Killing Pathogens: Intra-cellular and Extra-cellular

Question 1

What is the initial inflammatory response?

Answer 1

Barrier break, microbes enter

Microbes/injury activate sentinel cells

Sentinels secrete inflammatory mediators

Increased vascular permeability; fluid and proteins enter tissues

Complement, antibody and anti-microbial proteins kill microbes

Adhesion molecules and chemokines cause leukocyte migration into tissue

Phagocytosis and killing of microbes

Question 2

What happens after a microbe enters the system?

Answer 2

1. Initial inflammatory response
2. Dendritic cells activate T cells
3. T cells activate, divide and differentiate into effector cell

Question 3

What are the different Th cell subtypes?

Answer 3

Th1
Th2
Th17
Tfh

Question 4

Th1 cell properties?

Answer 4

Cytokines:
IFN-gamma

Target:
Macrophages

Immune reactions:
Macrophage activation

Host defence:
Intracellular pathogens
Extracellular bacteria

Immune pathology:
Autoimmunity: chronic inflammation

Question 5

Th2 cell properties?

Answer 5

Cytokines:
IL-4
IL-5
IL-13

Target cells:
Eosinophils

Immune reactions:
Eosinophils and mast cell activation
Alternative macrophage activation

Host defence:
Helminths

Immune pathology:
Allergy

Question 6

Th17 cell properties?

Answer 6

Cytokines:
IL-17
IL-22

Targets:
Neutrophils

Immune reaction:
Neutrophil recruitment and activation

Host defence:
Ectracellular bacteria and fungi

Immune pathology:
Autoimmunity- inflammation

Question 7

Tfh cell properties?

Answer 7

Cytokines:
IL-21
(Also IFN-gamma or IL-4)

Targets:
B cells

Immune reactions:
Antibody production

Host defence:
Extracellular pathogens

Immune pathology:
Autoimmunity (antobodies)

Question 8

What directs Th cell differentiation pathways?

Answer 8

Different cytokines direct Th cells down different differentiation pathways for different immune responses

Question 9

How does Th cell differentiation occur?

Answer 9

Signals 1 and 2 from APC activate T cell (recognised Ag + B7 danger signal)

Signal 3 directs T cell effector function (cytokines)
PRRs can tell to some degree what sort of pathogen is present
Certain cytokines released depending on microbe type

Question 10

What happens once Th cell has differentiated?

Answer 10

Th1/2/17 cells migrate to infection site, aid innate immune response

Tfh cells remain in Lymph node and give permission to B cells to activate

Question 11

What are “Plasma cells”

Answer 11

Activated antibody producing B cells

Question 12

What are antibody effector mechanisms?

Answer 12

Ab Ag site binds to microbe/Ag
Fc region interacts with innate cells/molecules to activate effector response

Neutralisation of microbes and toxins (antibodies alone)

Opsonisation and phagocytosis of microbes

Antibody dependent cellular cytotoxicity (NK cells)

Complement activation

Question 13

Benefit of antibody effector response?

Answer 13

Can give specificity to innate immune response

Question 14

What defines B cell effector functions?

Answer 14

The class of antibody it produces

Different Ab classes/isotypes interact with different innate molecules/cells so enhance different effector responses
Different isotopes deal with different infection types

Question 15

What are the properties of different antibody classes?

Answer 15

IgM:
Great complement fixer
Good opsoniser
First antibody made

IgA:
Resistant to stomach acid
Protects mucosal surfaces
Secreted in milk

IgG:
Ok complement fixer
Good opsoniser
Helps NK cells kill (ADCC)
Can cross placenta

IgE:
Defends against parasites l
Causes anaphylactic shock
Causes allergies

Question 16

Classical pathway of complement system?

Answer 16

Uses adaptive sensing as opposed to innate sensing used in alternative and lectin pathways

Ab (IgM, IgG) identifies pathogen with high specificity
Need an antibody response first so doesn’t work until there’s a lot of An for that specific microbe

Question 17

3 main effector functions of complement system?

Answer 17

Opsonisation for enhance phagocytosis (C3b)

Stimulates inflammation by recruiting and activating immune cells (C3a, C5a)

Lysing microbes and cells (C6-9)

Question 18

Complement mediated opsonisation process?

Answer 18

C3b binds microbe
Phagocyte C3b receptor recognises microbe
Phagocytoses and kills microbe

Question 19

Complement mediated inflammation process?

Answer 19

C3a, C4a, and C5a released during complement activation
Act locally similarly to inflammatory cytokines
-recruit cells to infection site
-can activate cells
Complement helps inflammatory response pull in effector cells

Question 20

Complement mediated cytolysis process?

Answer 20

Membrane attac complex (MAC) forms on bacterial membrane
Water rushes in, ions rush out, microbe bursts
Can also kill foreign/host cells
Killing of host cells makes it quite damaging

Question 21

Types of phagocyte?

Answer 21

Macrophage- generalist:
Resides in tissues
Can be recruited from blood monocytes
Talk to T cells

Neutrophils- killers on call:
Specialist killers (can be dangerous)
Kept in blood (50-70% of blood monocytes)
Recruited rapidly to infection site
Short lived to about collateral damage

Dendritic cells- talkers:
Specialised in activating naïve T cells
Don’t kill

Question 22

Steps of phagocytosis?

Answer 22

Detection of microbe via PRR, complement, or Ab

Membrane invaginates forming phagosome vesicle

Phagosome fuses with lysosome to form a phagolysosome containing toxic molecules to degrade microbes

Chemicals in phagolysosome activate and digest microbe

Question 23

Phagosome killing?

Answer 23

Vacuoles ATPases pump H+ ions into phagosome - acidify environment

Phagocyte oxidase and it’s cofactor NADPH oxidase initiate respiratory burst
Converts O2 into reactive oxygen species ROS (eg H2O2)

Neutrophils express myeloperoxidase (MPO) that makes HOCl (hypochlorite)
Also produce elastase that degrades bacteria

Macrophages produce Nitric Oxide via inductible Nitric Oxide Synthase (iNOS)

Phagosome also withhold pathogen nutrients (Lactoferrin and other scavenger proteins bind and transport out iron)

Also contain defensins - other directly microbicidal proteins

This makes and contains a v toxic environment for killing

Question 24

How do Ab increase phagocytosis efficiency?

Answer 24

IgG opsonises microbe

Phagocyte Fc receptors bind Ab Fc region

Fc receptor signals activate phagocyte

Microbe phagocytosed and killed

Question 25

What are Neutrophil extracellular traps?

Answer 25

NETs Neutrophil dies via NETosis Nucleus swells and bursts extruding DNA like a net DNA has anti microbial molecules attached (defensins, proteases) Traps and kills bacteria, fungi, and viruses (aggregate into pus)

Question 26

How do T cells enhance macrophage and neutrophil killing?

Answer 26

Th1 cells release IFN-gamma: Classical macrophage activation Th17 cells release IL-17: [According to diagram] causes epithelial cells to release chemokines, TNF, IL-1, IL-6, CSFs Causes inflammation and neutrophil recruitment/response

Question 27

How does neutralisation by antibodies work?

Answer 27

Antibody binds Ag on microbe Prevents penetration of microbe through epithelial barrier Prevents binding of microbe and infection of cells Prevents binding of toxin to receptor Bind multiple microbes and clumping them together (Ab has 2 binding sites), blocks entry through barrier

Question 28

Granulocytes?

Answer 28

Mast cells Basophils Eosinophils Neutrophils Pre-store effector molecules Ready to fire out when activated

Question 29

ADCC?

Answer 29

Antibody dependent cell-mediated cytotoxicity: Ab bind target cell/microbe Fc portion of Ab binds FcR on innate cell Cell activated if multiple FcR-Ab interactions occur Cell fires out granules at target Destructive process - collateral damage to cells around it Important for killing of intra- and extracellular microbes

Question 30

Killing of helminth via ADCC?

Answer 30

Too large to phagocytose :( IgE coats helminth Eosinophil binds IgE Then degranulates releasing toxic compounds

Question 31

Mast cell degranulation?

Answer 31

IgE coats resting mast cell Ag binds to multiple IgE and cross links them Mast cell activation Weep response: Liquid flows from tissue into lumen to push helminths off gut wall Sweep response: Histamine causes muscle contractions Push helminth out of gut with faeces after it has been detached by weep Causes wheeze in allergy Cytokine production: Amplifies inflammatory response

Question 32

How do Th2 cells help with helminth immunity?

Answer 32

Produce specific cytokines IL-4: B cells produce antibodies - mast cells and eosinophils need IgE IL-13 and IL-4: Weep and sweep responses IL-5: Activates eosinophils

Question 33

Benefits of being intracellular for microbes?

Answer 33

Hidden from effector mechanisms Ready suppply of resources (Nutrients, cellular machinery for viruses to hijack) Hitch ride around host

Question 34

Different strategies for cell entry?

Answer 34

Phagocytes: Have mechanisms to survive phagosome Others escape from phagolysosome into cytoplasm Nonphagocytic cells (eg epithelial): Bind cellular receptor and use that to enter

Question 35

Why infect immune cells?

Answer 35

Macrophages trying to eat you anyway If you can evade killing mechanisms then makes infection easier They migrate around host - perfect transporters Easier to manipulate immune pathways inside immune cell

Question 36

How are intracellular microbes detected?

Answer 36

PRRs inside cell Endosomes (eg phagosome): TLRs Cytosol: NOD-like receptors (eg NOD1 and NOD2) recognise bacteria RIG-Like receptors (eg RIG-1) recognise viral RNA

Question 37

How do microbes avoid macrophage killing mechanisms?

Answer 37

Legionella pneumophila: prevent s lysosome fusion with phagosome and ends up in rough-ER like vesicle where it proliferates until it lyses the vesicle and then the cell

Question 38

How do microbes escape phagosome before lysosome fusion?

Answer 38

Listeria monocytogenes: can break out of lysosome and then live and replicate in cytosol Listeriolysin O (LLO) disrupts phagosome membrane Bacteria then uses host actin to move within and between cells (actin rocket) Shigella also escapes phagosome

Question 39

How are macrophages helped with intracellular microbes?

Answer 39

Macrophage needs to be fully activated by IFN-gamma signal from NK cells IFN-gamma signal can also come from a T cell (CTL or Th1) Macrophage base MHC class II on surface so displays antigen Antigen + B7 danger signal causes IFN-gamma release from T cell

Question 40

What happens if Th1 and Th2 cells are activated at the same time?

Answer 40

Th1 cell releases cytokines which activate macrophages (IFN-gamma and TNF) Th2 cell releases cytokines that inhibit classical macrophage activation (IL-10, IL-4, IL-3) Th2 activation can inhibit the activation of macrophages when they’re needed, blocking Th1 action

Question 41

How does tuberculosis cause immune pathology?

Answer 41

Mycobacterium tuberculosis v good at evading phagocytosis Leads to chronic infection - continual IFN-gamma production and macrophage activation Leads to continual release of macrophage killing molecules (eg NO) Causes immune (? 🦑) pathology Granulomas form around infected macrophages to wall off infection

Question 42

What is the anti viral state

Answer 42

Viral infection stimulates production of Type 1 interferons (IFN-alpha, IFN-beta) Type 1 IFNs induce anti viral state in cell that protects from viral infection

Question 43

How do type 1 IFNs inhibit viral gene expression?

Answer 43

Part of anti viral state Block viral transcription and translation Degradation of viral DNA Autophagy - cell eats it’s own organelles so virus can’t use them

Question 44

How do type 1 IFNs cause apoptosis?

Answer 44

Part of anti viral state Misfolded proteins trigger unfolded protein response causing apoptosis Alters cells response to TNF-alpha from pro-inflammatory to apoptosis

Question 45

How do type 1 IFNs promote T cell and NK cell activation?

Answer 45

Sequester lymphocytes in LN Increase cytotoxicity of CTL and NK cells Promote Th1 differentiation Upregulates Class I MHC

Question 46

NK cells properties?

Answer 46

Target cells infected with bacteria viruses and Protozoa (and tumour cells) Innate counterparts to CTL Faster but less precise Important backup mechanism to deal with intracellular pathogens that evade CTL by inhibiting MHC class I IFN-gamma from Th1 cells and CTL is important NK cells source of IFN gamma to activate macrophages

Question 47

Necrosis?

Answer 47

Uncontrolled cell death (mechanical damage or microbe bursting cell) Cell ruptures releasing contents Highly inflammatory

Question 48

Apoptosis?

Answer 48

Programmed cell death Fragmentation of DNA, membrane blebbing Apoptotic cells cleared by phagocytosis Non-inflammatory

Question 49

What causes NK cell activation?

Answer 49

NK cells Express range of activating and inhibitory receptors Inhibitory receptors recognise ligands on healthy cells (eg MHC class 1 which is often downregulated by intracellular pathogens) Activating receptors recognise markers on infected or injured cells (stress markers) Activation depends on balance if these signals Lack of inhibitor (working MHC class I) or presence of activator (presented antigens) signal killing

Question 50

ADCC and NK cells?

Answer 50

Ab can enhance NK killing of microbe when Ag remains on cell surface (eg microbe leaving it behind as it enters cell, doesn’t include Ag presented by MHC class I and II) Fc of Ab interacts with FcR on NK - killing of antibody coated cell

Question 51

How do Th1 cells enhance CD8+ T cell differentiation?

Answer 51

CD4+ helper T cells produce cytokines which stimulate CTL differentiation from CD8+ T cells (IL-2, IFN-gamma) They also enhance abilities of APCs to stimulate CTL differentiation

Question 52

Benefits of adaptive CTL over innate NK?

Answer 52

Precisely identify infected cells More specific than NK cells - more accurate and efficient MHC class I expressed by all nucleated cells so can identify any cell that becomes infected

Question 53

How do CTL kill with high precision?

Answer 53

Engage target cell Form synapse with target where all interactions occur This allows killing to be precise as it’s done by the synapse Important as killing mechanisms are dangerous to healthy cells

Question 54

What are the CTL/NK cell killing mechanisms?

Answer 54

Perforin/granzyme mediated: Perforin creates pore in infected cells membrane Granzyme enters via pore, induces apoptosis Fas/FasL mediated: Target cells express Fas (eg hepatocytes) CTL/NK cells express FasL (Fas ligand) Fas activation signals apoptosis

Question 55

How are naïve CTL activated if DC is not infected?

Answer 55

All Naïve T cells (including CTL) need activation from DC If the DC itself isn’t infected then it will only be presenting Ag it picked up from extracellular environment on its MHC class II which CD8+ CTL don’t interact with In this case the DC can cross present the exogenous antigens on MHC class I instead of II- allowing for activation of naïve CTL

Question 56

How do viruses evade CTL and NK cells?

Answer 56

Disrupting MHC class I Ag presentation This is why downregulated MHC1 is detected by NK cells

Question 57

How do antibodies neutralise intracellular microbes?

Answer 57

Target them during their extracellular stages (eg moving through cells) Protects from further infection

Question 58

Effector functions of memory T and B cells?

Answer 58

Effector functions of memory cells are already established They won’t undergo differentiation when activated for a second time (unlike thei naïve counterparts which have yet to differentiate)