

Question 28

Which type of phagocytic cell is capable of killing cells by inducing the synthesis of nitric oxide at high concentrations?

Answer 28

macrophages

Question 29

Which type of phagocytic cell has a multitude of pore-forming molecules within their granules – these granules will fuse with the phagosome?

Answer 29

neutrophils

Question 30

Phagocytosis visual

Answer 30

Question 31

What amino acid are defensins rich in?

Answer 31

cysteine

Question 32

Are defensins a neutrophil or macrophage?

Answer 32

Neutrophil

Question 33

What form voltage-dependent pores in bacteria that are permeable to water?

Answer 33

Neutrophil granules

Question 34

Microbe killing via neutrophils

Answer 34

Neutrophil granules: Defensins are very rich in cysteine Form voltage-dependent pores in bacteria that are permeable to water Cause lysis Cathepsin - a type of protease Cathelicidins – another pore-forming molecule Causes lysis, multitude of different structures Lysozyme – a glycoside hydrolase Doesn’t require an acidic pH Found in a variety of glandular secretions Great at killing gram +(ve) bacteria Lactoferrin – iron-binding protein that interferes with iron metabolism in microbes Neutrophils can do a neat trick – when they’re in an environment with many bacteria (they’re “surrounded”) they can can lyse and release their DNA into the ECF Known as a NET – a neutrophil extracellular trap NETs are “sticky” – most bacteria are trapped in the chromatin Histones are toxic to many bacteria The granule contents will remain close to the NETs and help with killing bacteria, even after the neutrophil itself is dead

Question 35

What is an iron-binding protein that interferes with iron metabolism in microbes>

Answer 35

lactoferrin

Question 36

Which neutrophil granule is a glycoside hydrolase?

Answer 36

Lysozome Doesn’t require an acidic pH Found in a variety of glandular secretions Great at killing gram +(ve) bacteria

Question 37

Cathepsin is a type of what?

Answer 37

protease

Question 38

Which pore-forming molecule causes lysis, and has a multitude of different structures?

Answer 38

cathelicidins

Question 39

What is it called when a neutrophil can lyse and release their DNA into the ECF?

Answer 39

NET- a neutrophil extracellular trap NETs are “sticky” – most bacteria are trapped in the chromatin Histones (released after neutrophil activation) are toxic to many bacteria The granule contents (also released) will remain close to the NETs and help with killing bacteria, even after the neutrophil itself is dead

Question 40

Questions to know How do phagocytes recognize that something is a “target” for phagocytosis? What’s an opsonin and how are they involved in this process? How do phagocytes kill a phagocytosed pathogen? What is the role of: Lysosomes Free radicals (how are they produced? Which ones are involved?) Anti-microbial peptides How can a NET add to host protection beyond phagocytosis?

Answer 40

How do phagocytes recognize that something is a “target” for phagocytosis? The PRRs on phagocytes recognizes the PAMPS or DAMPS. What’s an opsonin and how are they involved in this process? opsonin is a super secreted PRR on the cell that enhances the effectiveness of phagocytosis How do phagocytes kill a phagocytosed pathogen? What is the role of: Lysosomes: use hydrolysis to break peptidoglycan wall which leads to lysis off bacterial cell wall. Also increasing acidity by lowering pH Free radicals (how are they produced? Which ones are involved?) produced by the NAPDH oxidase complex through oxidations —> respiratory burst—> free radicals which cause oxidative damage to microbe. Nitric oxide (Perioxinitrate generated) and superoxide (hypochlorite and hydroxyl radical generated). Macrophages also synthesize nitric oxide. Granule contents may be released into EC tissue i NOS (inducible nitric oxide synthase. MPO (myeloperixidase) ROS (reactive oxygen species) Anti-microbial peptides How can a NET add to host protection beyond phagocytosis? Once the neutrophil is dead, the net still surrounds it and the body ones are toxic (suicide bombers)

Question 41

Toll-like receptors

Answer 41

Cytokines secreted in response to TLR’s include: Inflammatory cytokines (IL-1b, IL-6, CXCL8, IL-12, TNFa) Cytokine – a (small) protein messenger, secreted by a vast array of cells, that can: Influence the differentiation of a wide variety of cells, including leukocytes Mediate – activate or inactivate – the activity of many cells, including leukocytes Increase or decrease the production of a wide variety of stem/hematopoietic cells More cytokine overview in the e-learning module Interferons Interferon (IFN) alpha, beta, and lambda (IFNa, IFNb, IFNl) Autocrine and paracrine signaling molecules that are effective in activating macrophages, NK cells, and inducing an antiviral state More on interferons in subsequent lectures

Question 42

What is a cytokine? What are its roles?

Answer 42

Cytokine – a (small) protein messenger, secreted by a vast array of cells, that can: Influence the differentiation of a wide variety of cells, including leukocytes Mediate – activate or inactivate – the activity of many cells, including leukocytes Increase or decrease the production of a wide variety of stem/hematopoietic cells

Question 43

What are interferons?

Answer 43

Interferon (IFN) alpha, beta, and lambda (IFNa, IFNb, IFNl) Autocrine and paracrine signaling molecules that are effective in activating macrophages, NK cells, and inducing an antiviral state

Question 44

Consequences of TLR signaling

Answer 44

The phenotype of individuals with specific gene mutations/polymorphisms can tell us about the overall importance and function of that gene Example: MyD88  An essential adaptor in TLR signaling Patients with MyD88 deficiency: Suffer frequent and severe bacterial infections Antiviral responses generally unaffected Patients with constitutively active MyD88: Develop various blood disorders and blood cancers: Overproduction or dysregulated production of IgM B cell lymphoma, marginal cell lymphoma

Question 45

What deficiency leads to patients suffering frequent and severe bacterial infections, while antiviral responses generally unaffected?

Answer 45

MyD88 deficiency

Question 46

What happens when a patient has constitutively active MyD88?

Answer 46

Develop various blood disorders and blood cancers: Overproduction or dysregulated production of IgM B cell lymphoma, marginal cell lymphoma

Question 47

Nod-Like Receptors

Answer 47

Family of intracellular receptors found in the cytoplasm that detect products derived from the intracellular degradation of phagocytosed pathogens (e.g. components of bacterial cell wall) Also recognize DAMPs associated with cellular stress activates expression of inflammatory cytokines

Question 48

Steps of Acute Inflammation (Step 1) What happens? What is released?

Answer 48

Alteration of vascular caliber - vasodilation Leads to increases in blood flow at the capillary bed due to arteriolar dilation, dilation of precapillary sphincters Nitric oxide and histamine release A variety of prostaglandins (PGI2, PGE2, PGD2) Platelet activating factor (at low concentrations – higher concentrations cause vasoconstriction) Complement -C5a and C3a stimulate histamine release

Question 49

At low concentrations, nitric oxide is a potent what? At high concentrations, it’s capable of what?

Answer 49

vasodilator (why Viagra is a profitable drug) destroying both microbes and host cells since it’s a free radical Higher concentrations produced by an inducible nitric oxide synthase in macrophages

Question 50

Vasodilation

Answer 50

Arterioles and pre-capillary sphincters dilate leading to vastly increased blood flow in inflamed tissue Vasodilation and fluid loss (due to increased permeability) lead to slower blood flow Known as vascular congestion This helps with margination of leukocytes

Question 51

Production of arachidonic acid metabolites and their roles in inflammation. Pathways FIGURE.

Answer 51

Question 52

Prostaglandins and leukotrienes are produced when?

Answer 52

PLA2 generates arachidonic acid from membrane phospholipids

Question 53

Which prostaglandins cause vasodilation and increase vascular permeability? What is their important role?

Answer 53

Different types of cyclooxygenases produce different types of prostaglandins from arachidonic acid important acute inflammatory mediators

Question 54

Different types of 5-lipoxygenase produce different types of leukotrienes from arachidonic acid that seem particularly important in what tissue?

Answer 54

lung

Question 55

LTB4 is an important ________?

Answer 55

chemotactic agent

Question 56

What increase vascular permeability and smooth muscle constriction (think asthma)?

Answer 56

Other LTs

Question 57

Lipoxins are generated from what? By what? To do what?

Answer 57

Lipoxins are generated from arachidonic acid by 12-lipoxygenase – they decrease inflammation

Question 58

Steps of acute inflammation: Step 2 What mediators are released during this step?

Answer 58

Enhancement of vascular permeability Capillaries and venules become more “leaky” with the release of a number of mediators Histamine and serotonin (released by activated platelets, a link between inflammation and clotting) Prostaglandins (PGD2 and PGE2) Leukotrienes (LTC4, LTD4, LTE4) Platelet activating factor C3a and C5a Bradykinin a wide variety of proteins and mediators can enter the interstitial space from the bloodstream

Question 59

Vascular permeability

Answer 59

Increased vascular permeability is due to contraction of endothelial cells Occurs mainly in venules Often short-lived Another mechanism is endothelial damage Can be caused by trauma, burns, microbial damage Can also be caused by leukocyte-mediated damage to the endothelium (often longer-lived) Increased transcytosis can also result in leakage of plasma components into the interstitial space

Question 60

Transcytosis: what is it? What can move across? How?

Answer 60

Active, vesicle-mediated transport across the capillary endothelial cell Large molecules can move across the endothelium via: 1.) Pinocytosis (caveolin pathway) 2.) Receptor-mediated endocytosis

Question 61

Mechanisms of increased vascular permeability CHART

Answer 61

Question 62

Lymphatics

Answer 62

As interstitial fluid accumulates during inflammation, pressure increases in the interstitial space and lymphatic drainage increases Normally only a small amount of interstitial fluid is produced in non-inflamed tissue Excess fluid, microbes, debris, and leukocytes all migrate into the lymph during inflammation The lymphatic vessels themselves can become inflamed – known as lymphangiitis

Question 63

Steps of acute inflammation: Leukocyte migration

Answer 63

Emigration and activation of leukocytes Neutrophils, monocytes, eosinophils, and basophils will all migrate from the circulation into inflamed tissue Steps: 1. Margination 2. Rolling 3. Adhesion 4. Diapedesis 5. Chemotaxis of leukocytes to sites of injury or infection 2 and 3 are mediated by binding of selectins and cellular adhesion molecules to their respective ligands on leukocytes

Question 64

Chemokine or cytokine?

Answer 64

Cytokine – a (small) protein messenger, secreted by a vast array of cells, that can: Influence the differentiation of a wide variety of cells, including leukocytes Mediate – activate or inactivate – the activity of many cells, including leukocytes Increase or decrease the production of a wide variety of stem/hematopoietic cells Chemokine – structurally-related family of small cytokines that: Bind to cell-surface receptors (usually leukocytes) Induce movement of leukocytes along the chemokine concentration gradient Mediate adhesion of leukocytes for the purposes of: Differentiation Inflammation/migration

Question 65

What are the 2 major chemokine families?

Answer 65

CXC CXC chemokines attract neutrophils, are angiogenic, and are very similar in structure The “X” indicates the location of a disulphide bond CC Act on/attract a wide variety of other leukocytes

Question 66

The chemokine receptor expression based on cell type CHART

Answer 66

Question 67

What step in leukocyte migration do leukocytes migrate towards vessel wall?

Answer 67

1.) margination

Question 68

Which step in leukocyte migration is the formation & dissociation of adhesion bonds between leukocytes and endothelial cells?

Answer 68

2.) rolling **Activation by chemokines presented on endothelial cells is required before the leukocyte can form stable adhesion

Question 69

Emigration and activation of leukocytes

Answer 69

Selectin-mediated rolling of leukocytes will enhance exposure to chemokines that are bound to, and presented by, proteoglycan-like molecules on the surface of the endothelial cell Mediated by binding of selectins and cellular adhesion molecules to their respective ligands on leukocytes

Question 70

What stimulates the activation of the leukocytes? (hint: endothelial cell presents it) What does this do?

Answer 70

Endothelial cell presents a chemokine that stimulates activation of the leukocytes This increases affinity of leukocyte integrin for it’s ligand, allowing stable/tight adhesion bonds to form

Question 71

In what step of leukocyte migration is the formation of tight/stable adhesion bonds between leukocytes and endothelial cells?

Answer 71

Step 3: stable/ tight adhesion

Question 72

What step of LM does the leukocyte migrates through endothelium ?

Answer 72

4.) Diapedesis/ Transmigration

Question 73

What step in LM do chemotaxis of leukocytes to sites of injury or infection?

Answer 73

Step 5: chemotaxis of leukocytes

Question 74

Putting it all together FIGURE

Answer 74

Question 75

Adhesion molecules and their ligands CHART

Answer 75

Question 76

Which inflammatory mediators increase the ability of leukocytes to migrate to a target?

Answer 76

Histamine, Thrombin Rolling Selectin expression by endothelial cells TNF & IL1 ICAM expression by endothelial cells Chemokines Increased integrin affinity

Question 77

Chemotactic agents

Answer 77

All of these agents are produced in higher concentrations at sites of cellular damage/pathogen invasion Leukotriene B4 Bacterial products containing N-formyl-methionine Activated complement (particularly C5a) More on this in the e-module Chemokines (IL-8, RANTES, eotaxin) Leukocytes can “follow the breadcrumbs” to the site of pathology via the chemotactic agent concentration gradient

Question 78

For leukocyte emigration QUESTIONS to answer. What molecules cause loose adhesion of leukocytes to the vascular endothelium? What binds to what? How about tight adhesion? What cells are integrins found on? How about ICAMS? What causes expression of the molecules above? What is the role of a chemokine in: Tight adhesion? Migration of a leukocyte to a site of inflammation? What is a chemotactic agent? Name a few

Answer 78

What molecules cause loose adhesion of leukocytes to the vascular endothelium? Selectins (E/P on endothelial cells, L on leukocytes) What binds to what? Leukocytes and endothelial cells How about tight adhesion? Integrins What cells are integrins found on? WBC How about ICAMS? Endothelial What causes expression of the molecules above? INTEGRINS: Histamine, thrombin, chemokines (IL-8) ICAMS: TNF & IL-1 What is the role of a chemokine in: Tight adhesion? Sends message from endothelial cell to activate WBC in order for WBC to put out a tighter receptor (integrin), induce formation of integrin by changing surface markers Role of chemokine Migration of a leukocyte to a site of inflammation? Via chemotaxis (follows breadcrumbs) What is a chemotactic agent? Any chemotactic agents that Produce more at damage or pathogen invasion. Name a few: leuk. B4, bacterial products with N-formyl-methionine, activated complement (c5a), chemokines (IL-8, RANTES, EOTAXIN)