L8 Neutrophils

Question 1

what are neutrophils?

Answer 1

Neutrophils are the first line of the cellular immune defense as first to arrive at site of infection or injury. Up to 70% of white blood cells are neutrophils so continuously released from bone marrow. 10^11 are produced everyday and released. Very short life span only 6-12 hours old. When recognise threat they extravasate (leave blood vessel) and migrate along chemotaxic gradient to site of infection to employ effector functions.

Question 2

key features of neutrophils?

Answer 2

Key feature is a lobulated nucleus so also called polymophonuclear (PMN) cells. They carry granules that contain antimicrobials so also called granulocytes that also comprise basophils and eosinophils. Further neutrophils are charcterised by producing lots of reactive oxygen species. Produce ros more than other immune cells.
Neutrophils have transcriptional and epigenetic variability. When subjected to methylation and rna sequencing- neutrophils had highest variability among individuals.

Question 3

neutrophil life cycle?

Answer 3

Steps of Granulopoiesis (Specific to Neutrophils):

Hematopoietic Stem Cells (HSCs):

Found in the bone marrow, these multipotent stem cells differentiate into all blood cell types.
Common Myeloid Progenitor (CMP):

HSCs give rise to CMPs, which are committed to producing myeloid lineage cells (e.g., granulocytes, monocytes, megakaryocytes, and erythrocytes).

CMPs further differentiate into GMPs (Granulocyte-Macrophage Progenitor ) , which are precursors for granulocytes and monocytes.

Development into neutrophils is driven by gsf: granulocyte colony stimulating factor. (a growth factor) Activaes cascade of diff lineage determining transcription factor. Neutrophils terminally diffeentiate/ only proliferate in bone marrow. Unlike t and b cells. Lots of morphalogical changes in development to cytoplasm and nucleus. Band shape- last immature progenitor. But can be released prematureley during acute infection/inflammation e.g: sepsis, cancer, autoimmune disease.
segmenter have secratory vesicles.
Expression of surface receptors change during
Development.
Stromal cells in bone marrow
release stromal cell derived factor (SDF-1) AKA
CXCL12.
Binds to CXCR4- expressed on immature neutrophil progenitors.
Retains neutrophils in bone marrow and controls
Recruitment of immune cells into tissue.

Question 4

life cycle cells?

Answer 4

myeloblast (primary granules) to promyelocyte to myelocyte (secondary granules) to metamyelocytes to band (tertiary granules) to segmenter (secratory vesicles)

Question 5

surface receptor expression?

Answer 5

When endothelial cells on bv are stiluated they release chemokines cxcl1,2,8 that bind to the cxcr 2 receptor release neutrophil from bone marrow into bloodstream. So surface receptor expression controls retention and release of neutrophils into bone marrow.

Question 6

after leaving bone marrow and returning?

Answer 6

After leaving bone marrow: freely circulating for 6-12 hrs.
Homing: return to bm for elimination. Only 50-70% follow this process. Follow a circadian rhythm. Have spatial plasticity (diff functions in diff organs) and temporal regulation. Morning released night eliminated.
30-50%= marginated pool so they stay in the microvasculator of specific tissues: lung, spleen, liver so neutrophils become older- up to 5 days. Do this to fulfil functions. In lungs reside for microbial effects. To be immediatley ready to attack pathogens, in spleen: specialised b cell helper neutrophils to give signals to b cells to proliferate and produce ab. In liver eliminated.

Question 7

efferocytosis?

Answer 7

Efferocytosis: neutrophils undergo apoptosis and are phagocytosed by macrophages. Macrophages produce low levels of il-12. Apoptosis suppresses il-23, which downregulates il-17 regulation and so g-csf regulation (the growth factor driving granulo…) so negative feedback if too many neutrophils are efferocytosed they will turn down new neutrophil porduction in the bone marrow.
Recruitment and activation is tightly regulated. Neutrophils good for bacterial infection but too many= cause damage to tissue and pathology.

Question 8

what are their different surface receptors?

Answer 8

Different surface receptors so recognise different cell surface receptors using different intracellular signalling pathways e.g: GPCR for- chemokines e.g cxcr 1 and 2 receptor, complement proteins and lipid regulators e.g: leukotriene ltb4.
Recognise antibodies by Fc receptor (gamma and alpha)
Recognise icams: intracellular cell adhesion modules expressed on endothelial cells, important for extravasation.
Recognise g-csf and gm-csf (growth factors) through type 1 and type 2 cytokine receptors.
Recognise pamps through prrs: tlr and c-type lectin. E.g: mannose, lps, ssrna by tlr-7 and tlr-8. Effect recruitment, activation and migration of effector functions

Question 9

extravasation?

Answer 9

Extravasation to reach the site of infection:
When endothelial cell activated e.g: through cytokines it upregulates regulation of P and E selectins. psgl-1 which bind to L-selectin on the nutrophil (also known as cd-62l). Interaction is loose, selectin bonds prone and break easily leads to rolling of neutrophil along the endothelium. Rolling can form membrane protrusions? Due to Selectin binding = intracellular signalling activated. Leads to clustering of integrin receptors: Lfa-1 and mac-1 bind to icam on endothelial cells, cause stop of cell, firm adhesion. Cytoskeleton rearranged. Cell forms lamellipodum. Cell Penetrates between gap junction of endothelial cells squeezes through blood vessel into tissue, secretory vesicles are mobalised. Neutrophils release enzymes to break the extracellular matrix and so can move into the tissue (transmigration) and move along a chemotaxic gradient.
Neutrophils recruitment is fast and tightly regulated. Neutrophil swarm as they arrive fast at site of infection.

Question 10

effector functions of neutrophils>

Answer 10

Effector functions of neutrophils: ros prod, phagocytosis and degranulation, net formation and release of cytokines inflammation.
Oxidative burst initiated by enzyme nox2 (NADPH oxidase 2) triggers cascade where different radicals of oxygen are produced. Nox2 is multisubunit enzyme complex assembles in membrane, nadph is its cofactor, nadph delivers a electron that is transferred through the membrane on a moleculcular oxugen leading to a super reactive superoxide. Immediatley react with proteins , ipids, dna , rna other radicals. Oxidate everything so destory function of molecule. Superoxide spontaneously or mediated by superoxide dis? To form h2o2 and mpo enzyme convert h2o2 to hocl.
Mpo: enzyme specific to neutrophils

Question 11

phagocytosis by neutrophils?

Answer 11

Phagocytosis by neutrophils
Sensing and Ingestion of
particles larger 0.5μm
* Phagosome maturation upon
sequential fusion with
granules - phagolysosome
* Delivery of antimicrobial
peptides, enzymes, chelators to phagosomal lumen
* enzymes can assemble in phagosomal membrane and cause Production of ROS and pump into phagosomal lumen
* pH regulation. Pump protons into phagosomal lumen. Make hostile environment for pathogen.
Phagocytic receptors insude phagocytosis. Opsonic receptors: recognise microbes that are opsonised, coated with complement proteins or antibodies.
Cr and fcyr can induce phagocytosis. Non opsonic receptors: prrs binding to pamps and c-type lectin receptors e.g: dectin-1 binds to fungal polysaccharides.

Question 12

granules?

Answer 12

Following phagocytosis. Granules needed to fuse with phagosomes and alos fuse with cell membrane of neutrophil an d be released extracellularly (degranulation).
Can also be produced by neutrophils in the bone marrow. Primary (asurophilic) secondary (specific) eritary (gelatinase) and secretory vesicles. Form by budding from the golgi apparatus and secretory vesicles form by endocytosis of the cell membrane. Immobalised in a sequence that couunters their prpduction in bone marrow i.e: what is produced last (secretory vesicles) are immobalised first. These vessels contain receptors such as fc receptors and complement receptors what neutrophil needs first to be activated? And react to pathogen. Tertiary next, contains enzymes to degrade extracellular matrix for neutrophil to move to tissue and reach infection site, then secondary specific have lactoferrin Binds to iron and deprives microbes of essential nutrients. Azurophillic, mpo enzyme (Myeloperoxidase)) plays role in ability to kill pathogens by reactive oxygen spcies as it is involved in oxidative burst.

Question 13

alpha defensives?

Answer 13

Know the alpha-defensives. present in azurophilic granules. Cationic (positively charged and) bind to negativley charged lipid membrane, or negatviely charged backbones of dna and rna. form pores in membrane. ll-37 also a small cationic peptide. Neutrophils elestas, calprotectin bind to zinc and membranes, depriving microbes.

Question 14

NET?

Answer 14

Net: trap, disarm and kill pathogens. Evolutionary conserved.
effective against mainly bacteria and fungi.
These nets are covered with antimicrobials so deliver high local concentration of antimicrobials. Trap to keep microbes in place.
Nets are composed of chromatin, and antimicrobials. The histones associated with chromatins are cytotixic, small cationic. Also form pore in membranes. Also have granule structures in membrane e.g: ellestas. Have mpo too. Composition depends on stimulus.

Question 15

NET inducers?

Answer 15

• bacteria
• fungi
• protozoa
• viruses
• activated platelets
• complement-derived
  peptides
• autoantibodies
• Urate/ cholesterol
  crystals
• cigarette smoke

Question 16

NETosis?

Answer 16

Process of net formation is called NETosis, takes about 2-5hrs. Naive neutrophil first, after 1hr chromatin decondensation,nucleus enlarhes, 2hrs ne dissassembles, chromatin mixes with granules and cytoplasmic materia,s 3 hrs the cell starts to rupture due to force of chromatin expansion.
Net formation mechanism: Essential role for ROS production, NE & MPO. NE and MPO synergise to mediate chromatin decondensation. In resting neutrophils these enzymes are in azurophillic granules. The granules hardest to mobalise. When neutrophil recieves stimulus to proruce reactive oxygen species, neutrophil elastase translocates to nucleus and protease starts to cleave histones inducing decondesnation of chromatin. Mpo also translocates to nucleus and both enzymes synnergise to mediate the cleavage of histones and decondensation of chromatin.

Question 17

NET in disease?

Answer 17

High local concentrations of antimicrobials released during inflammation can be toxic to host cells, particularly epithelial and endothelial cells. Histones, due to their strong cationic charge, disrupt membrane integrity and contribute significantly to this toxicity.

NETs (neutrophil extracellular traps) are inflammatory structures composed of DNA and antimicrobial proteins. These NETs are recognized by other immune cells, such as macrophages, which are activated to produce pro-inflammatory cytokines. The combination of DNA and antimicrobial components also triggers receptor-mediated responses that amplify inflammation.

Key roles of NETs:

Thrombosis: NETs can form scaffolds within blood vessels, trapping circulating platelets. Proteases present on NETs can activate the coagulation cascade, contributing to blood vessel occlusion during thrombosis.
Cancer: NETs can trap tumor cells, promoting metastasis by creating a microenvironment favorable for cancer spread.
Autoimmune Diseases: NETs are a source of autoantigens. For instance, RNA released during NETosis can be recognized by autoantibodies in autoimmune diseases such as systemic lupus erythematosus (SLE). These immune complexes can further stimulate neutrophils to form more NETs, creating a feedback loop that exacerbates the disease.
Gout: In gout, urate crystals that accumulate in joints can induce NET formation. NET-associated proteins, such as elastase, may cleave pro-inflammatory cytokines and potentially modulate inflammation under certain conditions.
In the lungs during influenza infection, neutrophils can guide T cells to the infection site by releasing vesicles containing CXCL12, which binds to CXCR4 receptors on T cells, aiding their migration.

Question 18

atherosclerosis and net?

Answer 18

Example: Atherosclerosis
High cholesterol levels. Driven by pro inf cytokine il-1beta. When neutrophils stimulated by cholesterol crystals, induce net formation deposited on crystals. Net components can be found in atherosclerosis lesions. Nets recognised by macrophages by tlr to activate macrophage to prod pro inf cytokines il-1beta. To recruit more neutrophils and make them more prone to net formation.

Question 19

crosstalk?

Answer 19

Neutrophil Crosstalk and Interaction with Immune Cells
Neutrophils are the first responders to infection sites and play a crucial role in coordinating immune responses by instructing other immune cells.

Key Interactions:
Chemokine Signaling:

CXCL8 (IL-8): Binds to CXCR2, recruiting additional neutrophils to the site of infection.
CCL2: Recruits monocytes, which can differentiate into macrophages or dendritic cells.
CXCL12: Activates immune cells such as T cells, guiding their migration to infection sites.
Cytokine Secretion:

Neutrophils produce IL-1, IL-18, TNF, and IL-12, which are pro-inflammatory cytokines that activate T cells and amplify the immune response.
Antimicrobial Molecules with Secondary Functions:

Neutrophil granules contain antimicrobial peptides and proteases like LL-37, cathepsin G (CG), and azurocidin, which also act as chemoattractants, recruiting immune cells to infection sites.
Lipid Mediator Production:

Neutrophil stem cells produce lipid mediators, such as:
PGE2, LTB4: Promote inflammation.
Lipoxins, resolvins, and protectins: Mediate anti-inflammatory and resolution-phase responses.
B Cell Support:

Neutrophils produce factors like BAFF (B-cell activating factor) and APRIL, which promote B cell proliferation and antibody production.
Immune Suppression:

Reactive Oxygen Species (ROS): Can suppress immune responses by reducing cytokine production in other immune cells, exhibiting immunosuppressive effects.
Arginase: Depletes arginine, an amino acid essential for T cell proliferation, thus suppressing T cell responses.
Additional Roles of Neutrophils:
Recruitment and Activation: Mobilize monocytes, dendritic cells (DCs), and T cells to infection sites.
Antigen Presentation: Support antigen presentation functions, aiding dendritic cells and macrophages.
Macrophage Reprogramming: Facilitate macrophage reprogramming through efferocytosis (clearance of apoptotic cells).
B Cell Activation: Enhance B cell proliferation and antibody production, particularly in the spleen.
T Cell Modulation: Can either activate or suppress T cell responses depending on the context.

Question 20

cross talk with t cells?

Answer 20

Neutrophil elestase can cleave and deactivate inflammatory cytokines like il-1beta or And il-6 and il-2. Arginase depletion and ros suppress t cell function. Neutrophilexpress immune checkpoint inhibitors like pdl-1 that bind to pd-1 on t cell. Activating: cscl-12 and il-12 activate and recruit t cells. Present mhc 1 and 2 so can present antigens to t cells. Net in disease? Can be recognised by prr and activate t cells.