2 - Neutrophils pt. 2 Flashcards
What is swarming?
- collective behaviour exhibited by cells, clime mounds, insects
- relies on positive feedback loops and self regulatory mechanisms
What was found about swarming in 2008?
Noted 2008 for neutrophils in mouse lymph nodes infected with protozoan parasites
Advances in swarming
- Since then, advances in cell imaging have enabled detailed characterization of swarms and their regulation, although control of resolution still largely unknown.
- ‘Pioneer’ neutrophils (NTs) react to stimulus and form small clusters, followed by large (several hundred) migration of other NTs. Swarms compete for NT recruitment
How can neutrophil swarm phenotypes be catergorised?
Neutrophil-swarm phenotypes can be broadly categorized into two forms: transient and persistent swarms.
Transient swarms
- Transient neutrophil swarms form small cell clusters for few minutes before quickly dispersing again
- Neutrophils perform chemotactic migration toward a neutrophil accumulation and migrate out again to join nearby growing swarm centres
- Individual neutrophils often move between competing swarms
Persistent swarms
Persistent swarms show a sustained neutrophil recruitment and form large-cell clusters that can remain stable and persist for hours.
Where are transient swarms found?
- lymph nodes
- lungs
- intestines
Where are persistent swarms found?
- skin
- liver
- spleen
- cornea
What did Kienle find about swarms (2021)?
- NTs secrete attractants, which act through cell surface-expressed G protein-coupled receptors (GPCRs) on adjacent cells
- high chemoattractant concentrations (positive feedback loop) lead to GPCR desensitisation at later stages of the swarm, mediated by GPCR kinases (esp. GRK2)
- desensitisation leads to optimal bacterial detection and containment
Killing mechanisms:
- phagocytosis - pathogens frequently opsonised e.g., by Ab or complement factors
- degranulation - extracellular release of anti-microbial molecules
- NETs
What happens after the killing mechanisms?
- Neutrophils cleared by apoptosis in liver, spleen or bone marrow
- Terminal trafficking – gut, to regulate commensals
- Reverse trafficking – migrate away from site
Reverse trafficking: possible mechanisms (in vitro cell culture)
a. chemoattractants act as chemorepellent at high concentrations
b. reverse transmigration through endothelial cell layers
c. reverse migration in microfluidic chambers depends on which signals a neutrophil encounters
Reverse trafficking: possible mechanisms (in vitro cell culture) described
a. Reverse neutrophil transmigration through endothelial cell monolayers has been reported; reverse transmigrated neutrophils have been characterised by high expression of intercellular adhesion molecule 1 (ICAM1) and low expression of CXC-chemokine receptor 1 (CXCR1)
b. In vitro analysis in microfluidics has identified factors that regulate neutrophil forward and reverse migration
c. The pro-resolving lipid mediator lipoxin A4 (LX4A) induces neutrophil reverse migration whereas zymosan induces neutrophil trapping.
How can neutrophil migration be a drug target?
- Failure to appropriately resolve inflammation - contributes to several inflammatory diseases, e.g. rheumatoid arthritis, pulmonary fibrosis, multiple organ failure
- Neutrophil behaviour - potential target for drug therapies
- Targeting forward migration: inhibit primary signals for neutrophil recruitment or amplification
- Targeting reverse migration: promote neutrophil reverse migration from local sites of damage
Examples of drugs in current clinical trials that specifically target neutrophil migration signals
- SCH 527123 targets CXCR2 - severe asthma, allergen induced asthma, COPD, psoriasis, and colon cancer
- Reparixin targets CXCR1 and CXCR2 - ischaemia-reperfusion injury, lung, pancreatic islet and kidney transplantation, and beast cancer
What are neutrophils classed as?
neutrophils are classed as ‘terminally differentiated’ cells
Can neutrophils transform?
No capacity to transform into other cell types
What are neutrophil subsets?
Now known that neutrophil subsets exist which show different phenotypes and functionality, and which can be characterized by expression of receptors/markers
Examples of different neutrophil phenotypes
Tumour-associated neutrophils: N1 (anti-tumorigenic and pro-inflammatory) and N2 (pro-tumourigenic and immunosuppressive)
Variations in neutrophil cell surface receptor expression include
-CXCR4 expression in aged/senescent neutrophils
-CD63 expression in the airways of cystic fibrosis patients
-Intercellular adhesion molecule 1 (ICAM1) expression associated with systemic inflammation and reverse migration
What has research suggested about neutrophils and B cells?
- Research has suggested neutrophils enhance B cell function and that this results from neutrophils being ‘reprogrammed’ by spleen endothelial cells
- Originally thought that neutrophils only occupy the spleen during bacterial infection, observations suggested that there was natural colonization in the spleen marginal zone
What has been proposed about neutrophils and B cells?
Proposed that interleukin 10 released from spleen cells ‘re-programmed’ neutrophils, which subsequently activated B cells and induced functions such as Ab production
- Defined existence of a neutrophil B helper cell sub-type, possessing a distinct phenotype (observable properties of a cell/organism)
Puga et al 2011
Model of reprogramming proposed:
T cell-independent responses by human MZ B cells
- T cell-independent (Tl) antigens are thought to enter the marginal zone (MZ) through the perifollicular zone
- Once in the MZ, they may be captured by neutrophil extracellular trap (NET)-like structures emanating from B cell-helper neutrophils (NBH cells)
- These cells may differentiate from circulating neutrophils as a result of the production of interleukin-10 (IL-10) by perifollicular sinus-lining cells and macrophages in response to microbial Toll-like receptor (TLR) ligands
- Antigen capture may also involve reticular cells, macrophages, sinus-lining cells and dendritic cells (DCs)
- In addition to making Tl antigens available to the B cell receptor (BCR) and TLRs on MZ B cells, antigen-capturing cells release B cell-activating factor (BAFF) and a proliferation-inducing ligand (APRIL), which engage transmembrane activator and CAML interactor (TACI) on MZ B cells. NBH cells also release IL-21, thereby inducing class-switch recombination, somatic hypermutation and antibody production in MZ B cells
- The generation of plasmablasts secreting IgM or class-switched IgG and IgA involves the production of IL-6, IL-10, IL-21 and CXC-chemokine ligand 10 (CXCL10) by antigen-capturing cells.
FIGURE: Arrows indicate the putative path followed by antigens through the spleen. IFN, interferon; PALS, periarteriolar lymphoid sheath; PRR, pattern-recognition receptor
Was the proposed model successful?
“Failure to Detect Functional Neutrophil B Helper Cells in the Human Spleen” Nagelkerke et al.
Main points of the lack of neutrophil B helper cells in the spleen paper
- Homogenous population of neutrophils detected in fresh human spleen samples, which did not differ in phenotype/function from blood neutrophils
- No phenotypic characteristics of costimulatory nature detected on neutrophils
- No reproduction of Ig production by splenic B cells in the presence
- of splenic neutrophils detected
- B cell function and neutrophil activity were normal
- Independent confirmation of a role for NBH cells is required.
Possible reasons for the failure to detect function neutrophil B helper cells
- Protocol differences for spleen cell collection and isolation
- Cell contamination – B cells consistently contaminated neutrophil cultures
- Neutrophils help by secreting cytokines, but may not have a different
phenotype?
Can metabolic changes alter neutrophil production, phenotype and function?
- Studies in mice suggestion diurnal changes in neutrophil phenotypes
- Conditions such as hyperglycaemia or hypercholesterolaemia now
believed to influence increased production of reactive oxygen species (ROS) from neutrophils - ROS essential for killing
-BUT Increased ROS linked to chronic inflammation
Neutrophil phenotypes and disease
Different NT phenotypes now identified and associated with conditions
including: gum disease, asthma, cancer, survival from trauma
Neutrophil phenotypes and COVID?
Different NT phenotypes recently linked with Covid severity, via examination of Covid patients and proteomic analysis of > 1700 NT proteins
- immunomodulatory receptors: significant differences in abundance in NT proteome
- migratory receptors/integrins: exclusively reduced abundance
What does neutrophil swarming involve?
Neutrophil swarming involves positive feedback loop for aggregation
and desensitization to limit swarms
What do killing mechanisms include?
Killing mechanisms include phagocytosis, degranulation and
extracellular trap formation
What do killing mechanisms include?
Killing mechanisms include phagocytosis, degranulation and
extracellular trap formation
Why is neutrophil life span/migration important?
Neutrophil life span/migration important in balance between acute and
chronic inflammation. Manipulation of migration of interest in drug
development
Do neutrophil phenotypes exist?
Neutrophil phenotypes exist, including reports of new ‘helper’ neutrophil sub-type in spleen and associations with many different pathologies
Why is ROS important?
Some phenotypes produce excess ROS: ROS needed for killing microbes, excess linked to numerous health conditions
What has neutrophils recently been linked to?
Recent report links NT phenotype to Covid outcomes
How does extravasation occur?
by paracellular or trans cellular routes
Phenotypes of neutrophils
Neutrophils possess different phenotypes that have been linked to
factors such as aging and disease conditions, as well as claims that
distinct sets of ‘B helper’ neutrophils exist
