14 - Cell Death and Immunity

Question 1

What is the process involving NETs referred to as?

Answer 1

Process referred to as:
NETosis - neutrophil extracellular trap formation
or ETosis - extracellular trap formation, as several immune cells undergo process

Question 2

What are NETs important in?

Answer 2

Important in antimicrobial immunity, conserved throughout evolution
- NETs detectable in pus – presence related to viscosity

Question 3

What are NETs associated with?

Answer 3

Associated with certain disorders:
- Appendicitis, bovine mastitis, murine model of necrotizing fasciitis, pre-eclampsia

Question 4

What can products ETs?

Answer 4

slime moulds produce ETs, even plants produce extracellular traps at the bottom of the growing root tips

Question 5

When were NETs discovered?

Answer 5

• discovered 2004 by Brinkmann et al.
• further characterised by Fuchs et al 2007

Question 6

Structure of NETs

Answer 6

• Extracellular chromatin structures, studded with globuli (30–50 nm diam.)
• NETs: flexible, surround the cell from which they originated and float in media

Question 7

How can NETs be activated?

Answer 7

• Can be activated with IL-8, lipopolysaccharide (LPS), bacteria, fungi or activated platelets.
• Less efficiently by Ab or Ab/Ag complexes
• activation triggered by different receptors
• primarily toll-like receptors, cytokine and Fc receptors

Question 8

What can NET activation pathway involve?

Answer 8

Activation pathway can involve different receptors, e.g. TLRs, cytokine & Fc receptors

Question 9

How can extracellular structures be dissolved?

Answer 9

Extracellular structures dissolved by addition of DNAse
- not instant, over the course of 60 to 90 minutes

Question 10

What proteins are in NETs?

Answer 10

Globules of protein in NETs
- flexible NETs - look like clouds in culture medium
- stimulated with IL-8

Question 11

What can neutrophil elastase do?

Answer 11

Neutrophil elastase – can cleave virulence factors of Shigella, Salmonella, Yersinia
- neutrophil elastase is a key protein on the NET

Question 12

How do neutrophils die?

Answer 12

Neutrophils die via a regulated form of cell death to release NETs

Question 13

Fuchs et al 2007 study

Answer 13

• 0-1hr: Nucleus loses lobules, chromatin decondenses, inner & outer NMs detach granules disintegrate
• > 1 hr: Nuclear envelope disaggregates, nucleoplasm/cytoplasm merges; Cell rounds, contracts, PM ruptures, cell interior ejected
• > 30 proteins quantifiable on neutrophil extracellular traps. Majority from granules, a few from nucleus, cytoplasmic – rare.
• Clearance: thought to be DNAse-1, other phagocytes

Question 14

What did the Fuchs et al 2007 study show about the formation of NETs

Answer 14

Disintegration of the nucleus and granules allows the formation of NETs

Question 15

Different morphologies displayed by apoptosis, necrosis and NETosis

Answer 15

• necrotic cell: nucleus is broken down
• can’t see the nuclear membrane in NETosis
• cresent typic of apoptotic cell
• apoptosis is characterised by very precise breaks in DNA (anti-fas antibody stimulate apoptosis - TUNEL assay quantifies apoptosis)
• necrosis induced by many things, e.g., S aureus toxin

Question 16

What is NET cell death independent of?

Answer 16

NET cell death is caspase-independent, not accompanied by DNA fragmentation

Question 17

Involvement of ROS in NET production

Answer 17

• PMA: activator of protein kinase C
• NADPH oxidase inhibitor: dephenylene iodonium (DPI) blocks NET formation
• when manipulating cells in vitro, DPI can block NET formation
• H2O2 is a strong ET stimulator
• enzyme inhibitor DPI can block ROS production
• if ROS blocked, H2O2 production can still be stimulated artificially

Question 18

How are H2O2 and catalase (ROS) involved in NET production

Answer 18

• H2O2 generate continuously using GO (glucose oxidase)
• DPI blocks NADPH oxidase, thus blocks ROS production (measured using chemiluminescence)
• generation of H2O2 via GO quantified as ROS production with/without DPI
• cell death (quantified by fluorescence microscopy using Sytox green strain which permeates dead cells) significantly less in presence of DPI, but not in presence of H2O2)

Question 19

Can neutrophils from CGD patients form NETs?

Answer 19

GDP patients lack NADHP oxidase
- with added GO neutrophils able to produce ETs

Question 20

What traps are formed that kill pathogens?

Answer 20

Expulsion of chromatin from neutrophils (NETosis) and other cells (ETosis) forms antibacterial mesh traps which kill pathogens

Question 21

What activating factors are there for ET formation?

Answer 21

Extracellular trap formation has numerous activating factors, including LPS, cytokines, PMA, and is ROS-dependent

Question 22

What are the ket proteins decorating the NET?

Answer 22

Key proteins decorating the net include histone and elastase

Question 23

How is ETosis distinct from apoptosis and necrosis?

Answer 23

ETosis distinct from apoptosis and necrosis, is caspase-independent, not accompanied by DNA fragmentation

Question 24

What is shown about NETs and microorganisms?

Answer 24

Microorganisms are killed by neutrophils stimulated to produce NETs, but microbicidal activity eliminated if NETs disrupted by treating activated neutrophils with DNases

Question 25

CGD patients and NETs

Answer 25

Neutrophils from CGD patients cannot form NETs unless stimulated downstream of NADPH oxidase, by using GO to generate H2O2