14 - Cell Death and Immunity Flashcards
What is the process involving NETs referred to as?
Process referred to as:
NETosis - neutrophil extracellular trap formation
or ETosis - extracellular trap formation, as several immune cells undergo process
What are NETs important in?
Important in antimicrobial immunity, conserved throughout evolution
- NETs detectable in pus – presence related to viscosity
What are NETs associated with?
Associated with certain disorders:
- Appendicitis, bovine mastitis, murine model of necrotizing fasciitis, pre-eclampsia
What can products ETs?
slime moulds produce ETs, even plants produce extracellular traps at the bottom of the growing root tips
When were NETs discovered?
- discovered 2004 by Brinkmann et al.
- further characterised by Fuchs et al 2007
Structure of NETs
- Extracellular chromatin structures, studded with globuli (30–50 nm diam.)
- NETs: flexible, surround the cell from which they originated and float in media
How can NETs be activated?
- 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
What can NET activation pathway involve?
Activation pathway can involve different receptors, e.g. TLRs, cytokine & Fc receptors
How can extracellular structures be dissolved?
Extracellular structures dissolved by addition of DNAse
- not instant, over the course of 60 to 90 minutes
What proteins are in NETs?
Globules of protein in NETs
- flexible NETs - look like clouds in culture medium
- stimulated with IL-8
What can neutrophil elastase do?
Neutrophil elastase – can cleave virulence factors of Shigella, Salmonella, Yersinia
- neutrophil elastase is a key protein on the NET
How do neutrophils die?
Neutrophils die via a regulated form of cell death to release NETs
Fuchs et al 2007 study
- 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
What did the Fuchs et al 2007 study show about the formation of NETs
Disintegration of the nucleus and granules allows the formation of NETs
Different morphologies displayed by apoptosis, necrosis and NETosis
- 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
What is NET cell death independent of?
NET cell death is caspase-independent, not accompanied by DNA fragmentation
Involvement of ROS in NET production
- 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
How are H2O2 and catalase (ROS) involved in NET production
- 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)
Can neutrophils from CGD patients form NETs?
GDP patients lack NADHP oxidase
- with added GO neutrophils able to produce ETs
What traps are formed that kill pathogens?
Expulsion of chromatin from neutrophils (NETosis) and other cells (ETosis) forms antibacterial mesh traps which kill pathogens
What activating factors are there for ET formation?
Extracellular trap formation has numerous activating factors, including LPS, cytokines, PMA, and is ROS-dependent
What are the ket proteins decorating the NET?
Key proteins decorating the net include histone and elastase
How is ETosis distinct from apoptosis and necrosis?
ETosis distinct from apoptosis and necrosis, is caspase-independent, not accompanied by DNA fragmentation
What is shown about NETs and microorganisms?
Microorganisms are killed by neutrophils stimulated to produce NETs, but microbicidal activity eliminated if NETs disrupted by treating activated neutrophils with DNases
CGD patients and NETs
Neutrophils from CGD patients cannot form NETs unless stimulated downstream of NADPH oxidase, by using GO to generate H2O2
