16 - ETosis Pathology and Disease Flashcards
Is the extruded DNA solely from a nuclear source?
Yousefi et al., 2009
- neutrophils primed with GM-CSF and stimulated by TLR-4 and C5a receptors generated extracellular traps comprising mitochondrial DNA
- mitochondrial DNA ROS-dependent, and occurs independently of cell death
NET formation vs NETosis
NET formation:
- mitochondrial DNA
- efficient innate immune response
- supports adaptive tune responses
- rapid resolution of inflammation
NETosis:
- cell rupture and nuclear collapse
- immunopathology
- excessive inflammation
- possible autoimmune responses
What happens in response to pathogens?
- Evidence that neutrophils can detect pathogen size and release traps against larger pathogens, demonstrated by Branzk et al (2014) using fungal cells
- ‘Decision’ regulated by competition for neutrophil elastase (NE)
Neutrophils that meet a microbe too large to be phagocytosed do not form phagosomes - NE released into cytosol without membrane fusion, and is then free to
translocate to the nucleus and drive chromatin decondensation
Does size selection operate in vivo? (Branzk et al, 2014)
- Mice infected via intra-tracheal route with wild-type Candida albicans (able to form yeast and hyphae) and with a C. albicans mutant unable to form hyphae (yeast-locked hgc1Δ )
- Lung tissue showed presence of traps only with wild-type C. albicans
- NETosis induced by small bacteria, but often species whose virulence creates large aggregates and/or interferes with phagosomal killing (e.g. S. aureus, Pseudomonas aeruginosa, Neisseria gonorrhoea)
Protection during pregnancy?
- Neutrophils collected from patients with intra-amniotic infection (infection of chorion, amnion, amniotic fluid, placenta, or a combination).
1. Maternal (peripheral blood) samples. - Unstimulated
2. Maternal (peripheral blood) samples. - Stimulated with PMA
3. Amniotic fluid neutrophils. - Unstimulated
- AF neutrophils: assumed to be of foetal origin, but may be mix of maternal/foetal
Pathogen evasion of ETs
- Inhibition of NET formation, e.g., by induction of IL-10 or production of inhibitory factors
- capsule: increases resistance to killing
- secretion of endonuclease
- conversion of NET-derived products into cytotoxic molecules
e.g., S. aureus adenosine synthase (a 5’,3’-nucleotidase) converts NET-derived nucleotides into dAdo, triggering apoptosis in macrophages
Role of lysins in pathogen evasion
- significant NET induction observed with/without pneumolysin
- S. pneumoniae, wild type, +ve pneumolysin
- S. pneumonia, mutant, -ve pneumolysin
What did pneumolysin do to the bacteria?
Pneumolysin reduced quantity of bacteria trapped on the NETs
Diseases/pathologies associated with extracellular traps
Pre-eclampsia, Cystic fibrosis, Gum disease, Thrombosis, Strokes, Pancreatitis, Antherosclerosis, Arthritis, Autoimmunity, Psoriasis, Cancer
What is human preeclampsia?
Severe disorder of late pregnancy, linked to placental abberancies, particularly elevated release of inflammatory syncytiotrophoblast debris
What are the symptoms of pre-eclampsia
Symptoms: hypertension, proteinuria, extreme cases lead to liver/kidney failure, or foetal hypoxia
Pre-eclampsia: in vitro and in vivo
- Known that pre-eclampsia associated with elevated foetal cell-free DNA
- Placenta-derived microparticles (syncytiotrophoblast microparticles: STBM) inducing NET formation in vitro
- In vivo – NETs in close contact with syncytiotrophoblast layer
Syncytiotrophoblast
Epithelial structure of placental villi that invades uterine tissue and determines which substances cross the placenta (e.g., nutrients and oxygen) and which do not (e.g., maternal hormones and certain
toxins)
Tissue sections of placenta from pre-eclampsia
- Normal placenta: NEts present within villi or adjacent to syncytiotrophoblast
- Pre-eclamptic: NEts diffuse throughout intervillous space
What is cystic fibrosis caused by?
Caused by mutations in cystic fibrosis transmembrane conductance regulator (CFTR) gene
What does cystic fibrosis code for?
Codes for an anion channel, resulting in altered transcellular chloride and bicarbonate transport
Characteristics of cystic fibrosis
- Disproportionate neutrophil abundance
- NETs abundant at sites of infection
- Thick mucus facilitates microbial lung colonization
- Persistent, neutrophil-rich inflammation
Cystic fibrosis disease research progression
- > 85% CF patients suffer premature mortality through respiratory complications
- Progress slow due to lack of animal models, currently pigs and ferrets being used as both species develop similar lung conditions
Normal lung vs CF lung
NORMAL:
- degranulation
- bacterial killing by phagocytosis
- phagocytosis of apoptotic neutrophils
- resolution of inflammation
CF LUNG:
- NET formation in response to bacteria
- frustrated inflammation
- possible lung damage from neutrophil contents
What does NE do?
Degrades lung structural proteins, reduces ciliary motility
Where are NETs present in CF?
NETs present in CF sputum - palliative treatment includes inhalation of recombinant DNase-1
Neutrophils in cystic fibrosis
- CF lung shows higher proportion of neutrophil survival
- Unclear whether this arises from primary neutrophil defect or a response to chronic inflammation
CF and apoptosis
- CF neutrophils show decreased apoptosis
- Delayed apoptosis related to a loss of cystic fibrosis transmembrane conductance regulator (CFTR) function
- Demonstrated by CFTR null neutrophils from CFTR-/- piglets (CF piglets)
What is prolonged cystic fibrosis neutrophil lifespan due to?
- Prolonged CF neutrophil lifespan is not caused by inflammation
- CF neutrophils show normal baseline apoptotic signalling of both Mcl-1
(antiapoptotic) and BAX (proapoptotic) proteins, implicated in inflammation-induced neutrophil survival
NET production: CF neutrophils vs healthy
CF neutrophils form more NETs than healthy controls when aged in culture conditions
Inflammatory responses in cystic fibrosis
- NETs stimulate an inflammatory response from macrophages, which is exaggerated in CF
- Demonstrated by co-culture of net-producing neutrophils and monocyte-derived macrophages (MDM), which showed increased production of cytokines IL-8 and TNF
NETs and gum disease
- NETs involved in periodontitis, caused by Porphyromonus
- Neutrophils recruited into gingival crevices
- Chronic periodontitis/hyper-coagulation associated with atherothrombosis
- Has been associated with abdominal aneuryism
NETs and cancer
- Evidence exists that circulating tumour cells trapped by NETs in vitro
- Murine model demonstrated microvascular NET deposition and trapping of lung carcinoma cells
- Trapping associated with increased formation of micrometastases at 48 hours and gross metastatic disease
- Effects were abrogated by DNAse or neutrophil elastase inhibitor
NETs & cancer associated thrombosis
- high level of association between cancer and thrombosis
- evidence that tumour cell vesicles (exosomes) stimulate NETs
NETs and heparin-induced thrombocytopenia (HIT)
- HIT: caused by pathogenic Ab (HIT Abs) specific for complexes of heparin and cytokine
- CXCL4 (aka PF4 - platelet factor 4)
- CXCL4 released in high concentrations at sites of platelet activation
- Binds to surface glycosaminoglycans (GAGs) on platelets, monocytes, endothelial cells, and neutrophils
NETs and COVID-19
“Patients with COVID-19: in the dark NETs of neutrophils” Ackerman et al.
- Neutrophilia directly correlates with disease severity in COVID-19
- Increased serum levels of NET degradation products (neutrophil-derived
- MPO-DNA, citrullinated histone H3) closely parallel lung distress and predict COVID-19 severity
- NETs abundant in circulation, and in lung and kidney tissues of COVID-19 patients
Possible therapeutic interventions of COVID-19
Possible therapeutic interventions:
- Heparin (neutralizes histones)
- Cytokine inhibitors
- DNAses
- Pro-inflammatory inhibitors
Cell death comparison
- NECROSIS: uncontrolled, passive, affects large areas of cells
- APOPTOSIS: controlled, energy dependent, occurs in single cells, clusters
- ET-OSIS: controlled, ROS dependent, primarily occurs in phagocytes but not exclusively
What are other forms of cell death (not apoptosis etc.)
Autophagy/autophagic cell death, pyroptosis, necroptosis
Stages of autophagic cell death
- phagophore - isolation membrane
- cytosolic proteins and organelles
- vacuoles appear in cell, no nuclear condensation
- autophagosome
- (+ acid hydrolyses in lysosome) autolysosome
Features of autophagy
- May be activated by starvation, cellular damage
- Removes protein complexes, damaged organelles or intracellular pathogens
- Can occur when apoptosis is blocked
- Can cause cell death – sporadic clearance of cell remains by phagocytes
Pyroptosis
- Novel, pro-inflammatory form of cell death induced by Salmonella and Shigella
- Uniquely dependent on caspase-1 (not involved in apoptotic cell death)
Role of caspase-1 in pyroptosis
- Caspase-1: processes pro-forms of inflammatory cytokines IL-1 and IL-18
- Caspase-1 activation in macrophages infected with Salmonella or Shigella results in processing of these cytokines and death of the host
cell - Observed caspase-1 activation/dependence during cell death in immune, CNS, and cardiovascular systems indicates significant role of pyroptosis in various biological system
Comparison of apoptosis and pyroptosis characteristics
- cell lysis (P)
- cell swelling (P)
- pore formation (P)
-membrane bedding (A) - nuclear condensation (A/P)
- DNA fragmentation (TUNEL positive) (A/P)
- caspase-1 activation (P)
- caspase 3 activation (A)
- caspase 7 activation (P)
- MOMP/ cytochrome C release (A)
- ICAD cleavage (A)
- PARP cleavage (A)
- glycolysis enzyme inactivation (A/P)
- inflammation (P)
What kind of cell death is HIV associated with?
Pyroptosis
Necroptosis
- Mediated via death receptor family proteins, e.g. Tumour Necrosis Factor (TNF), as well as several other PRRs
- Caspase-independent
- occurs independently of caspases, dependent on kinases, such as receptor- interacting protein kinase (RIP-1)
When does necroptosis occur?
Occurs when caspase-8 activation is absent
What is necroptosis dependent on?
Dependent on interaction between kinases:
- Receptor-interacting protein kinase (RIP) -1—-> RIP-3 —-> Mixed-Lineage Kinase domain-Like (MLKL)
What is necroptosis beneficial for?
Beneficial as ‘back-up’ e.g. viral inhibition of apoptosis
What is released in necroptosis (compare to apoptosis?
In NECROPTOSIS there is massive DAMP release and it is a strong inducer of inflammation
In APOPTOSIS there is limited DAMP release and weak inducer of inflammation
DAMP:
Damage-Associated Molecular Pattern e.g. cytokines (IL-1), alarmins,
nucleic acid, histones, heat-shock proteins
What are NETs beneficial in and detrimental to?
Neutrophil extracellular trap beneficial in anti-microbial defence, detrimental in certain disease conditions, e.g. pre-eclampsia, cystic fibrosis, gum disease, cancer, thrombosis and COVID-19
What do neutrophils in CF show?
Neutrophils in cystic fibrosis show decreased apoptosis, increased survival, increased NET production and stimulate the inflammatory response of Mφs
What does evidence suggest about NETs and cancer?
Evidence suggests extracellular traps may facilitate cancer development through metastases
