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AI - Innate Immunity > Cellular Killing Mechanisms > Flashcards

Cellular Killing Mechanisms Flashcards

1
Q

What is phagocytosis?

A

A form of receptor-mediated endocytosis, used by phagocytes to internalise pathogens.

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2
Q

What is the main difference between phagocytosis and other forms of endocytosis?

A

Phagocytosis allows uptake of larger materials.

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3
Q

Describe internalisation in endocytosis.

A

Membrane budding around the material to be internalised.

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4
Q

Describe internalisation in phagocytosis.

A

Relies on the membrane moving out towards the target and engulfing the target. Dependent on the actin cytoskeleton.

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5
Q

Give the two types of phagocytic receptors.

A

Opsonin receptors and microbe receptors.

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6
Q

What do opsonin receptors recognise?

A

Host-derived molecules that have been deposited onto the microbe, i.e. antibodies/complement.

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7
Q

Give examples of opsonin receptors.

A

FcR - binds antibodies.

CR3 - receptor for C3 complement protein.

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8
Q

What is the role of microbe receptors?

A

Bind microbial proteins found on the pathogen cell surface. Some can have both phagocytic and signalling functions.

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9
Q

Give examples of microbe receptors.

A

CD14 - binds LPS.

Dectin-1 - binds fungal glucans.

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10
Q

What is the nascent phagosome?

A

a very brief stage between the phagocytic cup and the early phagosome, in which the phagocytic cup first closes.

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11
Q

Give the stages of phagosome maturation.

A

Phagocytic cup, nascent phagosome, early phagosome, late phagosome, phagolysosome.

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12
Q

Why is the phagosome progressively modified?

A

To give an increasingly hostile environment which the microbe cannot survive in.

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13
Q

How is phagosome maturation achieved?

A

Through progressive fusion of the phagosome with other distinct intracellular vesicles.
NP fuses with EE -> EP which fuses with LE -> LP which fuses with lysosomes -> phagolysosome.

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14
Q

Describe the conditions of the phagolysosome.

A

pH 4.5, contains hydrolases, proteases, lipases and DNases, contains ROS-producing enzymes. High levels of v-ATPase activity.

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15
Q

What gives vesicle identity?

A

Different vesicle surface proteins and phosphatidylinositides.

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16
Q

What family of proteins are involved in vesicle identity?

A

Rab GTPases.

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17
Q

How many Rab GTPases are encoded in the human genome?

A

35

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18
Q

What is the partial vesicle code?

A

Different vesicle types are labelled by different Rab proteins - different Rabs are also found in different regions of the Golgi.

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19
Q

Describe the phagocytic cup/nascent phagosome.

A

Derived strictly from the plasma membrane.
pH 7.4 - same as extracellular fluid.
High levels of PI(3,4,5)P3 in membrane.

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20
Q

Describe the early phagosome.

A

Derived from fusion with early endosomes.
Has EEA1, Rab5 and PI3P.
Some v-ATPase present in membrane.
pH 6-6.5

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21
Q

What is the role of EEA1?

A

Allows recruitment of Rab5 to early endosomes.

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22
Q

What is the role of Rab5?

A

Recruitment of downstream maturation markers, and recruitment of Vps34.

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23
Q

Why does the early phagosome have PI3P rather than PI(3,4,5)P3?

A

Rab5 recruits Vps34 lipid phosphatase - removes phosphate groups from inositol head group.

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24
Q

What is the role of v-ATPase?

A

Uses ATP to move the proton gradient - decreasing the pH inside the phagosome.

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25
Q

What are recycling endosomes?

A

Endosomes that remove the transferrin receptor from the early phagosome, and carry it back to the plasma membrane.

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26
Q

Why must the transferrin receptor be removed from the early phagosome?

A

Transferrin receptor would allow transferrin and therefore iron uptake into the vesicle - removal of this from the phagosome deprives microbes of iron, causing microbe death.

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27
Q

Give an example of a bacterium that has evolved not to use iron.

A

Borrelia burgdorferi - the bacterium that causes lyme disease.

28
Q

Describe the late phagosome.

A 
Derived from fusion with late endosomes. 
Has Rab7 (replaced Rab5), LAMP1, M6PR and Lysobisphospatidic acid. 
Increased v-ATPase activity - pH lowered to 5-6.
29
Q

What markers is the phagolysosome positive for?

A

Rab7 and LAMP1.

30
Q

What is the outcome of phagosome maturation?

A

Progressive acidification and deposition of acid hydrolases - this is sufficient to kill most non-pathogenic bacteria.

31
Q

Why have we evolved to use cytokines to alter the phagolysosome conditions?

A

Some pathogenic bacteria are resistant to the normal conditions of the lysosome. Therefore, we evolved tp use cytokines to make the phagolysosome more efficient at bacterial killing.

32
Q

What are the signals that drive macrophages to adopt the M1 phagocytic programme?

A

TLR activation and IFNy released by T cells.

33
Q

What are the signals that drive macrophages to adopt the M2 phagocytic programme?

A

IL-4 and IL-13, released by T cells.

34
Q

What is the main biochemical difference between M1 and M2 macrophages?

A

M1 generate higher levels of ROS in their phagolysosome.

35
Q

Describe M1 macrophages.

A

Rapid ROS production due to rapid activation of NOX2 upon internalisation. Weaker v-ATPase activity.

36
Q

Describe M2 macrophages.

A

Lower activation of NOX2 (only acquired from plasma membrane), lower levels of ROS production, stronger v-ATPase activity. Still sufficient to kill many microbes.

37
Q

When are M1 macrophages used?

A

During serious infection.

38
Q

What is the cost of using M1 macrophages?

A

Increased tissue damage due to increased ROS production.

39
Q

When are M2 macrophages used?

A

During tissue repair.

40
Q

Why must NOX2 be rendered inactive across the plasma membrane?

A

Prevents oxidative stress.

41
Q

How and when is NOX2 activated?

A

By PI3P - only activated in the early phagosome.

42
Q

Describe the structure of NOX2.

A

gp91 catalytic subunit containing 2 b-type cytochromes, and the p22 subunit containing FAD. Requires accessory proteins -p40, p47 and p67 - for NADPH binding.

43
Q

What is the function of NOX2?

A

Reduces oxygen to superoxide, using NADPH as an electron donor.

44
Q

How are the accessory proteins recruited to NOX2?

A

p47 is phosphorylated during phagocytosis, exposing its SH3 domains to allow assembly with gp91/p22. Further level of control of NOX2 activity, where NOX2 can only bind NADPH upon phagocytosis.

45
Q

How can mitochondria synergise with the phagosome?

A

If TLRs 1, 2 or 4 are activated, mitochondria migrate to the phagosome, increasing ROS production in the phagosome of M1 macrophages.

46
Q

What do secretory granules provide the phagolysosome with?

A

Pore forming proteins and MPO.

47
Q

How are neutrophils recruited to the site of infection?

A

IL-8, released by macrophages.

48
Q

What are the killing mechanisms used by neutrophils?

A

Phagocytosis (similar to M1, with high NOX2 activity), and production of NETs.

49
Q

What do NETs consist of?

A

Nuclear DNA, histones, and toxic proteins from secretory granules.

50
Q

Are histones antimicrobial?

A

Yes - H2B and H4 are components of surfaceous gland secretion on the skin.

51
Q

Is extracellular DNA toxic?

A

Yes - it is a good chelator of cations and leads to disruption of the bacterial cell membrane, causing bacterial cell lysis.

52
Q

How long does it take neutrophils to release NETs?

A

2-3 hours.

53
Q

Why is NETosis thought to be a last resort action for neutrophils to take?

A

Neutrophils die in the process.

54
Q

Why is there controversy over whether or not neutrophils die during NETosis?

A

It has been claimed that neutrophils can release NETs in a non-lytic way, and remain viable and phagocytic (without a nucleus).

55
Q

What is the role of elastase in NET release?

A

Thought to be important in the decision to phagocytose or release NETs. Elastase is sequestered in the phagosome during phagocytosis, but triggers NET release if it translocates to the nucleus.

56
Q

Give examples of pathogens able to survive inside macrophages.

A

Legionella pneumophila and mycobacterium tuberculosis.

57
Q

Why have some pathogens evolved to live inside host cells?

A

Allows evasion of humoral immune defences and immune cells in circulation.

58
Q

How do pathogens manage to survive inside phagocytes?

A

Disruption of the vesicle maturation process.

59
Q

What is the main challenge faced by pathogens that escape vesicles altogether and live in the cytosol?

A

Inflammasome formation.

60
Q

How do some pathogens escape the phagosome to live in the cytosol?

A

By causing lysis of the phagosome.

61
Q

How can some pathogens induce phagocytosis in non-phagocytic cells?

A

By secreting effectors into epithelial cells that trigger the actin rearrangment that is required for internalisation.

62
Q

How does mycobacterium tuberculosis inhibit maturation at the early phagosome?

A

ManLAM - inhibits cytosolic Ca2+ fluctuations that are required for Vps34 activity.
SapM - dephosphorylates any PI3P produced, giving PI.

63
Q

How can macrophages still kill internalised mycobacterium tuberculosis, without further phagosome maturation?

A

If the macrophage is activated by IFNy, there will be enough ROS production to kill the bacterium in the early endosome.

64
Q

How does legionella pneumophila survive inside cells?

A

Lives in a modified vesicle (LCV) that acts like an element of the secretory pathway - 300 effectors secreted by dot/icm secretion system.

  • RaIF recruits Arf1 (normally found on Golgi)
  • LidA and DrrA recruit Rab1 (normally found on ERGIC)
  • VipD binds Rab5, preventing EEA1 recruitment.
65
Q

How does listeria monocytogenes live inside non-phagocytic cells?

A
  • lnlA and lnlB bind host cell surface proteins.
  • LLO forms pores in the membrane, triggering membrane uptake for repair.
  • PI-PLC degrades the vesicle once inside the cell.
  • ActA recruits actin, allowing intracellular motility of listeria.
66
Q

Why do listeria recruit actin for intracellular motility?

A

Thought to allow evasion of the inflammasome. Also thought to allow spreading between cells - but it is unclear if this happens in vivo.

AI - Innate Immunity (3 decks)

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